By RABIA JAVED - Higher Education...

Synthesis, Characterization and Applications of

ZnO and CuO Nanoparticles for Biological

Activities and Steviol Glycosides Production in

Stevia rebaudiana Bertoni

By

RABIA JAVED

DEPARTMENT OF BIOTECHNOLOGY

QUAID-I-AZAM UNIVERSITY

ISLAMABAD, PAKISTAN

2017

i

Synthesis, Characterization and Applications of ZnO and CuO

Nanoparticles for Biological Activities and Steviol Glycosides

Production in Stevia rebaudiana Bertoni

A thesis submitted to the

Department of Biotechnology,

Quaid-i-Azam University, Islamabad

In Partial Fulfilment of the Requirement for the Degree of

Doctor of Philosophy

In

Biotechnology

By

Rabia Javed

DEPARTMENT OF BIOTECHNOLOGY

QUAID-I-AZAM UNIVERSITY

ISLAMABAD, PAKISTAN 45320

2017

vi

DEDICATED

TO

MY PARENTS

iv

Acknowledgements

Thanks to Almighty ALLAH, the most gracious, merciful and compassionate WHO

bestowed man with intelligence and knowledge. I pay my gratitude towards HIM for

flourishing my thoughts with exaltation and enabling me to contribute something to mankind

and humanity.

I am sincerely thankful to my supervisor, Dr. Muhammad Zia, for his encouragement,

unprecedented guaidance, valuable and noteworthy suggestions and interest in my research.

He gave me a new insight in observing the things and broadened my horizon to see them in

different manners.

Many thanks to respected Dr. Zabta Khan Shinwari, Dean of Faculty of Biological Sciences,

and Dr. Muhammad Naeem, Chairman of Department of Biotechnology, Quaid-i-Azam

University, Islamabad, Pakistan, and all other faculty members for their helping and

encouraging behaviour during the whole research. I am also grateful to the administration

staff of department and university for their cooperative and nice behaviour.

I also wish to express my gratitude to Dr. Muhammad Usman, Department of Physics, Quaid-

i-Azam University, Islamabad, Pakistan for his generous help from the start to end of my

research. I am really very thankful to him for his greatness. Lots of appreciation to my

country lab fellows for their worthy cooperation and friendly behavior. My heartfelt thanks to

Saira Tabassum, Madiha Ahmad, Humaira Fatima, Hira Zafar, Attarad Ali and Bakht Nasir

for their moral support and caring attitude towards me during all my frustrations encountered

during the whole research period. Their immense companionship really meant a lot to me.

The section would be incomplete without mentioning the generous sponsorship of TUBITAK

because without their funding it was not possible for me to complete this research. My special

thanks and admiration to the Professor Dr. Ekrem Gurel who gave me chance to work in his

laboratory at Abant Izzet Baysel University, Bolu, Turkey for one year. I also express my

sincere gratitude to my Turkish lab fellows for their help and assistance in my research work.

It is my pleasure to mention the names of Dr. Buhara Yucesan, Yesim Yavuz, Aliyu

Mohamed, Gunce Sahin, Merve Arslan, Yunus Sahin, Cansu Cihangir, Baris Kilicsaymaz,

Tugce Illal and Ozge Kaya for their tireless cooperation and friendly behaviour with me in

Turkey.

v

In the end, I would like to deliver special thanks to my family; especially I owe great deal of

intellectual debt to my loving mother for her affection, love, support, encouragement and

belief in me. Her prayers and holy wishes made me able to complete this dissertation. My

feelings of indebtedness to my father, brothers and loving sister for their continuous support

that enabled me to complete this arduous task.

Rabia Javed

vi

Table of Contents

CHAPTER 1 ............................................................................................................................ 1

Introduction and Review of Literature .................................................................................. 1

1.1 Nanoparticles ............................................................................................................... 1

1.2 Metallic Oxide Nanoparticles...................................................................................... 1

1.3 Zinc Oxide (ZnO) Nanoparticles................................................................................. 1

1.3.1 Synthesis of ZnO Nanoparticles .......................................................................... 2

1.3.2 Capping of ZnO Nanoparticles ............................................................................ 3

1.3.3 Applications of ZnO Nanoparticles ..................................................................... 3

1.3.4 Biological Activities of ZnO Nanoparticles ........................................................ 4

1.4 Interaction of ZnO Nanoparticles with Plants ............................................................. 5

1.5 Copper Oxide (CuO) Nanoparticles ............................................................................ 6

1.5.1 Synthesis of CuO Nanoparticles .......................................................................... 7

1.5.2 Capping of CuO Nanoparticles ............................................................................ 8

1.5.3 Applications of CuO Nanoparticles ..................................................................... 8

1.5.4 Biological Activities of CuO Nanoparticles ........................................................ 8

1.6 Interaction of CuO Nanoparticles with Plants............................................................. 9

1.7 Characterization of Nanoparticles ............................................................................. 10

1.8 Stevia rebaudiana Bertoni ......................................................................................... 11

1.8.1 Structure of Steviol Glycosides ......................................................................... 11

1.8.2 Importance of Stevia .......................................................................................... 12

1.8.3 Tissue Culture of Stevia ..................................................................................... 13

1.9 In vitro Production of Steviol Glycosides ................................................................. 14

1.10 Aims and Objectives ................................................................................................. 16

CHAPTER 2 ........................................................................................................................... 17

Synthesis, Characterization and Bioassays of ZnO Nanoparticles and its Derivatives ... 17

2.1 Introduction ............................................................................................................... 17

2.2 Materials and Methods .............................................................................................. 18

2.2.1 Synthesis of ZnO, ZnO-PEG and ZnO-PVP Nanoparticles .............................. 18

2.2.2 Characterization of Synthesized Nanoparticles ................................................. 19

2.2.3 Biological Assays of Nanoparticles ................................................................... 19

vii

2.2.3.1 Antibacterial Assay ........................................................................................... 20

2.2.3.2 Antioxidant Activities ....................................................................................... 20

2.2.3.3 Antidiabetic Assay (α-Amylase Inhibition Assay) ........................................... 22

2.2.4 Statistical Analysis ............................................................................................. 22

2.3 Results ....................................................................................................................... 22

2.3.1 X-Ray Diffraction (XRD) .................................................................................. 23

2.3.2 Fourier-Transform Infra-Red Spectroscopy (FTIR) .......................................... 25

2.3.3 UV-Visible Spectroscopy .................................................................................. 26

2.3.4 Scanning electron microscopy (SEM) ............................................................... 27

2.3.5 Energy dispersive X-ray (EDX)......................................................................... 29

2.3.6 Antibacterial Activity of Nanoparticles ............................................................. 29

2.3.7 Antioxidant Activities of Nanoparticles ............................................................ 30

2.3.8 Antidiabetic Activity of Nanoparticles .............................................................. 30

2.4 Discussion ................................................................................................................. 32

CHAPTER 3 ........................................................................................................................... 34

Synthesis, Characterization and Bioassays of CuO Nanoparticles and its Derivatives .. 34

3.1 Introduction ............................................................................................................... 34


3.2.1 Synthesis of CuO, CuO-PEG and CuO-PVP Nanoparticles ............................. 35

3.2.2 Characterization of Synthesized Nanoparticles ................................................. 36

3.2.3 Biological Assays of Nanoparticles ................................................................... 36

3.2.3.1 Antibacterial, Antioxidant and Antidiabetic Assay .......................................... 36

3.2.3.2 Cytotoxicity Assay (Brine shrimp lethality assay) ........................................... 36

3.2.3.3 Antitumor Assay ............................................................................................ 37

3.2.4 Statistical analysis .............................................................................................. 37

3.3 Results ....................................................................................................................... 37

3.3.1 X-Ray Diffraction (XRD) .................................................................................. 38

3.3.2 Fourier-Transform Infra-Red (FTIR) Spectroscopy .......................................... 40

3.3.3 Scanning Electron Microscopy (SEM) .............................................................. 41

3.3.4 Energy Dispersive X-ray (EDX) Spectra ........................................................... 42

3.3.5 Antibacterial Activity of Nanoparticles ............................................................. 43

3.3.6 Antioxidant Activities of Nanoparticles ............................................................ 43

3.3.7 Cytotoxic Activity of Nanoparticles .................................................................. 44

viii

3.3.8 Antitumor Activity of Nanoparticles ................................................................. 45

3.3.9 Antidiabetic Activity of Nanoparticles .............................................................. 46

3.4 Discussion ................................................................................................................. 47

CHAPTER 4 ........................................................................................................................... 50

Tissue Culture and DPPH-free radical scavenging activity of Stevia rebaudiana Bertoni

.................................................................................................................................................. 50

4.1 Introduction ............................................................................................................... 50


4.2.1 Seed Germination............................................................................................... 51

4.2.2 Callus Induction ................................................................................................. 51

4.2.3 Shoot Organogenesis ......................................................................................... 52

4.2.4 Root Organogenesis ........................................................................................... 52

4.2.5 DPPH-free radical scavenging activity .............................................................. 52


4.3 Results ....................................................................................................................... 53

4.3.1 Callogenesis ....................................................................................................... 53

4.3.2 Shoot Organogenesis ......................................................................................... 56

4.3.3 Root Organogenesis ........................................................................................... 57

4.3.4 DPPH-free radical scavenging activity .............................................................. 59

4.4 Discussion ................................................................................................................. 60

CHAPTER 5 ........................................................................................................................... 63

Application of ZnO and CuO Nanoparticles to Callus Cultures of Stevia rebaudiana

Bertoni for Secondary Metabolites Production .................................................................. 63

5.1 Introduction ............................................................................................................... 63


5.2.1 Callusing under ZnO and CuO nanoparticles stress .......................................... 64

5.2.2 Extract preparation for antioxidant assays ......................................................... 64

5.2.3 Statistical analysis .............................................................................................. 65

5.3 Results ....................................................................................................................... 65

5.3.1 Physiology of Callus grown under ZnO and CuO nanoparticles stress ............. 65

5.3.2 Phytochemical Analysis of Callus under ZnO nanoparticles stress ................... 68

5.3.3 Phytochemical Analysis of Callus under CuO nanoparticles stress .................. 69

5.4 Discussion ................................................................................................................. 71

CHAPTER 6 ........................................................................................................................... 73

ix

Application of ZnO Nanoparticles to Micropropagated Shoots of Stevia rebaudiana

Bertoni for Steviol Glycosides Production ........................................................................... 73

6.1 Introduction ............................................................................................................... 73


6.2.1 Synthesis and Characterization of ZnO Nanoparticles ...................................... 74

6.2.2 Preparation of Medium containing ZnO Nanoparticles ..................................... 74

6.2.3 Growth Conditions of Plant Shoot Organogenesis ............................................ 74

6.2.4 Extraction of Steviol Glycosides ....................................................................... 75

6.2.5 Analysis of Steviol Glycosides .......................................................................... 75

6.2.6 Preparation of Extract for Anti-Oxidant Assays ................................................ 75

6.2.7 Antioxidant Assays ............................................................................................ 76


6.3 Results ....................................................................................................................... 76

6.3.1 Characterization of ZnO Nanoparticles ............................................................. 76

6.3.2 Determination of Physiological Parameters of Stevia rebaudiana .................... 80

6.3.3 Determination of Steviol Glycosides in Stevia rebaudiana ............................... 81

6.3.4 Determination of Anti-oxidant Activities of Stevia rebaudiana ........................ 82

6.4 Discussion ................................................................................................................. 83

CHAPTER 7 ........................................................................................................................... 85

Application of CuO Nanoparticles to Micropropagated Shoots of Stevia rebaudiana

Bertoni for Steviol Glycosides Production ........................................................................... 85

7.1 Introduction ............................................................................................................... 85


7.2.1 Synthesis and Characterization of CuO Nanoparticles ...................................... 86

7.2.2 Preparation of Medium having CuO Nanoparticles ........................................... 87

7.2.3 Growth Conditions of Shoot Organogenesis ..................................................... 87

7.2.4 Extraction and Analysis of Steviol Glycosides .................................................. 87

7.2.5 Preparation of Extract and Anti-Oxidant Assays ............................................... 87

7.2.6 Statistical Analysis ................................................................................................ 87

7.3 Results ....................................................................................................................... 87

7.3.1 Characterization of CuO Nanoparticles ............................................................. 88

7.3.2 Determination of Physiological Parameters of Stevia rebaudiana .................... 91

7.3.3 Determination of Steviol Glycosides in Stevia rebaudiana ............................... 92

7.3.4 Determination of Antioxidant Activities in Stevia rebaudiana ......................... 93

x

7.4 Discussion ................................................................................................................. 94

CHAPTER 8 ........................................................................................................................... 97

Application of PVP and PEG modulated CuO and ZnO Nanoparticles to

Micropropagated Shoots of Stevia rebaudiana Bertoni for Steviol Glycosides Production

.................................................................................................................................................. 97

8.1 Introduction ............................................................................................................... 97

8.2 Materials and methods ................................................................................................. 98

8.2.1 Shoots Development in Medium containing Nanoparticles .................................. 98

8.2.2 Extract Preparation and Steviol Glycosides Analysis ........................................... 98

8.2.3 Extract Preparation and Antioxidant Activities ..................................................... 99

8.2.4 Statistical Analysis ................................................................................................ 99

8.3 Results .......................................................................................................................... 99

8.3.1 Physiology Analysis of Stevia rebaudiana ........................................................ 99

8.3.2 Steviol glycosides Analysis in Stevia rebaudiana ................................................. 99

8.3.3 Antioxidant Activities Analysis of Stevia rebaudiana ....................................... 102

8.4 Discussion ............................................................................................................... 103

General Summary ................................................................................................................ 105

Future Recommendations ................................................................................................... 108

References ............................................................................................................................. 109

xi

List of Tables

Table 2.1: Particle size, lattice parameters (a, c) and c/a ratio of ZnO, ZnO-PEG and ZnO-

PVP nanoparticles. ................................................................................................................... 24

Table 2.2: Antibacterial activity of nanoparticles in terms of their zone of inhibition. .......... 30

Table 2.3: Antioxidant assays: flavonoid equivalent determination (FED), phenolic

equivalent determination (PED), and antioxidant activities including total antioxidant activity

(TAC), total reducing power (TRP) and DPPH percent inhibition of nanoparticles. .............. 31

Table 3.1: Particle size, lattice parameters (a, b, c) and angle γ of CuO nanoparticles. ......... 39

Table 3.2: Antibacterial activity of CuO, CuO-PEG and CuO-PVP nanoparticles in terms of

their zones of inhibition. .......................................................................................................... 43

Table 3.3: Phytochemical assays: phenolic equivalent determination (PED), flavonoid

equivalent determination content (FED), and antioxidant activities including total antioxidant

activity (TAC), total reducing power (TRP) and DPPH percent inhibition of CuO, CuO-PEG

and CuO-PVP nanoparticles. ................................................................................................... 45

Table 4.1: Comparison of physiological parameters in callus tissue developed from leaf

explants cultured on MS media supplemented with different hormonal combinations after 6

weeks of cultivation. ................................................................................................................ 53

Table 4.2: Comparison of physiological parameters in 4 weeks old shoots produced from

nodal explants on MS medium supplemented with different concentrations of cytokinins

………………………………………………………………………………………………..56

Table 4.3: Comparison of several physiological parameters in 4 weeks old regenerants

developed on MS medium supplemented with different concentrations of auxins ................. 58

Table 5.1: Comparison of physiological parameters in 6 weeks old callus produced from leaf

explants on MS medium supplemented with different concentrations of ZnO nanoparticles….

.................................................................................................................................................. 66


explants on MS medium supplemented with different concentrations of CuO nanoparticles….

.................................................................................................................................................. 66


nodal explants on MS medium supplemented with different concentrations of ZnO

nanoparticles. ........................................................................................................................... 80

Table 6.2: Comparison of phytochemical assays in 4 weeks old shoots produced from nodal

explants on MS medium supplemented with different concentrations of ZnO nanoparticles.

............................................................................................................................................. ….83

xii


nodal explants on MS medium supplemented with different concentrations of CuO

nanoparticles. ........................................................................................................................... 92


explants on MS medium supplemented with different concentrations of CuO nanoparticles..

.................................................................................................................................................. 94

Table 8. 1: Comparison of physiological parameters in 4 weeks old shoots produced from

nodal explants on MS medium supplemented with 1 mg/L and 10 mg/L of different

nanoparticles. ......................................................................................................................... 100

Table 8.2 : Comparison of phytochemical assays in 4 weeks old shoots produced from nodal

explants on MS medium supplemented with 1 mg/L and 10 mg/L of different nanoparticles.

................................................................................................................................................ 102

xiii

List of Figures

Figure 1.1: Structure of Rebaudioside A ................................................................................ 11

Figure 1.2: Structure of Stevioside ......................................................................................... 12

Figure 2.1: Systematic representation of attachment of polymers (PVP and PEG) on the

surface of ZnO nanoparticles ................................................................................................... 23

Figure 2.2: X-ray diffractogram of (a) ZnO, (b) ZnO-PEG and (c) ZnO-PVP nanoparticles 25

Figure 2.3: FTIR spectra of (a) ZnO, (b) ZnO-PEG and (c) ZnO-PVP nanoparticles............ 26

Figure 2.4: Reflectance spectrum of ZnO, ZnO-PEG and ZnO-PVP nanoparticles............... 27

Figure 2.5: SEM micrograph of ZnO, ZnO-PEG and ZnO-PVP nanoparticles, (left) high

resolution images (150,000 x) (Right) low resolution images (50,000 x) ............................... 28

Figure 2.6: Energy-dispersive X-ray (EDX) spectra of ZnO, ZnO-PEG and ZnO-PVP

nanoparticles ............................................................................................................................ 29

Figure 2.7: Comparison of antidiabetic activities of ZnO, ZnO-PEG and ZnO-PVP

nanoparticles against α-amylase enzyme.. ............................................................................... 31

Figure 3.1: Systematic representation of capping of PEG and PVP on the surface of CuO

nanoparticles ............................................................................................................................ 38

Figure 3.2: X-ray diffractogram of (a) CuO, (b) CuO-PEG and (c) CuO-PVP nanoparticles 39

Figure 3.3: FTIR spectra of (a) CuO, (b) CuO-PEG and (c) CuO-PVP nanoparticles ........... 40

Figure 3.4: SEM micrograph of CuO, CuO-PEG and CuO-PVP nanoparticles, (left) high

resolution images (150,000 x) (Right) low resolution images (50,000 x). .............................. 41

Figure 3.5: Energy-dispersive X-ray (EDX) spectra of CuO, CuO-PEG and CuO-PVP

nanoparticles ............................................................................................................................ 42

Figure 3.6: Antibacterial assays in terms of zone of inhibition of nanoparticles ................... 44

Figure 3.7: Cytotoxic activity of CuO, CuO-PEG and CuO-PVP nanoparticles determined by

brine shrimp lethality assay ..................................................................................................... 45

Figure 3.8: (a) Zone of inhibition against Streptomyces, (b) Comparison of antitumor

activities of CuO, CuO-PEG and CuO-PVP nanoparticles against Streptomyces. .................. 46

Figure 3.9: Comparison of antidiabetic activities of CuO, CuO-PEG and CuO-PVP

nanoparticles against α-amylase enzyme.. ............................................................................... 47

Figure 4.1: Comparison of amount of callus formed under different hormonal combinations.

.................................................................................................................................................. 55

Figure 4.2: Comparison of percent DPPH-free radical scavenging activity of calli grown

under different hormonal combinations and concentrations. ................................................... 55

Figure 4.3: Comparison of percent DPPH-free radical scavenging activity of shoots grown

under different cytokinin concentrations. ................................................................................ 57

Figure 4.4: Comparison of percent DPPH-free radical scavenging activity of plantlets

(rooted-shoots) grown under different auxin concentrations. .................................................. 58

xiv

Figure 4.5: Comparison of percent DPPH-free radical scavenging activity of callus, shoots

and plantlets (rooted-shoots).. .................................................................................................. 59

Figure 4.6: Stepwise diagram of callogenesis and direct plant organogenesis in Stevia

rebaudiana ............................................................................................................................... 60

Figure 5.1: Callogenesis of Stevia grown in MS medium containing different concentration

of ZnO nanoparticles................................................................................................................ 67

Figure 5.2: Callogenesis of Stevia grown in MS medium containing different concentration

of CuO nanoparticles. .............................................................................................................. 67

Figure 5.3: Comparison of antioxidant activities at different concentrations of ZnO

nanoparticles ............................................................................................................................ 68

Figure 5.4: Comparison of percent DPPH inhibition activity at different concentrations of

ZnO nanoparticles .................................................................................................................... 69

Figure 5.5: Comparison of antioxidant activities at different concentrations of CuO

nanoparticles ............................................................................................................................ 70


CuO nanoparticle ..................................................................................................................... 70

Figure 6.1: (a) X-ray diffraction pattern, (b) FTIR spectrum of the ZnO nanoparticles ........ 77

Figure 6.2: UV-Visible spectrum of ZnO nanoparticles ......................................................... 78

Figure 6.3: FESEM image of ZnO nanoparticles (a) High Resolution image, (b) Low

Resolution image. .................................................................................................................... 79

Figure 6.4: Energy dispersive X-ray profile of ZnO nanoparticles ........................................ 79

Figure 6.5: Effect of various concentrations of ZnO nanoparticles on the shoot formation in

Stevia rebaudiana. ................................................................................................................... 81

Figure 6.6: Effect of ZnO nanoparticles at different concentrations ranging between 0 to

1000 mg/L on rebaudioside-A and stevioside contents.. ......................................................... 82

Figure 7.1: (a) X-ray diffraction pattern, (b) FTIR spectrum of the CuO nanoparticles ........ 89

Figure 7.2: FESEM image of CuO nanoparticles (a) High Resolution image, (b) Low

Resolution image. .................................................................................................................... 90

Figure 7.3: Energy dispersive X-ray profile of CuO nanoparticles ........................................ 91

Figure 7.4: Shoot organogenesis of Stevia in MS basal medium containing different

concentrations of CuO nanoparticles. ...................................................................................... 92

Figure 7.5: Effect of CuO nanoparticles at different concentrations ranging between 0 and

1000 mg/L on rebaudioside A content and stevioside content.. .............................................. 93

Figure 8.1: Amount of percent SGs content at different nanoparticles of 1 mg/L

concentration. Rebaudioside-A and stevioside. ..................................................................... 101

Figure 8.2: Amount of percent SGs content at different nanoparticles of 10 mg/L

concentration. Rebaudioside-A and stevioside.. .................................................................... 101

xv

List of Abbreviations

2,4-D 2,4-dichlorophenoxy acetic acid

BAP 6-benzylaminopurine

DMSO Dimethylsulfoxide

DPPH 2,2-diphenyl-1-picrylhydrazile

EDTA Ethylene diamine tetraacetic acid

EDX Energy dispersive x-ray

FDA Food and development administration

FESEM Field emission scanning electron microscopy

FRSA Free radical scavenging activity

FTIR Fourier-transform infra-red spectroscopy

GRAS Generally regarded as safe

HPLC High performance liquid chromatography

IAA Indole acetic acid

IBA Indole butyric acid

KIN Kinetin

LS Linsmaier and Skoog

MS Murashige and Skoog

NA Nutrient agar

NAA α-naphthalene acetic acid

PEG Polyethylene glycol

PGRs Plant growth regulators

PVP Polyvinyl pyrrolidone

Reb-A Rebaudioside-A

ROS Reactive oxygen species

SEM Scanning electron microscopy

xvi

SGs Steviol glycosides

ST Stevioside

TAC Total antioxidant capacity

TDZ Thidiazuron

TEM Transmission electron microscopy

TFC Total flavonoid content

TPC Total phenolic content

TPPO Triphenylphosphine oxide

TRP Total reducing power

TSB Tryptic soy broth

WHO World health organization

XRD X-ray diffraction

xvii

Abstract

Background: Nanoparticles are small entities (nm in range) that behave as a whole unit and

exhibit physical, chemical and biological properties. In the context of plant biotechnology,

nanoparticles play a key role in regulating growth parameters, secondary metabolites

production and exhibit abiotic stress to plants. The objective of this study is to explore the

properties and importance of synthesized ZnO and CuO (uncapped and capped with PEG and

PVP) nanoparticles and analyze the nanoparticles effect on multiplication and secondary

metabolite production of Stevia rebaudiana under in vitro conditions.

Methodology: ZnO and CuO nanoparticles (uncapped) and capped with PEG and PVP have

been synthesized using co-precipitation method. The synthesized nanoparticles are

characterized by X-ray diffraction (XRD), Fourier transform Infra red (FTIR) spectroscopy,

UV-visible spectrometry, Scanning electron microscopy (SEM) and Energy dispersive x-ray

(EDX). Various biological assays such as antibacterial activity, antioxidant activity, cytotoxic

activity, antitumor activity and antidiabetic activity are investigated to explore biological

properties of nanoparticles. The experimental set-up for tissue culture (callogenesis and direct

shoot organogenesis) of the medicinally important plant, Stevia rebaudiana is developed

followed by studying the effect of ZnO and CuO nanoparticles on in vitro propagation and

production of steviol glycosides of S. rebaudiana.

Results: The reduction in size of ZnO and CuO nanoparticles is revealed as a result of

binding with capping agents, PEG and PVP. The physical characterization by XRD, FTIR,

UV-visible spectrometry, SEM and EDX confirm particles to be nm in size, and functional

group binding, band gap, morphology, and purity of nanoparticles. The smaller size results in

more surface area to volume ratio making nanoparticles more reactive and catalytic. PEG and

PVP capped ZnO and CuO are found to be more reactive and showed higher biological

activities as compared to bare ZnO and CuO nanoparticles. Furthermore, the protocols for

efficient callogenesis and direct shoot organogenesis of highly valuable antidiabetic plant,

Stevia rebaudiana are optimized to analyze the effect of ZnO and CuO nanoparticles on the

callogenesis and shoot organogenesis, accordingly. Regarding callogenesis of Stevia

rebaudiana, the growth dynamics and antioxidant activities are calculated. The best growth

parameters of callus are obtained at 1 mg/L and 10 mg/L of ZnO and CuO nanoparticles,

respectively. Whereas, the maximum amount of phytochemical activities are achieved at 100

mg/L of ZnO and 10 mg/L of CuO on respective basis, making CuO more toxic than ZnO in

xviii

context of antioxidant activities in callus. 1 mg/L ZnO nanoparticles and 10 mg/L CuO

nanoparticles are found most effective for the shoot organogenesis of Stevia rebaudiana as

the maximum threshold of growth parameters, steviol glycosides and antioxidant activities

are achieved at these concentrations of nanoparticles. The growth, steviol glycosides and

antioxidant activities of Stevia rebaudiana in the presence of capped nanoparticles are found

greater than uncapped nanoparticles.

Conclusions: Based on the findings it can be concluded that both ZnO and CuO

nanoparticles (either capped or uncapped) act as antibacterial, antioxidant, cytotoxic,

antitumor and antidiabetic agents, and are best candidates to be used as nanomedicines. The

highest amount of biological activities have been exhibited by PEG and PVP capped ZnO and

CuO nanoparticles as compared to the uncapped ones. Furthermore, biological activities also

conclude that these nanoparticles can also be used as carrier molecules for drug delivery and

diagnostic agents. The ZnO and CuO nanoparticles largely effect the Stevia rebaudiana

callogenesis and direct shoot organogenesis and all plant organs possess DPPH-free radical

scavenging activity. Alongwith the increased growth parameters, these nanoparticles enhance

the secondary metabolites production and antioxidant activities of callus and shoots of Stevia

rebaudiana up to a certain threshold level. Moreover, the capped ZnO and CuO nanoparticles

are found to be more reactive in tissue culture studies of Stevia rebaudiana. These studies

encourage further development in the field of study of nanoparticles effect on different

medicinal plants in vitro, and considered as preliminary studies for such experiments in

bioreactors for enhancement of important secondary metabolites at an industrial scale.

1

CHAPTER 1

Introduction and Review of Literature

1.1 Nanoparticles

The particles ranging between 1 to 100 nm in size fall under the heading of nanoparticles.

Nanoparticles possess significantly large surface area to volume ratio as compared to their

bulk counterparts. This unique property of nanoparticles makes them highly reactive and

catalytic (Ying and Jackie 2001). The functionalization of nanoparticles depends on their

surface coating. Multivalent and polymeric coating determines high stability, solubility and

targeting. Polar surface coatings are essential for biological applications of nanoparticles in

order to prevent nanoparticles aggregation and their high aqueous solubility. For example,

polyethylene glycol (PEG) is a capping agent that repels non-specific interactions by linking

to the terminal hydroxyl or methoxy groups (Liu et al. 2010).

1.2 Metallic Oxide Nanoparticles

Metallic oxide nanoparticles have been immensely used in research and development due to

their diverse applications in electronics, optoelectronics, biosensors, catalysis, surface

coatings, bioengineering, biodiagnostics, agriculture, etc. Size, shape, composition,

morphology and crystallinity are critical parameters in determining the intrinsic properties of

metallic oxide nanoparticles (Navale et al. 2015). Metallic oxide nanoparticles research has

gained momentum in recent years because of their unusual stability under high temperature

and pressure (Xie et al. 2011).

1.3 Zinc Oxide (ZnO) Nanoparticles

ZnO is an inorganic white powder that occurs naturally in the form of zincite having

impurities (Klingshirn 2007). However, most of ZnO is produced synthetically, devoid of any

impurities (Liedekerke 2006) in the form of nanostructures. ZnO crystals occur in two forms;

wurtzite (hexagonal) and zincblende (cubic). The most common and stable ZnO structure is

wurtzite. ZnO has a wide band gap of 3.3 eV at room temperature, and is a semiconductor of

II to VI semiconductor group. ZnO nanoparticles have numerous applications in rubber

manufacture, cement industry, medicine, food industry, etc. ZnO nanoparticles possess high

catalytic and photochemical properties due to their large surface area to volume ratio (Chen

and Tang 2007). Different morphological structures of ZnO nanoparticles can be obtained

depending upon their method of synthesis and few other parameters. For example, they exist

2

in the form of 1-dimensional (1D), 2-dimensional (2D) and 3-dimensional (3D)

nanostructures. 1D structures include the nanorods, needles and helices. Whereas, 2D and 3D

structures are comprised of ribbons, tubes, wires, and flower, dandelion and coniferous

structures (Kołodziejczak-Radzimska and Jesionowski 2014).

1.3.1 Synthesis of ZnO Nanoparticles

Different methods of ZnO nanoparticles synthesis have been utilized by various scientists.

Precipitation is the simplest and most widely used method to obtain ZnO nanoparticles. It

involves spontaneous reduction of zinc salt solution with reducing agent, and involves

thermal treatment to remove impurities followed by milling. pH, calcination temperature and

time of precipitation are the parameters for controlled particle size. Zn(CH3COO)2, Zn(NO3)2

and ZnSO4 are the zinc salts usually used as precursors to be reduced by a base like KOH,

NaOH and NH4OH. The structural morphology and particles diameter varies depending upon

different conditions of reaction (Xu et al. 2000; Hong et al. 2006; Cao et al. 2009;

Kołodziejczak-Radzimska et al. 2010; Wang et al. 2010; Jia et al. 2012; Khoshhesab et al.

2012; Kumra et al. 2013; Lanji et al. 2013). In order to prevent agglomeration of synthesized

ZnO nanoparticles, polymers like polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP),

triethanolamine (TEA), etc can be used under different reaction conditions to obtain specific

size and shape of nanoparticles (Wang et al. 2002; Li et al. 2005).

Sol gel method to obtain ZnO nanopowders is also very simple, economical and reliable. Two

mechanisms of sol-gel method exist. The first method involves hydrolysis of precursor

solutions leading to sol formation. Later on, this sol can be transformed in to zerogel film

followed by dense film as a result of spin-coating and dip-coating. The spin- and dip-coating

involves gelatination and evaporation of solvent of xerofilm by heat treatment, ultimately

leading to dense films. Second mechanism involves sol formation and then condensation of

sol particles leading to gel formation. Xerogel forms by an evaporation of solvent from gel

which converts in to dense ceramic after heat treatment. The precursors of this method are

zinc salts like Zn(CH3COOH)2 and an organic solvents like oxalic acid, ethanol and

methanol. Nanoparticles of different size and morphology form depending upon the choice of

precursors, time and temperature of reaction, calcination and heating (Ristic et al. 2005;

Mahato et al. 2009; Benhebal et al. 2013; Yue et al. 2013).

Solvothermal and Hydrothermal methods are simple and environment-friendly techniques

that do not involve the use of organic solvents and calcination. Autoclave is used for

3

formation of nanoparticles in which the substrates are heated at 100-300oC temperature and

left for several days. The well-known precursors of this reaction are Zn(CH3COO)2,

Zn(NO3)2, ZnCl2, zinc acetylacetonate, zinc oximate, etc. Nanoparticles of different size and

morphology are obtained depending upon the type and concentration of reaction starting

agents, and reaction temperature as well as its duration. Highly pure nanomaterials are

formed by this method (Chen et al. 2000; Chen et al. 2003; Hu et al. 2004; Ismail et al. 2005;

Music et al. 2005; Dem’Yanets et al. 2006; Schneider et al. 2010; Zhang et al. 2010).

1.3.2 Capping of ZnO Nanoparticles

ZnO nanoparticles possess specific physicochemical properties that make them applicable for

many industries. The phenomenon of agglomeration of nanoparticles directed the scientists

towards the invention of techniques that prevent agglomeration. The surface of nanoparticles

is modified (capped) to give better performance. The capped ZnO nanoparticles present a

transparent surface for protection against UV-radiation due to significantly large surface area

to volume ratio as compared to uncapped ZnO nanoparticles. Capped ZnO nanoparticles do

not agglomerate and are very stable.

The different methods of capping of ZnO nanoparticles involve the use of organic

compounds, inorganics such as SiO2, Al2O3 and metal ions as capping agents. The surface

area and size of ZnO nanoparticles alter as a consequence of capping. Moreover, the degree

of dispersion of ZnO nanoparticles improve and their photocatalytic action reduce (Xia and

Tang 2003; Yuan et al. 2005; Hong et al. 2006; Cao et al. 2009). The organic compounds

such as carboxylic acids and silanes are also used as capping agents. As a result of such

capping, the surface area and physicochemical properties alter, ZnO compatibility with an

organic matrix increase and ZnO dispersion in rubber mixtures also improves (Kotecha et al.

2006; Chen et al. 2012). Another method involves use of polymers such as polyethylene

glycol (PEG), polyvinyl pyrrolidone (PVP), polystyrene, chitin, etc as capping agents

resulting in improved thermal, electrical and optical properties of ZnO-polymer matrices

(Xiong et al. 2005; Chae and Kim 2006; Hong et al. 2006; Tang et al. 2006; Tang et al. 2007;

Pyskło et al. 2007; Shim et al. 2009; Wysokowski et al. 2013).

1.3.3 Applications of ZnO Nanoparticles

ZnO nanoparticles possess diverse applications in various industries. For example, ZnO

nanoparticles are used as fillers and activators of rubber compounds. These nanoparticles

have been found to form smallest agglomerates which have the ability to concentrate/absorb

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stress unlike other ZnO molecules. In textile industry, ZnO nanoparticles are used as

absorbers of UV-radiation. Moreover, they also have the ability of self-cleaning and water-

repulsion (Vigneshwaran et al. 2006; Uddin et al. 2008; Gao et al. 2009; Atienzar et al. 2010;

Lim et al. 2010; Tanasa et al. 2012; Gomez et al. 2013). Hence, ZnO nanowires are ideal

multifunctional coatings for textiles.

Regarding usage in pharmaceutical and cosmetic industry, ZnO nanoparticles are used as

components of ointments and creams due to their ability to absorb UVA and UVB radiation

(Cross et al. 2007), dusting and liquid powders, dental pastes, etc. ZnO nanoparticles are used

in various kinds of medicines due to their antibacterial effect. Moreover, it produces marked

effect in wound healing, alleviation of itching and inflammation, and for temporary fillings in

dentistry. Zinc supplements are also used as dietry components (Mason 2006; Mirhosseini

and Firouzabadi 2006; Liu et al. 2013).

ZnO nanoparticles are also used in photoelectronics, UV-lasers, field emitters, solar cells,

sensors, etc. Due to wide energy band gap (3.37 eV) and high bond energy (60 meV) at room

temperature, ZnO nanoparticles possess an ability to be used in electronic and

photoelectronic equipment (Aoki et al. 2000; Kong et al. 2001; Purica et al. 2001). ZnO

nanoparticles are used as photocatalysts in industry. They offer better stability and

crystallinity, hence visible spectrum for these nanoparticles should be extended by adding

other components (Bizarro 2010; Guo et al. 2011; Li et al. 2011).

1.3.4 Biological Activities of ZnO Nanoparticles

Different biological activities of ZnO nanoparticles have been reported, e.g.,

Antibacterial activity

Due to budding resistance of bacteria against antibiotics, metallic oxide nanoparticles have

been screened as the potent candidates against bacteria. ZnO nanoparticles possess

antibacterial activity against a wide range of bacterial species such as Escherichia

coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus

faecalis, Salmonella types, Campylobacter jejuni and Clostridium perfringens which

increase their applications in the food industry (Ragupathi et al. 2011; Xie et al. 2011; Singh

et al. 2012; Guo et al. 2015; Navale et al. 2015; Sirelkhatim et al. 2015; Dobrucka and

Dlugaszewska 2016; Stan et al. 2016). In vitro assessment of antibacterial activity can be

achieved by methods such as disc diffusion, well diffusion, broth dilution, agar dilution and

microtiter plate-based methods. The mechanisms behind antibacterial activity involve the

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direct contact of ZnO nanoparticles with the bacterial cell wall which disrupts bacterial cell

integrity. The Zn+2

ions release in to the cytoplasm and form reactive oxygen species (ROS).

Antioxidant and Antidiabetic activity

Excellent DPPH-free radical activity of ZnO nanoparticles have been reported by different

researchers (Das et al. 2013; Kumar et al. 2014; Nagajyothi et al. 2014; Singh et al. 2014;

Afifi and Abdelazim 2015; Dkhil et al. 2015; Lingaraju et al. 2016; Stan et al. 2016).

Diabetes mellitus is a disorder in metabolism that involves accumulation of high glucose in

the body. Antidiabetic effect of ZnO nanoparticles on Streptozotocin-induced diabetic rats

was studied and ZnO nanoparticles are found to be potent antidiabetic agents (Alkakadi et al.

2014; Umrani and Paknikar 2014; Nazarizadeh and Asri-Rezaie 2016).

Antitumor and Cytotoxic activity

ZnO nanoparticles are also found as promising antitumor/anticancer agents that selectively

attack the cancer cells, sparing the neighbouring healthy cells (Rasmussen et al. 2010;

Arakelova et al. 2014; Bisht andRayamajhi 2016). ZnO nanoparticles destroy T98G human

glioblastoma cells of brain (Wahab et al. 2013) and HNSCC carcinoma cell lines of neck

(Hackenberg et al. 2012). Moreover, ZnO nanoparticles are photo-stimulated and combined

with paclitaxel or cisplatin in human squamous cell carcinoma HNSCC cell lines to

determine their antitumor potential. Consequently, a synergistic cytotoxic and antitumor

effect was generated (Hackenberg et al. 2010). A strong anticancer activity of ZnO

nanoparticles against MCF7 (Breast cancer cell) and A549 (Lung cancer cell) have been

investigated (Selvakumari et al. 2015). ZnO nanoparticles play a double role, i.e., drug

carriers and as antitumor drugs in human breast cancer cell lines (Al-Ajmi et al. 2016).

Size-dependent and dose-dependent cytotoxicity against human cancer cells has been

achieved by few scientists (Ostrovskyet al. 2009; Das et al. 2013; Kang et al. 2013;

Nagajyothi et al. 2014; Namvar et al. 2015). Cytotoxicity is determined by the inhibition of

cell proliferation, membrane damage, and ROS generation.

1.4 Interaction of ZnO Nanoparticles with Plants

The positive as well as negative impact of ZnO nanoparticles on different crop plants has

been observed till date. For instance, ZnO nanoparticles at a particular concentration promote

root elongation in soybean (Lopez-Moreno et al. 2010). ZnO nanoparticles have been found

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to promote seed germination, root length and shoot length in the peanut plant (Prasad et al.

2012). ZnO nanoparticles improve plant biomass, root and shoot length, chlorophyll and

protein synthesis and other growth parameters when exposed to Cyamopsis tetragonoloba

(Raliya and Tarafdar 2013). Burman et al. (2013) compared the foliar application of ZnO

nanoparticles at 1.5 mg/L concentration and ZnSO4 in chickpea, and found an increase in

biomass. ZnO nanoparticles at lower concentration increased seed germination in wheat

(Ramesh et al. 2014). The effect of ZnO nanoparticles on the seedlings and stem explants of

Brassica nigra has been observed. Seed germination and seedling growth was adversely

affected by ZnO nanoparticles exposure, however, it was concentration dependent (Zafar et

al. 2016).

In contrary to its beneficial effects, ZnO nanoparticles also create toxicity (Lee et al. 2010;

Du et al. 2011; Lee et al. 2013; Wang et al. 2013; Zhao et al. 2013; Kauhi et al. 2014). Most

of studies reveal that oxidative stress by ZnO nanoparticles inhibits the growth and

development of plants (Hernandez-Viezcas et al. 2011; Bandyopadhyay et al. 2015). ZnO

nanoparticles affect the growth of bean seedlings in a dose-dependent manner. These

nanoparticles impaired root growth and promoted higher bioaccumulation of zinc ions in

shoots (Dimpka et al. 2014). Kouhi et al. (2015) studied the comparative effects of ZnO

nanoparticles, bulk ZnO and Zn+2

ions on rapeseed (Brassica napus L.). They found that the

ZnO nanoparticles are least toxic among all, followed by the bulk ZnO. However, the Zn+2

were found to be highly phytotoxic and their accumulation in plant parts conferred various

disorders. Wang et al. (2016) found that 200 mg/L and 300 mg/L ZnO nanoparticles reduced

the biomass accumulation in Arabidopsis thaliana by 20 to 80%. Similarly, the net rate of

photosynthesis was reduced up to >50% in 300 mg/L of ZnO nanoparticles treated

Arabidopsis plants. Considering only medicinal plants, the influence of ZnO nanoparticles

has been illustrated in the medicinal plant, Fagopyrum esculentum (Sooyeon et al. 2013).

1.5 Copper Oxide (CuO) Nanoparticles

The oxidative copper exists in two forms; Cu2O (Cuprous oxide) and CuO (Cupric Oxide).

Cuprous oxide is p-type direct band gap II –VI semiconductor with the band gap of 2 eV,

while cupric oxide is p-type semiconductor with an indirect band gap of 1.21 – 1.51 eV and

possess a monoclinic structure. CuO nanoparticles are brownish-black coloured powdered

materials that have huge catalytic applications. These are widely used in superconducting

materials, sensing materials, glass, ceramics and thermoelectric materials (Singh et al. 2016).

CuO nanoparticles possess excellent electrical, optical, physical and magnetic properties. Its

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different nanostructures can be synthesized in the form of nanorod, nanowire, nanoflower, etc

(Phiwdang et al. 2013).

1.5.1 Synthesis of CuO Nanoparticles

CuO nanoparticles have been prepared by different methods. Etefagh et al. (2013)

synthesized CuO nanoparticles by the sol gel method. Ethanol and deionized water are used

as a precursor solution. Copper nitrate [Cu (NO3)2⋅3H2O] is then added in this solution (1:1).

A green solution is obtained after 1 h of stirring at 40°C that is maintained under reflux at

100–110°C for 4 h. A wet gel is attained after vaporization of excess solvents. The black

powder finally obtained is calcined at 600°C for 1 h. Later on, its milling is carried out. Hong

et al. (2002) and Akhavan and Ghaderi (2010) also prepared CuO nanoparticles by sol-gel

method. Etefagh et al. (2013) synthesized CuO nanolayers by spray pyrolysis method. A

precursor solution, 0.05 M Cu (CH3COO)2.H2O dissolved in 100 ml distilled water is

prepared under continuous stirring for 30 min. Thereafter, CuO nanolayer deposits on the

glass substrates at a temperature of 400°C.

In precipitation method, copper chloride (CuCl2) and copper nitrate (Cu(NO3)2.3H2O) are

used as precursors. Both are separately dissolved in deionized water forming 0.1 M solution.

0.1 M NaOH is dissolved dropwise in above solution under vigorous stirring. When the pH

reached 14, black precipitates are obtained that are washed with deionized water or ethanol.

Later on, drying is carried out at 80°C for 16 h. Furthermore, calcination is performed at

500°C for 4 h (Phiwdang et al. 2013). Precipitation method has been used by many

researchers to form CuO nanoparticles (Chang and Zeng 2004; Kida et al. 2007; Rahnama

and Gharaghozlou 2012).

Sonochemical method has been applied to synthesize copper oxide nanoparticles using

copper (II) acetate as a precursor, urea and sodium hydroxide as reducing agent and

polyvinylpyrrolidone (PVP) as stabilizing polymer. It is followed by irradiation using high-

intensity ultrasound (Anandan et al. 2012). In microwave irradiation method, copper (II)-bis-

acetate is used as a starting material, NaOH as reducing agent and ethanol as solvent. Highly

pure and regular shaped CuO nanoparticles are obtained at the end (Wang et al. 2002). In

hydrothermal microwave method, 5 × 10−3

mol/L of CuCO3·Cu (OH)2 and 0.1 g of PEG

(MW 400) are added to 100 mL of deionized water. After stirring the solution for 15 min and

then adding 5 mL of NH4OH under constant stirring, an intense blue precipitate of

[Cu(NH3)4]+2 is obtained. The resulted solution is transferred into a sealed Teflon autoclave

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and placed in a domestic microwave. Finally, the reaction system is heated at 393 K for 1 h.

CuO flower-nanostructures are collected and washed with deionized water and then dried at

353 K on a hot plate (Volanti et al. 2008).

1.5.2 Capping of CuO Nanoparticles

Different scientists have performed capping of CuO nanoparticles with capping agents to

increase their stability and control their size by preventing agglomeration. CuO nanoparticles

have been capped by different compounds such as organic compounds and polymer moities.

CuO nanoparticles have been capped with carboxylic acids by simple solvent-less method

(Estruga et al. 2012). CuO nanoparticles capped with amino acids such as L-lysine-capped

CuO or L-glycine capped CuO nanoparticles have also been prepared. In context of

polymers, CuO nanoparticles have been capped with ethylene diamine tetra acetic acid

(EDTA) by a sol-gel method (Jayaprakash et al. 2014). Polyvinylpyrrolidone (PVP)-capped

and tripheny phosphine oxide (TPPO)-capped CuO nanoparticles have been synthesized by

Sharma et al. (2016), and Chitosan-capped CuO nanoleaves have been prepared by an

ultrasound sonication method (Abiraman et al. 2017).

1.5.3 Applications of CuO Nanoparticles

CuO nanoparticles have been used in rechargeable batteries, solar cells, gas sensing

(Suleiman et al. 2013), heat-transfer (Manimaran et al. 2014), catalysis including

electrocatalysis, photocatalysis (Sanjini et al. 2014) and gas-phase catalysis (Gavande et al.

2016), adsorption of organic dyes (Asl et al. 2016), burning rate catalyst in rocket propellant,

semiconductors and high-tech superconductors (Singh et al. 2016).

1.5.4 Biological Activities of CuO Nanoparticles

CuO nanoparticles possess the following biological activities:

Antibacterial and antioxidant activity

The antibacterial activity of CuO nanoparticles has been reported against Pseudomonas

aeruginosa, Eschericia coli, Klebsiella pneumonia, Enterococcus faecalis, Shigella flexneri,

Salmonella typhimurium, Proteus vulgaris and Staphylococcus aureus (Ren et al. 2009;

Azam et al. 2012; Das et al. 2012; Ahamed et al. 2014; Khashan et al. 2016). Free radical

scavenging activity of CuO nanoparticles against the stable free radical, DPPH has been

revealed by Das et al. (2012).

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Antitumor and Cytotoxic activity

The CuO nanoparticles exhibit excellent antitumor activity against two kinds of cancer cells,

i.e., AGS (human gastric carcinoma) cells and HeLa (human cervix carcinoma) cells (Germi

et al. 2014). Sankar et al. (2014) revealed CuO nanoparticles as an anticancer agent, and

anticancer activity has also been elucidated by CuO nanoparticles against human cervical

carcinoma cells (Nagajayothi et al. 2016).

Fahmy and Cormier (2009) described cytotoxicity of CuO nanoparticles against airway

epithelial cells. Cytotoxic and genotoxic activities of CuO nanoparticles have been revealed

against human skin keratinocyte cells (Alarifi et al. 2013; Semisch et al. 2014). Cytotoxic

response of CuO nanoparticles has been demonstrated against human hepatic carcinoma cells

(Siddiqui et al. 2013) and human chronic myeloid leukemia (CML) K562 cell lines (Shafagh

et al. 2015).

1.6 Interaction of CuO Nanoparticles with Plants

The interaction of CuO nanoparticles with different plants has been studied. The CuO

nanoparticles inhibitory effect has been noticed in duckweed, Landoltia punctata by

comparing the influence of soluble Cu in bulk solution and CuO nanoparticles (Shi et al.

2011). Johnson et al. (2012) investigated an interaction between an aquatic plant, Elodea

canadensis and CuO nanoparticles. Another study was conducted examining the growth and

photosynthetic effects of CuO nanoparticles on an aquatic macrophyte, Lemna gibba L

(Perreault et al. 2014). Moreover, CuO nanoparticles effect on aquatic macrophyte, Elodea

nuttallii has also been studied in the context of photosynthesis and oxidative stress (Regier et

al. 2015). The copper accumulation in different parts of plants results in a toxicity effect.

CuO negatively affects the hydrophyte, Lemna minor (Duckweed). Different concentrations

of CuO nanoparticles, bulk CuO and Cu+2

ions were exposed to this water plant. The

ecotoxicity of CuO nanoparticles was significantly higher than the bulk CuO and Cu+2

ions,

and formation of reactive oxygen species (ROS) is the cause of toxicity (Song et al. 2016).

The effect of CuO nanoparticles on the growth, photosynthesis and oxidative response has

been studied in the crop plant, Oryza sativa (Da Costa and Sharma 2016). Wang et al. (2016)

determined the toxological effects of CuO nanoparticles on Arabidopsis thaliana and found

highest toxicity conferred by CuO nanoparticles rather than the bulk CuO or Cu+2

ions. Van

et al. (2016) reported the phytotoxicity of CuO nanoparticles in Ipt transgenic cotton. CuO

nanoparticles at higher concentrations reduce plant height, root length, root hairs and shoot

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biomass of transgenic Ipt cotton. Moreover, the Fe and Na absorption in the roots increased

and production of some phytohormones was prohibited. Regarding medicinal plants, the

influence of CuO nanoparticles on stevioside production and enzymatic antioxidant activities

of Stevia rebaudiana callus was deciphered (Hendawey et al. 2015).

1.7 Characterization of Nanoparticles

Nanoparticles can be characterized by different techniques such as X-ray diffraction (XRD),

Fourier-transform Infra-red spectroscopy (FTIR), UV-Visible Spectroscopy, Scanning

Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX). XRD is an analytical

technique used for phase identification of different crystalline materials including

nanoparticles. It determines unit cell dimensions. The result obtained from XRD analysis is

called “Diffractogram”. XRD analysis generates an x-ray diffraction pattern that provides a

fingerprint of crystals found in the sample. This fingerprint allows identification of the

crystalline form of sample by comparing with the standard reference patterns and

measurements. FTIR is an analytical technique for measurement of absorption or emission of

solid, liquid or gas in the form of infrared spectrum (Griffiths and Hasseth 2007). It converts

raw data in to actual spectrum and gives high resolution data over a wide range of spectrum.

It usually measures in the mid and near infra-red (IR) regions, and is very sensitive and

speedy instrument (Ali et al. 2016; Javed et al. 2016).

The absorption spectrometry or reflectance spectrometry in the UV-visible spectral region is

known as UV-visible spectroscopy or spectrophotometry. The principle of UV-visible

absorption is that the non-bonding electrons are excited to their higher anti-bonding

molecular orbitals by absorption of ultraviolet or visible light. Spectroscopic analysis is

commonly carried out in solutions, and according to Beer-Lambert’s law, the absorbance of

solution is directly proportional to the concentration of absorbing species in the solution. The

instrument used in UV-visible spectroscopy is called “UV-visible spectrophotometer”. SEM

is a microscopy technique that produces an image of sample by scanning it with a focussed

beam of high energy electrons. The electrons interact with sample surface atoms and

ultimately produce signals that confer sample morphology. The characteristic 3-D image of

the sample appears by using narrow beam of electrons. Hence, the surface structure of sample

is visualized clearly using a scanning electron microscope. EDX is an analytical technique

used for the analysis of different elements in a chemical sample. The principle of EDX is that

the x-rays are excited from their source to the sample and then the unique atomic structure of

11

every element gets drawn in the form of peaks in an electromagnetic emission spectrum.

Hence, the percent purity of chemical samples is determined by EDX (Ali et al. 2016; Javed

et al. 2016).

1.8 Stevia rebaudiana Bertoni

Stevia rebaudiana is a plant species of genus Stevia, belonging to Asteraceae (Sunflower)

family. It is commonly known as “candyleaf” or “sugarleaf” because of its sweet taste

(Mohamed and Alhady 2011). Stevia is indigenous to South America; Brazil and Paraguay

(Soejarto 2002). It favours humid and wet climate for its growth, and being cultivated as a

commercial crop in China, Japan, Thailand, Malaysia, etc. Stevia has gained enormous

importance due to its sweet leaves. The bioactive compounds present in its leaves are known

as steviol glycosides (mainly Rebaudioside A and Stevioside) which are sold under various

trade names in the market. Steviol glycosides have been found to confer 300 times more

sweetness than sugar (Chalapathi and Thimmegowda 1997).

1.8.1 Structure of Steviol Glycosides

The major steviol glycosides are rebaudioside A and stevioside. The chemical formula of

rebaudioside A is C44H70O23, and its structural formula contains a total of four glucose

molecules, out of which the central glucose is bonded with the hydroxyl group of steviol and

carboxyl group of another glucose molecule with an ester bond as shown in Figure 1.1.

Figure 1.1: Structure of Rebaudioside A.

The chemical formula of stevioside is C38H60O18, and its structural formula is shown below:

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Figure 1.2: Structure of Stevioside.

1.8.2 Importance of Stevia

This sweet herb has many health benefitial phytochemical compounds that function in

controlling blood pressure, blood sugar and cholesterol (Anton et al. 2010). Nowadays, the

demand for low-calorie food additives is rising so this zero calorie sweetener has gained

paramount importance in this regard (Wang et al. 2012). The sweetening compounds of

Stevia are rebaudioside A-E, stevioside, steviolbioside and dulcoside (Yadav and Guleria

2012). Besides that, it is non-fermentative (Abdullatif and Osman 2012). Furthermore, Stevia

plant has many sterols and antioxidant compounds like phenols, flavonoids, triterpenes,

tannins, kaempferol, quercetin, riboflavin, β-carotin, caffeic acid, chlorogenic acid, etc

(Konoshima and Takasaki 2002). Kaempferol reduces the risk of pancreatic cancer by 23%.

Chlorogenic acid reduces absorption of glucose in the gut and also prevents enzymatic

conversion of glycogen to glucose. The glycosides in Stevia extract function to dilate blood

vessels, and increase sodium excretion and urine output. Stevia helps to reduce dental caries

by preventing the growth of Streptococcus mutans bacteria in the mouth (Liu et al. 2003; Dey

et al. 2013). It has antifungal and antiviral properties. Additionally, Stevia contains many

vital vitamins and minerals that are not found in artificial sweeteners. Stevia leaf extracts

have long shelf life and high temperature tolerance. Moreover, Stevia extracts have been used

for the treatment of swelling of legs, wound infections and inflammatory conditions (Ahmad

et al. 2011). It also acts as a weight-reducer and as an anti-depressant (Jain et al. 2007). It can

be used in powdered form or in the form of syrup in tea, sauce, soft drinks, jams, ice creams,

desserts, etc.

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1.8.3 Tissue Culture of Stevia

Tissue culture is an important alternative to conventional methods of plant propagation to get

products on industrial scale in bioreactors. Different methods of tissue culture of Stevia have

been devised to get true-to-type progenies of mother plants (Rafiq et al. 2007; Aman et al.

2013; Yucesan et al. 2016).

Callogenesis

Uddin et al. (2006) performed callus establishment of S. rebaudiana using leaf, nodal and

inter-nodal segments as explant material on MS medium supplemented with different

concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D). Internodal segments are

considered better for callogenesis than leaf or nodal segments. MS media with 3 mg/L of 2,4-

D produced maximum amount of callus. Gupta et al. (2010) induced callogenesis using

nodes, leaves and roots explant on MS medium fortified with different concentrations of 2,4-

D, NAA, IBA and KIN. NAA is also used in combination with 2,4-D and it produced

maximum amount of callus, whereas the 2,4-D used alone produced the poorest callus. Leaf

explants are declared best for callogenesis within 3 weeks as they produce green and shiny

callus. On the other hand, callus obtained from root and nodal explants is hard and brown.

Shoot and Root Organogenesis

Yang et al. (1981) used nodal explants for in vitro multiple shoot induction of Stevia

rebaudiana. Murashige and Skoog (1962) medium supplemented with different cytokinins

has been used for shoot induction and auxins for root formation. Tamura et al. (1984) used

the stem tip culture for in vitro clonal propagation of S. rebaudiana. He also used leaf

primordia on Linsmaier and Skoog (1967) medium supplemented with 10 mg/L of kinetin

(KIN) for this purpose. The number of shoots formation was dependent on the size of stem

and the number of leaf primordia. They obtained multiple shoots within 2 months of

culturing.

Hwang (2006) reported adventitious shoots formation from nodal explants on basal media

supplemented with different concentrations and types of cytokinins. The best rooting results

were obtained in the presence of indole butyric acid (IBA). The rooted plants were

acclimatized in the field and produced a similar quantity of steviol glycosides as the mother

plant. Ahmed et al. (2007) used nodal explants for in vitro multiple shoot induction by

axillary shoot proliferation that was reported by augmenting the medium with 6-

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benzylaminopurine (BAP) and kinetin (KIN). Different concentrations of indole acetic acid

(IAA), indole butyric acid (IBA) and α-naphthalene acetic acid (NAA) were used for the

purpose of rooting. Rafiq et al. (2007) used nodular stem sections as an explant and reported

maximum shoot formation on MS media augmented with 2 mg/L BAP.

Alhady (2011) used stem node segments as an explant source and tested different cytokinin

and auxin combinations for shoot proliferation. He found 2 mg/L BAP and 0.5 mg/L KIN to

be the best plant growth regulators (PGRs) for shoot organogenesis. Shoot multiplication was

promoted by BAP, whereas elongated shoots were produced by KIN. In the case of root

organogenesis, IBA and NAA were assessed and significantly more roots were produced by

IBA as compared to NAA. Das et al. (2011) reported micropropagation of S. rebaudiana

through shoot tip culture and used MS medium supplemented with 2 mg/L kinetin for shoot

proliferation. They found MS medium without PGRs to be effective for root formation.

1.9 In vitro Production of Steviol Glycosides

In vitro production of steviol glycosides in Stevia rebaudiana is affected by different mineral

components, carbon sources, plant growth regulators (PGRs) and different stress conditions.

Effect of different concentrations of MS medium and its components were studied in detail

by Ibrahim et al. (2008). Shoot induction and chlorophyll content were found to be effected

by the concentration and composition of microelements (Jain et al. 2012). Copper is also

found to be effective in increasing chlorophyll content and plant biomass (Jain et al. 2009).

Sugars in medium act as a source of carbon and energy making plant independent of

photosynthesis. Bonderav et al. (2003) studied the effect of sugar type and concentration on

the shoot development and steviol glycosides production in bioreactors. Plant growth

regulators are the compounds regulating the biochemistry of plants for their proper growth in

changing environmental conditions. Different scientists studied the effects of cytokinins

(BAP, KIN) and auxins (IAA, IBA and NAA) on plant biomass and steviol glycosides

production (Bondarev et al. 2001, 2003; Sivaram and Mukundan 2003; Jain et al. 2009;

Anbazhagan et al. 2010; Taware et al. 2010; Yadav et al. 2011; Thiyagarajan and

Venkatachalam 2012; Hassanen et al. 2013). The presence of stevioside was not documented

in callus cultures or shoot buds by Swanson et al. (1992). Few scientists reported that both

rebaudioside A and stevioside are not produced by the callus or cell suspension cultures, and

they can only be produced in the whole plant (Bondarev et al. 2001).

15

The effect of two plant growth retardants; chlorocholine chloride (CCC) and paclobutrazol

(PBZ) have been investigated for antioxidant activities, growth and steviol glycosides

formation of Stevia rebaudiana. Although both function as anti-gibberellins, PBZ improved

SGs production, antioxidant capacity and plant growth (Karimi et al. 2014). The chemical

stress induced by proline and polyethylene glycol (PEG) enhanced the production of SGs in

callus and suspension culture of S. rebaudiana (Gupta et al. 2015). Recently, the chilling

stress induced by salicylic acid, hydrogen peroxide (H2O2), 6-benzylaminopurine (BAP) and

calcium chloride (CaCl2) resulted in significantly higher production of rebaudioside A and

stevioside (Soufi et al. 2016).

16

1.10 Aims and Objectives

Nanotechnology involving nanoparticles plays a vital role in the field of biotechnology as

being reported for the past few years. This study was designed to fulfil the major aims and

objectives that are described below:

Synthesis and characterization of uncapped and capped ZnO and CuO nanoparticles

using a cost-friendly method.

Study and comparison of different biological and chemical activities of synthesized

nanoparticles.

Establishment of efficient protocols for callogenesis, and direct organogenesis of

medicinal plant, Stevia rebaudiana followed by the study of antioxidative response of

callus and regenerated plantlets.

Elicitation of antioxidant activities and physiological parameters alongwith steviol

glycosides production by imposing ZnO and CuO nanoparticles (capped and

uncapped) stress to the callogenesis and shoot organogenesis of Stevia rebaudiana,

respectively.

17

CHAPTER 2

Synthesis, Characterization and Bioassays of ZnO Nanoparticles

and its Derivatives

Article Published:

Javed R, Usman M, Tabassum S, Zia M. (2016) Effect of Capping Agents: Structural, Optical

and Biological Activities of ZnO Nanoparticles. Applied Surface Science. 386: 319-326.

http://dx.doi.org/10.1016/j.apsusc.2016.06.042

2.1 Introduction

Nanoparticles having a size range of 1-100 nm possess versatile and distinct physical and

chemical properties attributed to large surface area to volume ratio as compared to their bulk

counterparts (Yadav 2013; Sirelkhatim et al. 2015). Methods of nanoparticles synthesis play

a key role in depicting their unique behavior, and once synthesized, characterization is critical

to understand the properties of nanoparticles (Kołodziejczak-Radzimska and Jesionowski

2014). Metallic oxide nanoparticles, specifically nano-scale ZnO, have gained considerable

importance in recent years due to their wide range of applications in various fields of science

notably biotechnology and pharmacology (Ashe 2011). ZnO nanoparticles have been

regarded as biocidal agents/disinfectants because of their safety, low toxicity and

biocompatibility towards humans (Zhou et al. 2006; Dutta et al. 2012; Bogutska et al. 2013).

Due to the ability of UV absorption and high photostability, role of ZnO nanoparticles has

been extensively exploited in the formation of sunscreen lotions and cosmetics, and studies

are being conducted to use ZnO nanoparticles as antimicrobial (Premanathan et al. 2011),

antioxidant (Das et al. 2013), and anticancer agents (Selvakumari et al. 2015). Moreover,

ZnO nanoparticles have also been used as cleansing agents for treatment of wastewater in

bioremediation (Wang et al. 2008).

Since bacterial pathogens have been found to develop resistance against antibiotics,

utilization of ZnO nanoparticles have gained momentum in the past decade for providing

broad spectrum resistance against bacteria (Adams et al. 2006; Jones et al. 2008). The

mechanism for bactericidal property of ZnO has been described as the production of reactive

oxygen species (ROS) that internalize the bacterial cell envelope and damage it leading to

subsequent cell death (Singh et al. 2014). ZnO nanoparticles have been regarded as promising

candidates for cancer treatment as they induce cytotoxicity against tumor cells while sparing

http://dx.doi.org/10.1016/j.apsusc.2016.06.042

18

the neighbouring normal cells (Chung et al. 2015; Namvar et al. 2015; Vinardell and Mitjans

2015). Zinc is an essential mineral for diabetic patients because of its involvement in glucose

metabolism and insulin integrity so the antidiabetic property of ZnO nanoparticles is required

to be immensely studied and elucidated in diabetic rats and eventually human trials (Alkaladi

et al. 2014; Umrani and Paknikar 2014).

ZnO nanoparticles are stable and their synthesis is inexpensive but agglomeration of

chemically synthesized nanoparticles due to high surface energy results in their size increase

as well as instability. Its ultimate solution is capping with polymeric modifying agents or

surfactants like polyethylene glycol (PEG), polyethylene oxide (PEO) and polyvinyl

pyrrolidone (PVP) (Ravichandran et al. 2010; Yuliah and Bahtiar 2013; Gutul et al. 2014;

Arakelova et al. 2014). Capping approach results in a significant size reduction of

nanoparticles contributing to their stability. A mechanism involves formation of strong

covalent bond between polymeric chains and surfaces of nanoparticles that is responsible for

stearic hindrance by which nanoparticles remain stable for months (Lopez-Serrano et al.

2014). Furthermore, size of nanoparticles has been found to be inversely proportional to their

reactivity and hence resulting in enhancement of biological activities (Nair et al. 2009).

The aim of this study is to synthesize and characterize the PEG- and PVP-capped ZnO

nanoparticles by an economical and easy method, and observe the comparative effects of

capped and uncapped ZnO nanoparticles on different biological activities.

2.2 Materials and Methods

2.2.1 Synthesis of ZnO, ZnO-PEG and ZnO-PVP Nanoparticles

ZnO nanoparticles have been synthesized using a co-precipitation method that involves the

controlled precipitation of nanoparticles from the solution of precursors (Sabir et al. 2014).

Zinc acetate di-hydrate (Zn(CH3COO)2∙2H2O; 99.5%) and sodium hydroxide (NaOH) are

purchased from Sigma Aldrich and used as received. A transparent 0.06 M aqueous zinc

acetate dihydrate is prepared in 20 mL distilled water under vigorous stirring. Separately, 1 M

aqueous NaOH dissolution is carried out in 300 mL of distilled water followed by subsequent

stirring at room temperature. Then aqueous 1.0 M NaOH is added drop by drop to zinc

acetate dihydrate solution until pH 12 is attained at room temperature and continuously

stirred until its color changes from transparent to milky white. After that, this milky white

solution is filtered using Whatman filter paper No. 1 to get the precipitates of ZnO that are

subjected to washing with distilled water and ethanol thrice. The precipitates are heated at

19

100°C until dried. The grinding of ZnO nanoparticles is performed to make a fine powder.

Afterwards, calcination is done at 350°C for 4 h.

In order to prevent agglomeration of ZnO nanoparticles, these are capped with PEG and PVP

surfactants. The process of capping helps in the stability of nanoparticles. The capping

process of ZnO nanoparticles with PEG and PVP is carried out separately during synthesis by

mixing the capping agents with precursor salt, i.e., zinc acetate dihydrate in 1:1 ratio and later

on following the similar procedure as was done for preparing uncapped ZnO nanoparticles.

2.2.2 Characterization of Synthesized Nanoparticles

Analytical methods of X-ray diffraction (XRD), Fourier-transform Infra-red spectroscopy

(FTIR), UV-visible spectroscopy, scanning electron microscopy (SEM) and EDX are

performed for characterization of synthesized nanoparticles. Powder X-ray diffraction (XRD)

provides the phase identification of crystalline materials and measures the size of samples.

XRD measurements are performed at room temperature over the range 2θ = 20−80° with a

PANalytical Empyrean diffractometer using Cu Kα radiation (λ = 1.5406 Å) and

Bragg−Brentano θ-θ configuration. FTIR spectroscopy is an effective technique to reveal the

composition of sample, and identification of functional group. The FTIR spectra is recorded

from Tensor 27 Bruker (Germany) OPUS Data Collection Program with a resolution of 1 cm-

1. The FTIR spectra of the capped and uncapped ZnO nanoparticles are recorded over 4000–

500 cm-1

.

Diffuse reflectance spectroscopy (DRS) is employed in order to study the optical properties

of all the prepared samples, and to find the band gap. Reflectance spectra are taken at room

temperature in the range of 250–850 nm on Lambda-950 PerkinElmer with integrating sphere

attachment. Moreover, morphological studies of nanoparticles are done by scanning electron

microscope (SEM, Nova NanoSEM 450) operated at an accelerating voltage of 10 kV, and

energy dispersive X-ray (EDX, Oxford Aztec) is utilized to determine the elemental

composition (purity) of prepared ZnO nanoparticles.

2.2.3 Biological Assays of Nanoparticles

Different bioassays involving antibacterial assay, antioxidant assays and antidiabetic assay

are performed.

20

2.2.3.1 Antibacterial Assay

Antibacterial activity is determined by a method (Haq et al. 2012) against two Gram positive

strains, Staphylococcus aureus (ATCC 6538) and Bacillus subtilis (ATCC 6633), and two

Gram negative strains, Escherichia coli (ATCC15224), and Acetobacter using disc-diffusion

method. All bacterial strains are grown on 2% (w/v) NA (Nutrient agar, pH 7) at 25°C and

preserved at 4°C in a refrigerator. 100 μl of tryptic soy broth (TSB) medium is spread on petri

plates containing NA medium in which the respective bacterial colonies are allowed to grow

by swabbing with cotton plugs. Disc loaded with 50 μl of test samples, (1 mg/mL in dH2O to

make 50 μg/mL final concentration) are placed on the surface of media. The plates are then

incubated at 37°C for 24 h. The media supplemented with dH2O and Cefaxime (1 mg/mL)

are used as negative and positive controls, respectively. Bacterial growth is determined by

measuring linear growth (mm) using a Vernier calliper.

2.2.3.2 Antioxidant Activities

Different types of antioxidant activities are performed including phenolic equivalent

determination, flavonoid equivalent determination, total antioxidant capacity, total reducing

power and DPPH-free radical scavenging activity.

Determination of Phenolic Equivalent

Phenolic equivalent is estimated according to previously described procedure (Jafri et al.

2014) after slight modification. An aliquot of 20 µL from 4 mg/mL dH2O stock solution of

each test sample is transferred in a respective well of 96 well plate followed by an addition of

90 µL of Folin–Ciocalteu reagent. The plate is incubated for 5 min after which 90 µL of

sodium carbonate is added to the reaction mixture. Gallic acid is used as standard and

samples are analyzed at 630 nm using microplate reader. The results are expressed as mg

gallic acid equivalent (µg GAE/mg).

Determination of Flavonoid Equivalent

For flavonoid equivalent determination, aluminium chloride colorimetric method is employed

with slight modifications according to system suitability (Jafri et al. 2014). An aliquot of 20

µL from 4 mg/mL dH2O stock solution of each test sample is transferred in respective well of

96 plates to which 10 µL of 10% aluminium chloride, 10 µL of 1.0 M potassium acetate and

160 µL of distilled water is added. The resulting mixture is kept at room temperature for 30

min. Later on, absorbance of samples is measured at 630 nm using a microplate reader and

21

quercetin is used as a standard sample.The results are expressed as µg quercetin equivalent

per mg (µg QE/mg).

Total Antioxidant Capacity Assay

Total antioxidant activity of nanoparticles is evaluated by the method of Haq et al. (2012)

with slight modification. An aliquot of 100 μL from stock solution of each sample (4 mg/mL

in dH2O) is mixed with 900 µL reagent solutions comprising of 0.6 M sulfuric acid, 4 mM

ammonium molybdate and 28 mM sodium phosphate. The reaction mixtures are incubated at

95oC for 90 min followed by cooling at room temperature and absorbance was measured at

695 nm using a microplate reader. Dimethyl sulfoxide (DMSO) was used as a negative

control. For calibration curve, ascorbic acid is used as a positive control. The resultant total

antioxidant capacity is expressed as µg ascorbic acid equivalent per mg (µg AAE/mg).

Reducing Power Assay

The reduction potential is investigated according to previously described procedure (Haq et

al. 2012). Briefly, 100 µL of each sample (4 mg/mL nanoparticales in dH2O) is mixed with

200 µL of phosphate buffer (0.2 M, pH 6.6) and 250 µL of 1% w/v potassium ferricyanide.

The resulting mixtures are incubated for 20 min at 50oC. After that, the reaction was acidified

with 200 µL of 10% w/v trichloroacetic acid. The resultant mixtures are centrifuged at 3000

rpm for 10 min and supernatant layer (150 µL) is mixed with 50 µL of 0.1% w/v ferric

chloride solution. An optical density is measured at 630 nm. Ascorbic acid is maintained as

positive control and results are expressed as µg ascorbic acid equivalent per mg (µg

AAE/mg).

DPPH-Free Radical Scavenging Assay

2,2-diphenyl-1-picryl hydrazyl (DPPH) reagent is employed for the determination of free

radical scavenging activity according to the method of Haq et al. (2012) with slight

modification. Briefly, 10 µL of nanoparticles (4 mg/mL) is mixed with 190 µL of DPPH

(0.004% w/v in methanol). The reaction mixture is incubated in the dark for 1 h. Then optical

density is measured at 515 nm using microplate reader. Ascorbic acid is employed as positive

standard while distilled water as negative control.

𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡𝑒𝑠𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 = % 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = (1 −𝐴𝑏𝑠𝐴𝑏𝑐) × 100

22

Where Abs is the absorbance of DPPH solution with sample, and Abc indicates the absorbance

of negative control (containing the reagent and solvent only). The IC50 is calculated by using

Table curve software 2D Ver. 4.

2.2.3.3 Antidiabetic Assay (α-Amylase Inhibition Assay)

α- amylase inhibition assay is performed using a method previously reported (Kim et al.

2010) with slight modification. The activity is performed in 96 well microplate, and to each

well included in the test, 15 µL phosphate buffer, 25 µL α- amylase enzyme, 10 µL of sample

and 40 µL starch is added in subsequent steps. Incubation is done at 50°C for 30 min; it is

followed by an addition of 20 µL of 1 M HCl and finally 90 µL iodine solutions. Suitable

wells are assigned for blank, positive and negative control. Blank contained buffer solution,

starch and dH2O while negative and positive control wells contained the respective dH2O and

acarbose instead of test sample. Results are noted using a microplate reader and readings are

taken at 540 nm. Percent enzyme inhibition is calculated using the following formula:

100%

yz

yx

ODOD

ODODinhibitionEnzyme

ODx, ODy and ODz are absorbance values of sample, negative control and blank respectively.

2.2.4 Statistical Analysis

All the assays are performed in triplicate and results are presented as mean with standard

deviation. Further the mean is statistically analyzed using least significant difference (LSD)

analysis at significance level 0.05.

2.3 Results

ZnO nanoparticles are synthesized by co-precipitation method as shown by chemical reaction

below. Zinc acetate first converts into zinc hydroxide that upon treatment with alkali reduces

to form ZnO nanoparticles. Presence of polymers in the surrounding medium attach on the

surface of nanoparticles. A systematic diagram of attachment of polymers on the surface of

nanoparticles is shown in Figure 2.1. The formation of ZnO nanoparticles and attachment of

PVP and PEG is confirmed by analytical techniques.

23

Zn(CH3COOH)2 . 2H2O ∆ → aq medium

Zn (OH)2 + 2CH3COOH

Zn(OH)2Na+OH−

↔ (Zn(OH)4)−2 + Na+

Zn(OH)2H+OH−

↔ (Zn(OH)4)−2 + H+

(Zn(OH)4)−2↔ ZnO + H20 + 2OH

−

Figure 2.1: Systematic representation of attachment of polymers (PVP and PEG) on the

surface of ZnO nanoparticles.

2.3.1 X-Ray Diffraction (XRD)

The crystal structures of prepared samples is confirmed using X-ray diffraction (XRD).

Figure 2.1 shows X-ray diffractograms of the capped and uncapped ZnO nanoparticles. The

diffraction peaks of all samples are identified as having a hexagonal or wurtzite structure in

accordance with PCPDFWIN card no. 891397. The strong and sharp diffraction peaks

indicate that ZnO nanoparticles are well crystallized (Xie et al. 2008, 2009; Thirugnanam

24

2013). Later on, the crystallite size of ZnO nanoparticles is calculated from the peak

broadening of diffraction peaks using Scherrer’s formula (Cullity 1978).

B

kD

cos

In this equation, λ represents the wavelength of X-ray radiation, β is the full width at half

maximum of diffraction peaks (in radians) and θB the Bragg’s angle. The average crystallite

size of uncapped ZnO and capped ZnO-PEG and ZnO-PVP is listed in Table 2.1.

Table 2.1: Particle size, lattice parameters (a, c) and c/a ratio of ZnO, ZnO-PEG and ZnO-

PVP nanoparticles.

Samples Particle

Size (nm)

Lattice Parameter

a (Å) c (Å) c/a

ZnO 34 3.25 5.21 1.603

ZnO-PEG 26 3.25 5.20 1.601

ZnO-PVP 32 3.24 5.19 1.602

The crystal structure of ZnO is Wurtzite, in which the oxygen atoms are arranged in a

hexagonal close packed type, with Zn atoms occupying half of the tetrahedral sites. Zn and O

atoms are in tetrahedral coordination to each other. The lattice parameter of ZnO hexagonal

structure and the plane spacing d is related to the lattice constant a, c, and the Miller indices

by (Cullity 1978).

2

2

2

22

2 3

41

c

l

a

khkh

d hkl

Using above equation along with the Bragg’s law (2dhklsinθ=nλ), we calculate the values of

“a” and “c”. The lattice constants calculated from 100 and 002 peaks along with the ratio

c/a are listed in Table 2.1.

25

Figure 2.2: X-ray diffractogram of (a) ZnO, (b) ZnO-PEG and (c) ZnO-PVP nanoparticles.

2.3.2 Fourier-Transform Infra-Red Spectroscopy (FTIR)

The FTIR spectra of the synthesized ZnO nanoparticles are shown in Figure 2.3. All samples

exhibit broad absorption band in the range of 3400–3450 cm-1

is assigned to the O H

stretching vibration and O-H bending of the hydroxyl group at 574 cm-1

is observed. Peaks

between 2850–2930 cm-1

are due to the C H stretching vibration. The peaks observed at 1576

and peak 1420 cm-1

are due to the asymmetrical and symmetrical stretching of the zinc

carboxylate, respectively (Chithra et al. 2015). The bond at 1025 cm-1

is due to the C O

stretching vibration. The absorption at 857 cm-1

is due to the formation of tetrahedral

coordination of Zn (Sharma et al. 2014). In Figure 2.3(c) PVP-capped samples clear

absorption bands positioned at around 2860 cm-1

is resulted from characteristic peak of PVP.

Symmetric C=O stretching at 1620 cm-1

and C H out of plane bending group at 1116 cm-1

are observed (Thirugnanam 2013).

26

Figure 2.3: FTIR spectra of (a) ZnO, (b) ZnO-PEG and (c) ZnO-PVP nanoparticles.

2.3.3 UV-Visible Spectroscopy

Figure 2.4 illustrates the typical reflectance spectra of ZnO samples with absorption edge

around 360–380 nm. The diffuse reflectance ‘R’ of the sample is related to the Kubelka–

Munk function F(R) by a relation

R

RRF

2

1)(

2

27

Where, ‘R’ is the absolute value of reflectance (Tahir et al. 2009; Tahir et al. 2013). The band

gap energy of the capped and uncapped ZnO is calculated from their diffuse-reflectance

spectra by plotting the square of the Kubelka–Munk function F2(R) as a function of photon

energy in eV. The linear part of curve is extrapolated to F2(R) = 0 to get the direct band gap

energy. Calculation of band edge value from K-Munk Function of PEG-capped ZnO sample

is shown in the inset of Figure 2.4. Band gap edge values of all other samples are calculated

in the same way. The values of band edge are 3.229, 3.239 and 3.249 eV for ZnO, ZnO-PEG

and ZnO-PVP, respectively.

Figure 2.4: Reflectance spectrum of ZnO, ZnO-PEG and ZnO-PVP nanoparticles.

2.3.4 Scanning electron microscopy (SEM)

Morphology of the sample is investigated using scanning electron microscope. Specimens are

prepared by sticking ZnO nanoparticles to the carbon tape, and the excess powder blown

away with compressed air. Then these specimens are sputter coated with a thin Au-Pd layer

of about 3 nm thickness in vacuum to avoid the charging. Typical SEM micrographs for

28

prepared nanoparticles without/with surfactant (ZnO, ZnO-PEG and ZnO-PVP) are shown in

Figure 2.5. The SEM image of uncapped ZnO nanoparticles (Figure 2.5(a)) shows irregular

shaped morphology (the obtained structural rectangle, radial hexagonal and spherical shapes).

The size of the particles is found in the range of 30-50 nm. The SEM image of capped ZnO-

PEG samples shows the hexagonal and spherical shaped morphology and size lies in the

range of 25-40 nm. A SEM image of ZnO-PVP with a low magnification (Figure 2.5(b))

displays that the sample is composed of microstructure. A SEM image with high

magnification (Figure 2.5(a)) reveals that the ZnO microstructure has a complex

superstructure assembled by elongated hexagonal disks. The width of these disks is 25-35 nm

and length 50-100 nm.

Figure 2.5: SEM micrograph of ZnO, ZnO-PEG and ZnO-PVP nanoparticles, (left) high

resolution images (150,000 x) (Right) low resolution images (50,000 x).

29

2.3.5 Energy dispersive X-ray (EDX)

The Energy dispersive X-ray diffractive (EDX) study is carried out for the synthesized

uncapped and capped ZnO nanoparticles (Figure 2.6) to know about the elemental

composition of samples. The EDX data show almost the same peaks for all samples. EDX

patterns indicate that the prepared ZnO nanoparticles are composed of only zinc and oxygen.

No evidence of other impurities is found and the ZnO nanoparticles are nearly stoichiometric.

This observation is in good agreement with XRD results, which confirmed the phase purity of

ZnO nanoparticles.

Figure 2.6: Energy-dispersive X-ray (EDX) spectra of ZnO, ZnO-PEG and ZnO-PVP

nanoparticles.

2.3.6 Antibacterial Activity of Nanoparticles

All three types of nanoparticles are tested for their antibacterial potential. Results are given in

Table 2.2, which reveal that all nanoparticles show antibacterial activity. However, it is

30

evident that more activity is possessed by ZnO nanoparticles against Gram-positive bacteria

(Staphylococcus aureus and Bacillus subtilis) as compared to the Gram-negative (Escherichia

coli and Acetobacter) bacterial strains. A significant effect of capping agents, i.e., PEG and

PVP is observed against all of the bacterial strains resulting in enhancement of antibacterial

activity against all strains, yet no significant difference in activities between ZnO-PEG and

ZnO-PVP is observed. However, antibacterial activity is observed to be highest (17 mm)

against Bacillus subtilis by ZnO-PVP nanoparticles.

Table 2.2: Antibacterial activity of nanoparticles in terms of their zone of inhibition against

bacterial strains. The small letters marked on each values represent significant difference

among the values at p<0.05 using LSD.

Test

Sample

Zone of inhibition (mm) against bacterial strains

Staphylococcus

aureus

Bacillus

subtilis

Escherichia

coli Acetobacter

Cefixime

(Standard)

ZnO 10±0.70c 13±1.11

c 7±0.72

c 9±0.10

c 21±1.22

ZnO-PEG 12±0.10b 16±1.10

ab 11±0.71

a 14±0.10

a 24±1.45

ZnO-PVP 14±0.75a 17±1.15

a 10±0.10

b 12±0.70

b 26±1.13

2.3.7 Antioxidant Activities of Nanoparticles

The data of antioxidant assays depict that capping agents result in significant rise of activities

of ZnO nanoparticles (Table 2.3).

The most premier quantity of flavonoids equvilance is revealed by ZnO-PVP (19.2 µg

QE/mg), while phenolics equvilance is found to be more significant in ZnO-PEG (22.9 µg

GAE/mg). ZnO-PEG exhibit highest total antioxidant capacity (22.8 µg AAE/mg). It is well-

known that the colour of the sample changes either to green or blue depending on the

reducing power of test samples and eventually higher absorbance confirms higher reducing

power. The most significant reducing power is found in ZnO-PVP (15.08 µg AAE/mg) while

the percentage DPPH scavenging potential is calculated to be similar in both ZnO-PEG and

ZnO-PVP (13.66% and 13.75%, respectively).

2.3.8 Antidiabetic Activity of Nanoparticles

The results of α-amylase inhibition assay in Figure 2.6 reveal that capping agents produce

significant enhancement of antidiabetic activity. Maximum enzyme inhibition (77.03%) is

31

achieved with ZnO-PEG nanoparticles followed by ZnO-PVP (74.74%), and the minimum α-

amylase inhibition (58.65%) is revealed by ZnO.

Table 2.3: Antioxidant assays: flavonoid equivalent determination (FED), phenolic

equivalent determination (PED), and antioxidant activities including total antioxidant activity

(TAC), total reducing power (TRP) and DPPH % inhibition of nanoparticles. The small

letters marked on each values represent significant difference among the values at p<0.05

using LSD.

Test

Sample

Antioxidant Activities (mg/mL) of Nanoparticles

FED (µg QE/mg)

PED (µg GAE/mg)

TAC (µg AAE/mg)

TRP (µg AAE/mg)

% DPPH

Inhibition

ZnO 4.55±0.01c 13.2±1.12

c 13.1±1.11

c 6.64±0.05

c 9.66

b

ZnO-PEG 11.7±1.11b 22.9±2.24

a 22.8±1.55

a 13.5±1.13

b 13.66

a

ZnO-PVP 19.2±2.11a 20.3±2.00

ab 19.1±1.64

b 15.1±1.65

a 13.75

a

Figure 2.7: Comparison of antidiabetic activities of ZnO, ZnO-PEG and ZnO-PVP

nanoparticles against α-amylase enzyme. The small letters marked on each bar represent

significant difference among the values at p<0.05 using LSD.

c

a b

0

10

20

30

40

50

60

70

80

90

ZnO ZnO-PEG ZnO-PVP

α-a

myl

ase

inh

ibit

ion

(%

)

32

2.4 Discussion

The study encompasses the synthesis of ZnO, ZnO-PEG and ZnO-PVP by co-precipitation,

and the effect of PEG and PVP to produce a smaller crystallite size of ZnO nanoparticles as it

is revealed by the results of XRD. The peaks of XRD are exactly similar to the standard

confirming the formation of ZnO, ZnO-PEG or ZnO-PVP nanoparticles. PEG and PVP are

absorbed onto the surface of ZnO nanoparticles and could thus inhibit diffusion of growth

species from the surrounding solute atoms onto the surface to growing particles. FTIR reveals

the functional groups of capped and uncapped ZnO nanoparticles. UV–visible reflectance

spectrum provides a convenient way to investigate the band gap of materials. The band gap

increases with the decrease in particles size (Tahir et al. 2009). The SEM analysis shows

morohology of nanoparticles, and the purity of ZnO, ZnO-PEG and ZnO-PVP nanoparticles

is confirmed by EDX.

Regarding antibacterial activity of nanoparticles, the results support the previous reports that

indicate more prominent antibacterial effect of ZnO nanoparticles against Gram-positive

bacteria (Brayner 2006; Jin et al. 2009; Premanathan et al. 2011; Tanna et al. 2015) as

compared to Gram-negative bacteria. The possible mechanism behind antibacterial activity

lies in the formation of reactive oxygen species (ROS), lipid peroxidation and protein

oxidation causing bacterial cell membrane dissipation and toxicity. Presence of strong

antioxidant activities by ZnO nanoparticles have been realized (Singh et al. 2014) because of

free activation energy or other structural properties, and all antioxidant activities have been

found to be far less in uncapped ZnO nanoparticles in comparison with the capped ZnO

nanoparticles (Das et al. 2013).

Reports on the use of ZnO nanoparticles as potent antidiabetic agents in rats are available

(Alkaladi et al. 2014; Umrani and Paknikar 2014), and the results are in good agreement with

the previous reports in which it is realized that the capping agents enhance biological

activities by reducing the size and increasing the surface area of nanoparticles. More surface

area results in more electrostatic interactions with the corresponding living cell membranes

leading to a rise of activities (Nair et al. 2009).

In summary, ZnO nanoparticles are synthesized and capped with surfactants using a simple

chemical method of co-precipitation. The characterizations of ZnO, ZnO-PEG and ZnO-PVP

nanoparticles is carried out that reveals a decrease in size of ZnO nanoparticles after capping.

XRD patterns of samples show that all samples exhibit wurtzite structure with no secondary

33

phases. The band gap energy is found to increase with the decrease in particle size. The

antibacterial, antioxidant and antidiabetic activities of nanoparticles depict a significant

increase possessed by ZnO-PEG and ZnO-PVP nanoparticles as compared to the uncapped

ZnO nanoparticles. All biological assays reveal highest activities in capped ZnO

nanoparticles as compared to the uncapped ZnO nanoparticles. The antibacterial activity is

more prominent against Gram-positive bacteria. The highest antibacterial activity has been

exhibited by ZnO-PVP while highest antioxidant and antidiabetic activities have been

conferred by ZnO-PEG.

In conclusion, the capping of ZnO results in improvement of physiological, chemical and

biological properties. The polymeric ZnO nanoparticles can be further exploited for carrier of

drug molecules etc.

34

CHAPTER 3

Synthesis, Characterization and Bioassays of CuO Nanoparticles

and its Derivatives

Article Published:

Javed R, Ahmad M, Haq IU, Nisa S, Zia M. (2017) PVP and PEG Doped CuO Nanoparticles

are More Biologically Active: Antibacterial, Antioxidant, Antidiabetic and Cytotoxic

Perspective. Materials Science and Engineering C. 79: 108-115.

https://doi.org/10.1016/j.msec.2017.05.006

3.1 Introduction

Nanomaterials have gained an ever-increasing interest in recent years because of their unique

properties and a wide range of applications (Balazs et al. 2006). Metal oxide nanoparticles

are used for a large variety of applications including catalysis, sensors, optoelectronic

materials and environmental remediation (Aprile et al. 2010). Controlled synthesis of metal

oxide nanoparticles is essential for several applications, and solution phase methods provide

a large degree of control over the synthesis products (Dahl et al. 2007). Different metallic

oxide systems have been investigated so far from both a fundamental and an application

point of view; iron oxide (Hematite Fe3O4, Maghemite Fe2O3), zinc oxide (ZnO), tin dioxide

(SnO2), magnesium oxide (MgO), alumina (Al2O3), titanium dioxide (TiO2) and cupric oxide

(CuO) to name a few (Arakha et al. 2015; Seabra and Duran 2015; Vinardell and Mitjans

2015). The surface morphology, size, shape, and crystal structure of nanomaterials are

important parameters that influence chemical, optical, electrical, and even biological

properties. Like other metallic oxide nanoparticles, CuO has gained notable importance in

recent years owing to fundamental and vast applications in different fields of science

specifically engineering and pharmacy (Hsieh et al. 2003; Ben-Moshe et al. 2009; Ren et al.

2009; Seo et al. 2011; Tran and Nguyen 2014).

Copper oxide (CuO) is a p-type semiconductor with narrow indirect band gap of about 1.2

eV and it has been recognized as an industrially important material for a variety of practical

applications, such as catalysis, batteries, magnetic storage media, antibacterial composites,

solar energy conversion, and gas sensing and field emission devices (Xu et al. 2004). The

most challenging step in synthesis of CuO nanoparticles is that their average size continues

to increase due to self-agglomeration which can only be prevented by the use of capping

https://doi.org/10.1016/j.msec.2017.05.006

35

agents (polymers). These capping agents prevent nanoparticle aggregation and are likely to

play an important role in stabilization of the nano-system. Coating by polymers cause

electrostatic repulsion between nanoparticles by stearic effect and cause size and structural

stability (Tran and Nguyen 2014). The mechanism involves covalent bond formation

between the nanoparticles surface and polymeric chains that result in nanoparticles stability

for months (Lopez-Serrano et al. 2014).

Nowak et al. (2014) studied the antibacterial activities of CuO on the growth of Gram

negative bacteria, Klebsiella pneumonia and Salmonella typhimurim, and Gram positive

bacteria, Enterococcus faecalis and Sarcinalutea. They found that the Gram negative bacteria

showed a fast reaction at very low concentration while for the Gram positive bacteria the

effect was satisfactory after a longer time. Strong antibacterial activity of CuO nanoparticles

against Gram-positive bacteria (Staphylococcus aureus) has been reported by Germi et al.

(2014). CuO nanoparticles against eight bacterial strains were tested and showed excellent

antimicrobial activities of nanoparticles (Ahamed et al. 2014). The copper oxide

nanoparticles have also been tested for antitumor activity. These particles showed a very

good result after 48 h against two kinds of cancer cells, AGS (human gastric carcinoma) cells

and HeLa (human cervix carcinoma) cells (Vinardell and Mitjans 2015; Germi et al. 2014).

The current study involves synthesis of CuO, CuO-PEG and CuO-PVP nanoparticles by

simple co-precipitation method, and characterization by means of X-ray diffraction (XRD),

Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) and

Energy dispersive X-ray spectra (EDX). Thereafter, various biological assays comprising

antibacterial, antioxidant, antitumor, cytotoxic, and antidiabetic activities have been

performed. Hence, we present a systematic study of the structural, morphological and

biological properties of cupric oxide nanoparticles.


3.2.1 Synthesis of CuO, CuO-PEG and CuO-PVP Nanoparticles

The promising method of co-precipitation is utilized for synthesis of CuO nanoparticles. The

method of Ahamad et al. (2014) is followed with slight modifications. Copper acetate

monohydrate (Cu (CH3COO)2. H2O; 98%) and sodium hydroxide (NaOH) were purchased

from Sigma Aldrich and used as received. 600 mL solution of 0.2 M copper acetate

monohydrate is prepared and stirred at 100°C using magnetic stirrer. 2 ml of glacial acetic

acid (CH3COOH) is dropwise added in it. Separately, 30 ml of 6 M aqueous NaOH solution

36

is prepared and dissolved in the above solution until a homogeneous solution is obtained. The

color change occur from blue to green and then brown. Black color is achieved at pH 6-7.

This blackish solution is then filtered using Whatman filter paper No. 1 to get the precipitates

of CuO that are subjected to washing with distilled water and ethanol thrice. The precipitates

are heated at 100°C until dried. The grinding of CuO nanoparticles is performed to make fine

powder. Afterwards, calcination is performed at 500°C for 4 h.

In order to prevent self-agglomeration of CuO nanoparticles, the capping of CuO

nanoparticles with PEG and PVP is carried out separately during synthesis by mixing the

capping agents with precursor salt, i.e., copper acetate monohydrate in 1:1 ratio, and later on

following the similar procedure of CuO nanoparticles synthesis.

3.2.2 Characterization of Synthesized Nanoparticles

CuO, CuO-PEG and CuO-PVP nanoparticles are characterized by the analytical methods of

X-ray diffraction (XRD), Fourier-transform Infra-red spectroscopy (FTIR), Scanning electron

microscopy (SEM) and Energy dispersive X-ray (EDX) as described in Chapter 2.

3.2.3 Biological Assays of Nanoparticles

Different biological assays involving antibacterial, phytochemical and antioxidant,

cytotoxicity, antitumor and antidiabetic assay are performed.

3.2.3.1 Antibacterial, Antioxidant and Antidiabetic Assay

Antibacterial, antioxidant and antidiabetic assays are performed according to the

methodology described in Chapter 2.

3.2.3.2 Cytotoxicity Assay (Brine shrimp lethality assay)

The cytotoxicity assay is executed by a method of Bibi et al. (2011) with slight modification.

The basic purpose of this assay is the assessment and subsequent quantification of percentage

of surviving nauplii. The eggs of Artemia salina are hatched in an unequal, perforated and bi-

partitioned tank filled with artificial sea water at 30oC. The larger compartment of the tank

containing eggs is covered with aluminium foil. After two days, the hatched shrimps are

collected while being gathered towards the illumination source placed over the smaller

compartment. The mature nauplii are harvested with the help of Pasteur pipette in a small

beaker. Various dilutions of stock solution of each sample are tested for lethality

determination at concentrations of 50, 25 and 12.5 µg/mL. These dilutions are transferred to

each well containing 10 nauplii and 150 µL of sea water supplemented with dried yeast (6

37

mg/L). Positive control includes 4 mg/mL of standard drug, doxorubicin while negative

control contains distilled H2O. The final volume in each well is raised to 300 µL with sterile

sea water. After 24 h incubation period, dead nauplii are counted in each well and LC50 for

the samples with ≥ 50% mortality is calculated accordingly using table curve 2D Ver.4

software.

3.2.3.3 Antitumor Assay

This assay is performed according to the method of Yao et al. (2011) with slight

modifications. It involves the use of refreshed culture of Streptomyces 85E strain, 100 µL of

which is applied to lawn minimal ISP4 plates. Whatmann filter paper discs of 6 mm diameter

are impregnated with 5 µL of test sample aliquots (4 mg/mL) and placed on freshly-seeded

plates. Surfactin and distilled H2O impregnated discs are kept as positive and negative

controls, respectively. Incubation period of 72 h is given to allow the bacterial hyphae

development to take place. The development of clear or bald zone around the disc provides

an indication of inhibition of phosphorylation (inhibition of spores and mycelia). The bald

inhibition zone of 12 mm is considered significant. Development of clear zone represents

cytotoxicity of samples by killing Streptomyces while development of bald zone signifies

inhibition of hyphae formation.

3.2.4 Statistical analysis

All assays are performed in triplicate and the mean with standard deviation is calculated

followed by their statistics analysis at the significance level of 0.05 using least significant

difference (LSD) analysis.

3.3 Results

The CuO nanoparticles have been successfully synthesized by co-precipitation method as

shown by chemical reaction below. Copper acetate upon heating and addition of basic

medium turns into blackish solution defining formation of CuO nanoparticles.

Cu(CH3COOH)2 . H20 ∆ → aq medium

Cu(OH)2 + 2CH3COOH

Cu(OH)2Na+OH−

↔ (Cu(OH)4)−2 + Na+

Cu(OH)2H+OH−

↔ (Cu(OH)4)−2 + H+

(Cu(OH)4)−2↔CuO + H20 + 2OH

−

38

The presence of PEG and PVP surfactants in the medium hypothetically binds on the surface

of nanoparticles by simple hydrogen bonding or electrostatic forces as described in Figure

3.1. The confirmation of binding and other physio-chemical properties are studied by

different techniques.

Figure 3.1: Systematic representation of capping of PEG and PVP on the surface of CuO

nanoparticles.

3.3.1 X-Ray Diffraction (XRD)

The powder patterns are recorded with the use of Empyrean PANalytical X-ray

diffractometer with Bragg-Brentano geometry using Cu Kα radiation (λ = 1.54 Å). The step-

scan covers the angular range 20-80º with the step of 0.02º. Figure 3.2 shows the XRD

pattern of all the prepared nanoparticles. The diffraction data reveal that the material is

composed of crystalline monoclinic cubic cupric oxides. The position of all peaks are in good

agreement with the PCPDFWIN data card 895899. The crystallite size is determined using

the Scherrer’s equation (Cullity 1978).

B

kD

cos

Where D is the crystallite size, k is a constant (~ 0.94 assuming that the particles are

spherical), λ is the wavelength of X-ray radiation, β is the line width at half maximum

intensity of the peak and θB is the angle of diffraction. The particles size obtained from the

39

XRD data is listed in table 3.1. The size of the nanoparticles decreased due to the surfactant.

The lattice parameter of CuO monoclinic structure and the plane spacing dhkl is related to the

lattice constants and the Miller indices (hkl) (Cullity 1978)

ac

hl

c

l

b

k

a

h

d hkl

cos2sin

sin

112

2

2

22

2

2

22

Using above equation along with Bragg’s law (2dhklsinθ=nλ), the values of lattice parameters

are calculated. Monoclinic CuO crystal has the lattice constants a, b, c and angle α as listed in

table 3.1. A significant change in the lattice constant b and c is observed due to decrease in

the particles size.

Table 3.1: Particle size, lattice parameters (a, b, c) and angle γ of CuO nanoparticles.

Samples Particle

Size (nm)

Lattice Parameter

a(Å) b (Å) c (Å) Γ

CuO 47 4.690 3.418 5.122 99.57º

CuO-PEG 27 4.685 3.419 5.156 99.57º

CuO-PVP 27 4.685 3.423 5.156 99.57º

Figure 3.2: X-ray diffractogram of (a) CuO, (b) CuO-PEG and (c) CuO-PVP nanoparticles.

40

3.3.2 Fourier-Transform Infra-Red (FTIR) Spectroscopy

FTIR spectroscopy is an effective technique to reveal the composition of sample. Figure 3.3

shows the FTIR spectra of CuO nanoparticles. Various well-defined peaks are observed at

3419, 2920, 2847, 2094, 1411, 1052, 869, 594 and 535 cm-1

. The absorption peak around

3419 cm-1

is assigned to the O H stretching vibration, and the peak around 2094 cm−1

is due

to the existence of CO2 molecules in air. The bond at 1052 cm-1

is due to the C O stretching

vibration (Zhao et al. 2012). Origin of two well-defined absorption bands at 1411 cm-1

are

due to the CH3 group, and CH3 asymmetrical stretching mode present on the surface of CuO

nanostructures. The bands at 2847 and 2920 cm-1

are assigned to –CH2 and C-H stretching

mode. The appearance of the peaks at 535, 594 corresponds to the characteristic stretching

vibrations of Cu-O bonds in the monoclinic crystal structure of CuO (Zheng and Liu 2007;

Ethiraj and Kang 2012) and 869 cm-1

corresponds well to Cu-O-H vibration (Park and Kim

2004; Yu et al. 2012).

Figure 3.3: FTIR spectra of (a) CuO, (b) CuO-PEG and (c) CuO-PVP nanoparticles.

( c )

( b )

Tr

an

sm

itta

nc

e

4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0

38

31

53

5

34

19

57

4

10

52 8

69

14

11

20

94

28

47

29

20

W a v e N u m b e r ( c m- 1

)

( a )

37

28

41

3.3.3 Scanning Electron Microscopy (SEM)

Morphology of the sample is investigated using scanning electron microscope. Specimens are

prepared by sticking CuO nanoparticles to the carbon tape, and blow away the excess of

powder with compressed air. Then these specimens are sputter coated with a thin Au-Pd layer

of about 3 nm thickness in vacuum to avoid charging. Typical SEM micrographs for prepared

nanoparticles without/with surfactant (CuO, CuO-PEG and CuO-PVP) are shown in Figure

3.3. The SEM micrograph of CuO and CuO-PEG clearly shows irregular shaped

morphologies of nanoparticles. The average size of the nanoparticles lies between 40-100 nm

and 25-70 nm for CuO and CuO-PEG, respectively. The morphology of the CuO-PVP is

more or less spherical, and average particles size is in the range of 25-90 nm.

Figure 3.4: SEM micrograph of CuO, CuO-PEG and CuO-PVP nanoparticles, (left) high

resolution images (150,000 x) (Right) low resolution images (50,000 x).

42

3.3.4 Energy Dispersive X-ray (EDX) Spectra

The EDX spectrum of all the prepared (CuO, CuO-PEG and CuO-PVP) nanoparticles is

given in Figure 3.5. The EDX results show that there are no other elemental impurities

present in the prepared nanoparticles.

Figure 3.5: Energy-dispersive X-ray (EDX) spectra of CuO, CuO-PEG and CuO-PVP

nanoparticles.

0 2 4 6 8 1 00

1

2

3

4

5

6E lem en t W t% E rro r

C 2 .4 2 0 .8 8

O 7 .6 7 0 .1 9

C u 8 6 .1 0 .8 3

P d 1 .1 3 0 .1 5

A u 2 .6 9 0 .2 2

T o ta l 1 0 0

C

Cu

Cu

Pd

Au

Cu

OK

a

C u O -P V P

CP

S/e

V

E n erg y (k eV )

0

3

6

9

1 2E lem en t W t% E rro r

C 5 .0 4 1 .1

O 2 1 .3 3 0 .3 5

C u 7 0 .1 1 0 .1 5

P d 1 .0 4 0 .1 5

A u 2 .4 8 0 .2 3

T o ta l 1 0 0

Cu

Cu

Pd

Au

CO

Cu

C u O -P E G

CP

S/e

V

0

1

2

3

4

5C u O

Cu

Cu

Pd

Au

Cu

OC

CP

S/e

V

E lem en t W t% E rro r

C 3 1 .1 3

O 1 1 .3 2 0 .2 7

C u 8 3 .2 6 1 .0 2

P d 0 .9 0 .1 6

A u 1 .5 1 0 .2 2

T o ta l 1 0 0

43

3.3.5 Antibacterial Activity of Nanoparticles

The antibacterial potential of CuO, CuO-PEG and CuO-PVP nanoparticles was tested. Table

3.2 shows antibacterial activity (radial diameter of the inhibition zone) exhibited by all

nanoparticles in this study. Zone of inhibition against two Gram positive bacterial strains,

Staphylococcus aureus and Bacillus subtilis, and two Gram negative bacterial strains,

Escherichia coli and Acetobacter are shown in the Fig. 3.6.It clearly indicates that the Gram-

positive bacteria (Staphylococcus aureus and Bacillus subtilis) possess significantly greater

antibacterial activity compared to Gram-negative bacteria (Escherichia coli and Acetobacter).

The highest antibacterial activity (22 mm) is revealed by CuO-PVP against Bacillus subtilis.

Moreover, the CuO-PEG and CuO-PVP nanoparticles have shown more antibacterial

activity than the uncapped CuO nanoparticles due to the larger size of the later.

Table 3.2: Antibacterial activity of CuO, CuO-PEG and CuO-PVP nanoparticles in terms of

their zones of inhibition. The small letters marked on each value represent significantly

difference at p<0.05 using LSD.

Test

Sample

Zone of inhibition (mm) against bacterial strains

Staphylococcus

aureus Bacillus

subtilis Escherichia

coli Acetobacter

CuO 11±0.5c 14±0.76

c 9±0.45

c 7±0.35

c

CuO-PEG 14±0.28

b 20±0.5

b 13±0.66

a 10±0.75

b

CuO-PVP 16±0.28

a 22±0.57

a 12±0.32

b 11±0.15

a

Cefixime

(Standard) 21±1.22 24±1.45 26±1.13 22±1.10

3.3.6 Antioxidant Activities of Nanoparticles

Table 3.3 shows the antioxidant activities exhibited by CuO, CuO-PEG and CuO-PVP

nanoparticles. This data clearly depicts a significant difference between the activities of

uncapped CuO and CuO-PEG or CuO-PVP nanoparticles.

The most premier quantity of phenolics equvilance (30.7 µg GAE/mg) and flavonoids

equvialnce (26.4 µg QE/mg) is obtained by CuO-PVP. Similarly, the most significant

antioxidant activity (32.44 µg AAE/mg) and reducing power (17.38 µg AAE/mg) is also

44

found to be greater in CuO-PVP. However, the percentage DPPH scavenging potential

(34.14%) is estimated to be highest in CuO-PEG.

Figure 3.6: Antibacterial assay: Zone of inhibition against two Gram positive bacterial

strains, Staphylococcus aureus and Bacillus subtilis, and two Gram negative bacterial strains,

Escherichia coli and Acetobacter.

3.3.7 Cytotoxic Activity of Nanoparticles

In vitro cytotoxic assays against brine shrimp (Artemia salina) have been performed to

preliminary screen the toxicity of test samples. According to figure 3.7, all CuO

nanoparticles are found to be cytotoxic, however, the maximum cytotoxicity is achieved at

50 µg/mL nanoparticle concentration because it exhibits 100% cytotoxicity. After that, 25

µg/mL concentration of nanoparticles shows lethality of shrimps, and the least cytotoxic

effect is determined at 12.5 µg/mL of CuO nanoparticles. Percent mortality of brine shrimps

is depicted significantly higher by the CuO-PEG and CuO-PVP nanoparticles as compared

to uncapped CuO nanoparticles because of high surface to volume ratio of the former.

45

Table 3.3: Phytochemical assays: phenolic equivalent determination (PED), flavonoid

equivalent determination content (FED), and antioxidant activities including total antioxidant

activity (TAC), total reducing power (TRP) and DPPH % inhibition of CuO, CuO-PEG and

CuO-PVP nanoparticles. The small letters marked on each value represent significantly

difference at p<0.05 using LSD.

Test

Sample

Antioxidant Activities of CuO Nanoparticles

PED (µg GAE/mg)

FED (µg QE/mg)

TAC (µgAAE/mg)

TRP (µgAAE/mg)

% DPPH

Inhibition

CuO 19.3±0.2c 14.9±0.47

c 18.94±0.57

c 7.10±0.3

c 13.79

c

CuO-PEG 26.3±0.24b 22.2±0.13

b 27.42±0.24

b 16.64±0.2

b 34.14

a

CuO-PVP 30.7±0.3a 26.4±0.25

a 32.44±0.1

a 17.38±0.15

a 28.36

b

Figure 3.7: Cytotoxic activity of CuO, CuO-PEG and CuO-PVP nanoparticles determined by

brine shrimp lethality assay.

3.3.8 Antitumor Activity of Nanoparticles

The antitumor activity of uncapped and capped (CuO-PEG and CuO-PVP) nanoparticles is

evaluated by calculating the zone of inhibition of nanoparticles against Streptomyces. Figure

3.8 illustrates that maximum zone of inhibition (14 mm) is observed by CuO-PEG

nanoparticles, followed by the CuO-PVP (10 mm) and CuO (8 mm) nanoparticles.

46

3.3.9 Antidiabetic Activity of Nanoparticles

The antidiabetic activity of CuO, CuO-PEG and CuO-PVP nanoparticles is determined by the

use of α-amylase enzyme. The figure 3.9 clearly shows that uncapped CuO nanoparticles

exhibit lesser activity on comparative basis. CuO-PEG and CuO-PVP nanoparticles reveal

32.23% and 37.75% α-amylase inhibition, respectively. However, the uncapped CuO

nanoparticles achieve 19.17% of inhibition.

Figure 3.8: (a) Zone of inhibition against Streptomyces, (b) Comparison of anti-tumor

activities of CuO, CuO-PEG and CuO-PVP nanoparticles against Streptomyces. The small

letters marked on each bar represent significantly difference at p<0.05 using LSD.

c

a

b

0

2

4

6

8

10

12

14

16

CuO CuO-PEG CuO-PVP

Zon

e of

Inh

ibit

ion

(m

m)

47

Figure 3.9: Comparison of antidiabetic activities of CuO, CuO-PEG and CuO-PVP

nanoparticles against α-amylase enzyme. The small letters marked on each bar represent

significantly difference at p<0.05 using LSD.

3.4 Discussion

Uncapped CuO and capped CuO (CuO-PEG and CuO-PVP) nanoparticles are obtained as a

result of synthesis and capping processes. The characterization performed by XRD, FTIR,

SEM and EDX clarified that the nanoparticles obtained are purely CuO, CuO-PEG and CuO-

PVP. The XRD determines crystallinity, FTIR measures functional group, SEM defines

morphology and EDX reveals purity of nanoparticles (Ali et al. 2016).

The highest antibacterial activity (22 mm) is revealed by CuO-PVP against Bacillus

subtilis. It is because the PVP on the surface of CuO binds more efficiently to the Gram-

positive bacterial cell wall. The mechanism involves change in cell membrane

permeability of Gram-positive bacteria which results in penetration of CuO-PVP into the

bacterial cell cytoplasm, ultimately leading to cell death (Javed et al. 2016). The CuO-PEG

and CuO-PVP nanoparticles have shown to be more antibacterial than the uncapped CuO

nanoparticles due to the larger size of the later. This significant variation is also due to the

combined effect of CuO and either PEG or PVP attached to its surface or may be due to

the different morphology of the nanoparticles. The PEG and PVP are highly antibacterial

long chain polymerizing agents having carbonyl functional groups that play a key role in

conferring the transport and penetration of nanoparticles, hence killing the bacteria (Saggers

c

b

a

0

5

10

15

20

25

30

35

40

45

CuO CuO-PEG CuO-PVP

α-a

myla

se i

nh

ibit

ion

(%

)

48

and Stewart 1964; Chirife et al. 1983; Lewis 2000; Tiller et al. 2001). CuO nanoparticles

have shown marked scale antibacterial activity (Das et al. 2013; Concha-Guerrero et al.

2014; Germi et al. 2014; Purkayastha et al. 2014; Meghana et al. 2015). Gram-positive

bacteria (Staphylococcus aureus and Bacillus subtilis) possess significantly greater

antibacterial activity compared to Gram-negative bacteria (Escherichia coli and

Acetobacter). The similar results are depicted in case of ZnO nanoparticles in few other

studies (Brayner et al. 2006; Jin et al. 2009; Premanathan et al. 2011; Tanna et al. 2015;

Javed et al. 2016). The most probable reason for this prominent variation among Gram-

positive and Gram-negative bacteria is the difference in composition and thickness of their

cell walls. The cell membranes and cell walls of both groups of bacteria possess different

functional groups on their surface (Rizvi et al. 2013; Javed et al. 2016).

CuO nanoparticles have shown strong antioxidant activities (Das et al. 2013; Purkayastha et

al. 2014) and it is interesting to notice greater amount of antioxidant activities in CuO-PEG

and CuO-PVP nanoparticles as compared to the uncapped ones. This behavior is similar to

the behavior of ZnO, ZnO-PEG and ZnO-PVP nanoparticles (Javed et al. 2016), and it is due

to the presence of carbonyl group and ester linkages in both PEG and PVP that confer

antioxidative activities (Juarez-Moreno et al. 2015). Thus, capping of these surfactants to

CuO contributes in significant enhancement of antioxidant activities of CuO nanoparticles.

Both capped and uncapped CuO nanoparticles exhibited significant cytotoxic activity against

brine shrimps. The capped are more cytotoxic as compared with uncapped, furthermore,

cytotoxicity increases by increase in concentration. CuO nanoparticles have shown

significant cytotoxicity in human cells in dose- and time-dependant manner (Fahmy and

Cormier 2009; Alarifi et al. 2013; Shafagh et al. 2015; Vinardell and Mitjans 2015). The

mechanism behind cytotoxic activity against different human cell lines such as cancer cells

is described to be the blockage of cellular oxidative defences and apoptosis through

generation of reactive oxygen species (ROS) (Vinardell and Mitjans 2015).

CuO nanoparticles exhibit strong antitumor activity against different cancer cells, i.e., AGS

(human gastric carcinoma) and HeLa (human cervix carcinoma) as previously described

(Germi et al. 2014). The mechanism involves perforation of excessive Cu+2

ions and reactive

oxygen species (ROS) into the cell cytoplasm where it disrupts homeostasis by interacting

with biomolecules due to their large surface area and electron density. This oxidative stress

causes disruption of oxidation and anti-oxidation processes and finally these ions enter in to

the nucleus resulting in DNA damage (Chang et al. 2012). Antitumor effects of CuO

49

nanoparticles on subcutaneous melanoma and metastatic lung tumors have been studied in

mice models (Vinardell and Mitjans 2015).

The mechanism behind enhancement of antidiabetic activity in CuO-PEG and CuO-PVP

nanoparticles involves the reduction in size and significant movement of atoms towards the

outer surface of capped nanoparticles as a result of more surface area (Usman et al. 2013). Cu

nanoparticles have been found antidiabetic (Ghosh et al. 2015).

Summing up, CuO nanoparticles with PEG and PVP as capping agents have been

synthesized by co-precipitation method. Characterization of CuO nanoparticles by XRD,

FTIR, SEM and EDX declares good crystallinity, high purity and different morphology.

XRD data reveal a decrease in the size of capped CuO as compared to the bare counterpart.

The attachment of capping agent; PEG/PVP results in smaller size, large surface area and

hence more reactivity. A significant amount of antibacterial, antioxidant, cytotoxic,

antitumor and antidiabetic activity is conferred by all of these nanoparticles. However, all

biological activities are greater in capped CuO nanoparticles as compared to the uncapped

ones. A substantial change in size and shape of nanoparticles cause a difference in their

biological reactivity. The higher activity of these synthetic capped CuO nanoparticles for

α-amylase inhibition in vitro provide strong scientific evidence for antidiabetic potential

of capped CuO nanoparticles.

50

CHAPTER 4

Tissue Culture and DPPH-free radical scavenging activity of

Stevia rebaudiana Bertoni

Article Submitted:

Javed R, Zia M, Yucesan B, Gurel E. (2017) Differential Effect of Plant Growth Regulators

(PGRs) Stress on Physiology, Steviol Glycosides Content, and Antioxidant Capacity in

Micropropagated Tissues of Stevia rebaudiana Bertoni. Biologia.

4.1 Introduction

Stevia rebaudiana, commonly known as “sugar leaf” or “candy leaf” belongs to the family

Asteraceae (Mohamed and Alhady 2011). It is a zero calorie sweetner having no

carbohydrates. This perennial sweet herb is found in humid and wet environments, and is

indigenous to Brazil and Paraguay (Soejarto 2002). The bioactive compounds of Stevia are

steviol glycosides (SGs) comprised of rebaudioside A, rebaudioside C and stevioside as basic

constituents that have 250-300 times greater sweetness than sugar (Chalapathi and

Thimmegowda 1997). These active constituents are mainly found in its leaves which are

edible and also used in different foods. For example, tea of Stevia leaves offers excellent

stomach relief from pain (Goyal et al. 2010). This natural high intensive sweetener causes

blood glucose and insulin regulation, and imposes positive effects on food intake and satiety

in human beings (Anton et al. 2010). The speciality of Stevia is its use in treatment of

diseases like type II diabetes, obesity, hypertension and dental caries (Liu et al. 2003; Dey et

al. 2013). It is reported that SGs also possess anti-oxidant, anti-inflammatory, anti-

carcinognic, anti-microbial, anti-diarrheal and non-mutagenic activities (Ahmad et al. 2011;

Dey et al. 2013). Despite of anti-bacterial use of S. rebaudiana extracts, rebaudioside A (Reb-

A) and stevioside (ST) have been found to create no significant influence in human intestinal

microflora (Gardana et al. 2003). Stevia leaves and extracts are used in weight-loss

programmes because of their ability to reduce the craving for sweet and fatty foods (Jain et

al. 2007).

S. rebaudiana plants produce seeds that are tiny and show poor germination due to infertility

(Goettemoeller and Ching 1999; Yadav et al. 2011). Vegetative cutting is another way of

Stevia propagation which is also limited because it require large amount of stem cuttings to

51

propagate (Tamura et al. 1984; Khalil et al. 2014). These factors limit the efficient production

of such an economically and commercially beneficial medicinal plant leads us towards

biotechnological approaches, mainly tissue culture for mass production of Stevia in a

minimum period of time using single mother plant producing true-to-type progeny (Rafiq et

al. 2007). Moreover, enhanced SGs production can also be achieved by micropropagation

techniques using different PGRs and agar concentrations in different stages of plant

development (Aman et al. 2013).Various factors like selection of explant, sucrose and media

concentration, type and concentration of PGRs and different genotypes of Stevia play a

pivotal role in micropropagation efficiency (Yucesan et al. 2016). Different protocols have

been optimized for enhanced production of secondary metabolites in Stevia grown under

different environmental pressures, hence producing clones having large amount of essential

pharmaceutical compounds (Thiyagarajan and Venkatechalam 2012).

This study involves regeneration of Stevia plantlets by optimizing conditions using different

plant growth regulators (PGRs) for its direct shoot organogenesis, root organogenesis and

callogenesis. The purpose of this work is also to compare free-radical scavenging activity by

1, 1-diphenyl-2-picrylhydrazyl (DPPH) in different plant parts, grown in-vitro.


4.2.1 Seed Germination

The seeds of S. rebaudiana were obtained from Polisan Tarim, Istanbul, Turkey. For

germination, the seeds are washed in running tap water and surface-disinfected under aseptic

conditions by 100 mL of 0.1% (w/v) mercuric (II) chloride (HgCl2). A magnetic stirrer plate

is used for this purpose at 300 rpm for 2 min. Later on, rinsing is done repeatedly with sterile

distilled water. The culture medium is prepared using MS medium (Murashige and Skoog

1962) and 3% (w/v) of sucrose. After adjusting 5.7–5.8 pH of medium using 0.1 N HCl or 0.1

N NaOH, it is solidified with 0.8% (w/v) plant agar. All culture media are autoclaved at

121°C and 1.06 kg cm-2

pressure for 15 min. The seeds are inoculated on MS medium and

shifted to growth room. The shoot nodes and leaf explants of plantlets are used as explants

for in vitro studies.

4.2.2 Callus Induction

For callus induction, ten treatments are prepared using a combination of different auxins and

cytokinins. It involves different combinations of 6-benzyl aminopurine (BAP; 0.5 mg/L to

2.0 mg/L), α-naphthalene acetic acid (NAA; 2.0 mg/L), 2,4-dichlorophenoxy acetic acid (2,4-

52

D; 0.5 mg/L, 2.0 mg/L), kinetin (KIN; 0.5 mg/L) and a treatment having no PGRs taken as

control. MS basal media is used supplemented with 3% (w/v) sucrose, 0.8% (w/v) agar and

growth regulators. In each treatment, 15 leaf explants (0.5 cm2) are cultured in Petri plates for

callogenesis. The culture plates containing leaf explants are then incubated at 24±1°C in 16 h

light: 8 h dark photoperiod (provided by cool-white fluorescent light, irradiance 35 μmol m-

1s

-1) at 55–60%. relative humidity. Data of callogenesis is scored in triplicate, and

documented after 6 weeks. The data involve amount, color, weight and callogenic response of

callus.

4.2.3 Shoot Organogenesis

For shoot organogenesis, a total of seven treatments are prepared by an augmentation of

different cytokinins in MS medium from shoot nodes. These treatments consisted of different

concentrations of 6-benzyl aminopurine (BAP; 1.0 or 2.0 mg/L), kinetin (KIN; 1.0 or 2.0

mg/L), thidiazuron (TDZ; 1.0 or 2.0 mg/L), and a control group without PGRs. The culture

plates containing shoot nodes are then incubated at 24±1°C in 16 h light: 8 h dark

photoperiod (provided by cool-white fluorescent light, irradiance 35 μmol m-1

s-1

) at a relative

humidity of 55–60 %. Data of shoot organogenesis were scored in triplicate, and documented

after 4 weeks of inoculation. The data involve mean percentage of shooting, mean length of

shoots, and mean number of nodes and leaves for shoot organogenesis.

4.2.4 Root Organogenesis

After shoot organogenesis, few shoots are selected to be transferred to separate jars (Magenta

B-cap, Sigma-Aldrich, USA) containing ten treatments prepared by augmenting different

auxins in MS medium. Indole acetic acid (IAA; 0.25 mg/L to 1.0 mg/L), indole butyric acid

(IBA; 0.25 mg/L to 1.0 mg/L), α-naphthalene acetic acid (NAA; 0.25 mg/L to 1.0 mg/L) and

control are used for root formation. The jars were kept in a growth room having 24±1°C

temperature, 35 μmol m-1

s-1

irradiance, 55–60% humidity and 16 h photoperiod. The data of

experiment conducted in triplicate were recorded after 4 weeks and different parameters

comprising of mean percentage of rooting, mean root and plantlet length, and mean number

of roots, leaves, and nodes are measured.

4.2.5 DPPH-free radical scavenging activity

All of the regenerated tissues, i.e., callus, shoots and rooted-shoots are exposed to DPPH (a

constant and stable free radical) and DPPH-free radical scavenging activity is calculated

according to the method of Haq et al. (2012). First of all, the extracts are prepared from dried

53

tissues and 0.1 g of fine powder was dissolved in 500 μl methanol. It is vortexed for 5 min

and then sonicated for 30 min. Then, the centrifugation at 10,000 rpm is performed and

supernatant is collected. 10 µL (4 mg/ml) extract is mixed with 190 µL of DPPH (0.004%

w/v in methanol). The reaction mixture is incubated in the dark for 1 h. Optical density of all

samples is measured at 515 nm wavelength using a microplate reader. Ascorbic acid is used

as positive standard while dimethyl sulfoxide (DMSO) as negative control. Purple color of

DPPH changes to colorless, and the free radical scavenging potential of extracts is

determined as percentage of DPPH discoloration using the following equation:-

% inhibition of test sample = % scavenging activity = (1- Abs/ Abc) x 100

Where Abs indicates the absorbance of DPPH solution with extract, and Abc is the absorbance

of negative control (containing the DPPH solution only). The IC50 is calculated using Table

curve software 2D Ver. 4.


All micro-propagation experiments are performed in triplicates and their design is completely

randomized. Thereafter, the data were statistically analysed using SPSS, Version 17.0 (SPSS

Inc., Chicago, IL, USA). Statistical difference is determined by ANOVA, and Duncan’s

multiple range test at p< 0.05 is used to calculate the significance of difference between

means ± SE (standard error) values.

4.3 Results

The seedlings obtained by the germination of seeds serve as explants for callogenesis and

direct shoot and root organogenesis. The leaves are used as explants for callogenesis and

shoot nodes as explants for shoot organogenesis. The results depict that callogenesis can be

performed using different auxins and cytokinins combination, while shoot organogenesis can

even be performed without an addition of cytokinins. The root organogenesis can be carried

out by inoculating different auxins into the rooting medium using in vitro cultured shoots.

4.3.1 Callogenesis

Table 4.1 shows different protocols for callus formation from leaf explants, and compares

different physiological parameters like callus color, weight (g) and callogenic response (%)

given by different hormonal combinations (auxins and cytokinins) for S. rebaudiana.

Figure 4.1 shows that the highest amount of callus (2.75 cm) is obtained by the combination

of 2 mg/L 6-benzyl aminopurine (BAP) and 0.5 mg/L 2,4-dichlorophenoxy acetic acid (2,4-

54

D) after 6 weeks of culture. However, its DPPH scavenging potential is much lower (61.15%)

as shown in Figure 4.2. Although all hormonal combinations produce callus from leaf

explants of Stevia, the highest free-radical scavenging ability (83.99%) is conferred by 0.5

mg/L BAP and 2.0 mg/L of α-naphthalene acetic acid (NAA) combination. It produces 2.37

cm of callus, the similar quantity of callus is obtained by combining 1 mg/L BAP and 2 mg/L

NAA, and 1.5 mg/L BAP and 0.5 2,4-D, that containes 76.9% and 62.99% DPPH-free radical

scavenging ability, respectively.

Table 4.1: Comparison of physiological parameters in callus tissue developed from leaf

explants cultured on MS media supplemented with different hormonal combinations after 6

weeks of cultivation.

±,standard error of the mean; G: green, B: brown, Y: yellow, GY: yellowish green; the means

with the same letter within the columns are not significantly different according to Duncan’s

multiple range test at P<0.05. PGRs: plant growth regulators, BAP: 6-benzyl aminopurine,

2,4-D: 2,4-dichlorophenoxyacetic acid, NAA: α-naphthalene acetic acid.

Concentration of

PGRs (mg/L)

Response

(%)

FW per

explant (g)

DW per

explant (g)

Color

No PGRs 0 0 0 -

0.5 BAP + 0.5 2,4-D 84.2 0.78±0.05c 0.06±0.01

c GY

0.5 BAP + 2.0 2,4-D 81.1 0.40±0.01g 0.04±0.01

d B

1.0 BAP + 0.5 2,4-D 85.5 0.64±0.01e 0.06±0.01

c GY

1.0 BAP + 2.0 2,4-D 83.3 0.23±0.01h 0.02±0.00

e Y

1.5 BAP + 0.5 2,4-D 86.5 0.57±0.02f 0.05±0.01

c GY

2.0 BAP + 0.5 2,4-D 89.0 0.68±0.02d 0.07±0.01

bc Y

0.5 BAP + 2.0 NAA 87.5 1.13±0.10b 0.08±0.01

b G

1.0 BAP + 2.0 NAA 86.4 1.59±0.05a 0.13±0.02

a G

55

Figure 4.1: Comparison of amount of callus formed under different hormonal combinations.

Standard deviations of the mean value (SD) represented with bar lines. Bars are significantly

different at confidence interval level of 95%.

Figure 4.2: Comparison of % DPPH-free radical scavenging activity of calli grown under

different hormonal combinations and concentrations. Standard deviations of the mean value

(SD) represented with bar lines. Bars are significantly different at confidence interval of 95%.

bc

d

bc c

a

d

bc b

c

0 0

0.5

1

1.5

2

2.5

3

0.5 BAP+ 2 NAA

0.5 BAP+ 2 2,4-D

1 BAP +2 NAA

1 BAP +2 2,4-D

2 BAP +0.5 2,4-D

1 BAP +0.5 2,4-D

1.5 BAP+ 0.52,4-D

0.5 BAP+ 0.52,4-D

0.5 KN +0.5 2,4-D

Control

Am

ou

nt

of

Call

us

(cm

)

PGRs (mg/L)

a

a

bc

b

e

d

e

c

a

0 20 40 60 80 100

0.5 BAP + 2 NAA

0.5 BAP + 2 2,4-D

1 BAP + 2 NAA

1 BAP + 2 2,4-D

2 BAP + 0.5 2,4-D

1 BAP + 0.5 2,4-D

1.5 BAP + 0.5 2,4-D

0.5 BAP + 0.5 2,4-D

0.5 KN + 0.5 2,4-D

Control

DPPH Free Radical Scavenging activity (%)

PG

Rs

(mg/L

)

56

4.3.2 Shoot Organogenesis

Table 4.2 shows different physiological parameters of shoot organogenesis optimized after

treating the nodal shoot explants with different kinds and concentrations of cytokinins. Best

shooting response is obtained from control medium which is consistent with the previous

findings (Ibrahim et al. 2008; Yucesan et al. 2016).


nodal explants on MS medium supplemented with different concentrations of cytokinins.

± standard error, the means with the same letter within the columns are not significantly

different according to Duncan’s multiple range test at P<0.05; + sign represents degree of

callusing. PGRs: plant growth regulators, BAP: benzyl aminopurine, KN: kinetin, TDZ:

thidiazuron.

The DPPH-free radical scavenging potential of control medium is lowest (48.03%) as shown

by Figure 4.3. The maximum mean length of plantlet (5.5 cm), mean number of nodes (4.66)

and mean number of leaves (12) are attributed to MS containing 1 mg/L kinetin (KIN). Its

DPPH-scavenging activity is 60.1 %; much lower than others. The highest free radical

scavenging ability (74.02%) is exhibited by the shoots grown on MS having 2 mg/L BAP. It

produce 4.5 cm mean length of plantlet, 3.66 mean number of nodes and 10.6 mean number

of leaves. Thidiazuron (TDZ) shows the least effective results in either 1 mg/L or 2 mg/L

concentration.

PGRs

(mg/L)

Response

(%)

Mean shoot

length

(cm)

Mean no.

of nodes

per

explant

Mean no. of

leaves per

regenerated

shoot

No PGRs 95.0 4.1±0.2b 4.6±0.4

a 13.0±1.1

a

1.0 BAP 89.0 5.2±0.7a 2.3±0.3

c 6.6±0.6

c

2.0 BAP 84.2 4.5±0.3ba

3.6±0.8ba

10.6±1.3ba

1.0 KN 93.6 5.5±1.0a 4.6±0.3

a 12.0±1.1

a

2.0 KN 91.7 3.3±0.3c 3.6±0.3

b 12.0±1.1

a

1.0 TDZ 86.5 3.2±0.6cd

3.3±0.3b 8.0±1.2

c

2.0 TDZ 83.3 2.6±0.4d 2.3±0.3

c 6.6±1.7

c

57

Figure 4.3: Comparison of % DPPH-free radical scavenging activity of shoots grown under

different cytokinin concentrations. Standard deviations of the mean value (SD) represented

with bar lines. Bars are significantly different at confidence interval level of 95%.

4.3.3 Root Organogenesis

Table 4.3 shows different physiological parameters obtained after optimization of root

organogenesis by treating the shoots with different types and concentrations of auxins. Best

rooting response is shown by 0.5 mg/L of indole butyric acid (IBA). The highest mean length

of plantlets (18 cm) and mean length of roots (4.5 cm) is obtained by 0.25 mg/L of indole

acetic acid (IAA). However, the mean number of roots (7) is much less compared to other

hormones. Our data clearly indicate that a greater number of roots are formed by an

augmentation of IBA instead of IAA or NAA in the MS medium.

The highest DPPH-free radical scavenging activity (64.44 %) has been evaluated by

supplementing 0.25 mg/L of indole acetic acid (IAA) to MS growth medium after 4 weeks of

sub-culture, as shown in Figure 4.4.

b

a

c

b

d

bc

e

0 10 20 30 40 50 60 70 80 90

1 BAP

2 BAP

1 KIN

2KIN

1 TDZ

2 TDZ

Control

DPPH Free Radical Scavenging Activity (%)

PG

Rs

(mg/L

)

58

Table 4.3: Comparison of several physiological parameters in 4 weeks old regenerants

developed on MS medium supplemented with different concentrations of auxins.

Conc.

(mg/L)

Response

(%)

Mean

shoot

length

(cm)

Mean root

length

(cm)

Mean no. of

roots per

regenerated

shoot

Mean no. of

nodes per

regenerated

shoot

Mean no. of

leaves per

regenerated

shoot

Control 87.0 13.6±1.7b 2.8±0.8

c 7.6±1.4

d 7.0±1.0

b 17.3±1.3

ab

0.25 IAA 85.5 18.0±1.0a 4.5±0.8

a 7.0±1.3

d 7.4±0.4

ab 15.0±0.4

c

0.5 IAA 84.3 14.3±0.3b 3.4±0.3

b 6.2±0.8

d 8.2±0.6

a 16.8±1.2

bc

1.0 IAA 83.1 16.9±0.9a 4.1±0.5

ab 4.8±0.4

e 7.4±0.2

ab 13.8±0.6

d

0.25 IBA 90.1 13.3±1.2b 2.8±0.1

c 14.0±0.6

c 7.0±0.6

bc 18.0±0.6

ab

0.50 IBA 94.0 11.0±1.0c 2.6±0.1

c 19.1±2.6

a 6.0±0.5

c 17.3±0.7

b

1.0 IBA 91.4 16.0±2.0ab

4.3±0.8ab

16.0±0.6b 6.6±0.3

bc 19.0±0.5

a

0.25 NAA 88.9 15.5±0.6b

5.5±1.2a

12.6±1.4c

9.0±2.0a

18.0±2.0a

0.50 NAA 92.8 15.6±1.2b 2.6±0.6

c 18.3±2.0

ab 8.3±0.9

a 16.3±2.3

bcd

1.0 NAA 90.5 15.0±1.5b 4.0±0.6

b 16.0±3.0

b 9.6±1.4

a 17.6±1.3

ab

±: standard error, the means with the same letter within the columns are not significantly

different according to Duncan’s multiple range test at P<0.05.

Figure 4.4: Comparison of % DPPH-free radical scavenging activity of plantlets (rooted-

shoots) grown under different auxin concentrations. Standard deviations of the mean value

(SD) represented with bar lines. Bars are significantly different at confidence interval level of

95%.

a

b

d

b

cd

cd

c

b

cd

b

0 10 20 30 40 50 60 70

0.25 IAA

0.5 IAA

1 IAA

0.25 IBA

0.5 IBA

1 IBA

0.25 NAA

0.5 NAA

1 NAA

Control

DPPH Free Radical Scavenging activity (%)

PG

Rs

(mg/L

)

59

4.3.4 DPPH-free radical scavenging activity

The natural foods or medicinal plants have the ability to produce free radical species and are

utilized as antioxidants (Hou et al. 2003; Galvez et al. 2005). Different antioxidants produced

by the leaf extracts of Stevia like phenolics and flavonoids that play an important role in

preventing DNA damage during oxidative stress, and these natural antioxidants scavenge free

radicals due to their high antioxidant potential (Saqib et al. 2015). In the current study, the

highest antioxidant activity regarding DPPH is found in callus, followed by shoots, and lastly

the rooted-shoots/plantlets as shown in Figure 4.5. This is in agreement with the results of

Ahmad et al. (2011) who also found hightest DPPH-FRSA in callus cultures rather than the

shoots or roots of S. rebaudiana.

Figure 4.5: Comparison of % DPPH-free radical scavenging activity of callus, shoots and

plantlets (rooted-shoots). Standard deviations of the mean value (SD) represented with bar

lines. Bars are significantly different at confidence interval level of 95%.

a

b

c

0

10

20

30

40

50

60

70

80

90

100

Callus Shoots Plantlets

DP

PH

Sca

ven

gin

g a

ctiv

ity (

%)

60

Figure 4.6: Schematic diagram of callogenesis and direct plant organogenesis in Stevia

rebaudiana: a) inoculation of leaf explants, b) callus formation, c) inoculation of shoot nodes,

d) formation of shoots, e) formation of shoots, f) formation of plantlet having shoots and

roots.

4.4 Discussion

S. rebaudiana is successfully in vitro cultured for callus induction and shoot organogenesis

from leaf and node explants, respectively. Presence and concentration of plant growth

regulators in MS media significantly varies responses. The callogenic response is more

pronounced in the presence of 2,4-D with BAP, however, callus morphology was more

attractive when 2,4-D is replaced with NAA. The results also demonstrate that free radical

scavenging activity is not dependent upon concentration of growth regulators present in the

media. However callus color seems to have some relation with free radical scavenging

activity. Different hormones at different concentrations have been reported to be effective for

callogenesis and organogenesis. Ojha et al. (2010) found highest amount of callus by the

combination of 1 mg/L BAP and 0.5 mg/L 2,4-D. Contrarily, BAP and 2 mg/L 2,4-D have

been documented to be the best for callus induction (Uddin et al. 2006). According to Ahmad

et al. (2011), the best callogenic response is shown by the combination of 2 mg/L BAP and 2

mg/L 2,4-D using flowers as explants. 3 mg/L 2,4-D is also found to be the best callogenesis

inducer (Uddin et al. 2006; Ali et al. 2010). Gupta et al. (2010) reports NAA and 2,4-D as

best PGRs for callus induction. Moreover, combination of BAP and NAA produces

61

promising results on half strength of MS medium, whereas KIN and 2,4-D combination, and

KIN and NAA produce better results (Das et al. 2006).

Direct shoot regeneration is observed in the presence of cytokinins at different concentratins

from nodal explants. Although shoot emergence percent response from nodal explants is

maximum in hormone free MS basal medium, however, number of shoots per explant, mean

number of nodes per plant and leaf per plant are maximum when KIN is augmented in MS

medium followed by BAP containing medium. It is further observed that DPPH based radical

scavenging activity is not dependent upon percent response or plantlet characteristics.

Organogenesis is a complex process and it exerts oxidative stress. Maximum oxidative stress

is observed in the presence of BAP which is the second best hormone to induce shooting

from explanst. Previous findings declare BAP and KIN as best shooting hormones for Stevia

(Jitendra et al. 2012). But the highest amount of shoots are obtained by supplementing the

MS basal medium with 2 mg/L BAP according to Rafiq et al. (2007), Ojha et al. (2010) and

Ahmad et al. (2011) which is consistent with our finding. However, Ali et al. (2010)

describes 1 mg/L BAP as the best shooting hormone for Stevia. Moreover, 2 mg/L BAP and

0.5 mg/L KIN is found to be the best hormonal combination for shoot organogenesis by

Mohamed and Alhady (2011).

Good rooting response of regenerated shoots is observed on hormone free MS medium.

However, incorporation of rooting hormones (IAA, NAA and IBA) at one end stimulates the

rooting response and on the other end results in healthier plants. Jitendra et al. (2012), and

Mathur and Begum (2015) reported best rooting response with 1 mg/L IBA which is in

agreement with our results. Debnath (2008) and Preethi et al. (2011) found 2 mg/L IBA as the

best rooting hormone. Furthermore, Mohamed and Alhady (2011) found 1 mg/L and 2 mg/L

IBA as the best for rooting. Contrarily, Ojha et al. (2010) declared 0.5 mg/L IAA to be the

best in highest amount of plantlet formation. In addition to this, Ali et al. (2010) and Rafiq et

al. (2007) found 1 mg/L NAA and 0.5 mg/L NAA to be the best rooting hormones.

DPPH-free radical scavenging activity of Stevia leaves and callus is elucidated by Tadhani et

al. (2007). The aqueous leaf extracts of Stevia are investigated for DPPH activity (Shukla et

al. 2012) and this activity from different types of solution extracts is calculated from Stevia

leaves of Bangladesh (Jahan et al. 2010). Two Stevia rebaudiana varieties cultivated in

Mexico are analysed for DPPH-FRSA (Ruiz et al. 2015). All of these studies declare high

antioxidant activity of Stevia plant parts against the DPPH free radical assay.

62

This study contributes to the regeneration protocols of Stevia using various types and

concentrations of PGRs. Additionally, DPPH-free radical scavenging activity of different

cultures of Stevia including callus, shoots and rooted-shoots/regenerants has been performed.

The antioxidants found in medicinal plants play an important role in scavenging of free

radicals but the amount of this ability differs in different plant cultures. Hence, different rate

of antioxidant effect elucidated by different organs of Stevia has been noticed.

63

CHAPTER 5

Application of ZnO and CuO Nanoparticles to Callus Cultures of

Stevia rebaudiana Bertoni for Secondary Metabolites Production

Article Submitted:

Javed R, Yucesan B, Zia M, Gurel B. Elicitation of Antioxidant activities in Callus cultures

of Stevia rebaudiana Bertoni grown under ZnO and CuO nanoparticles stress. Sugar Tech

(Accepted).

5.1 Introduction

Nanoparticles within the size range of 100 nm have garnered great interest nowadays due to

their small size per volume ratio in contrast to their bulk states (Nagajyothi et al. 2014). The

properties of nanoparticles are strongly dependent on their method of preparation,

temperature and surfactants used for capping. Among various methods adopted for the

synthesis of nanoparticles, co-precipitation is the most promising due to its simple operation

and cost-effectivity (Shahmiri et al. 2013). The biological properties depend on the

biocompatibility of nanoparticles with the biological agent (Sharma et al. 2010). Although all

nanoparticles have been used in biotechnology, metal oxide nanoparticles are considered the

safest because of their stability and other salient features (Pandurangan and Kim 2015). ZnO

is an inorganic, wide band gap semiconductor and has various applications owing to its large

surface area and high catalytic activity (Chen and Tang 2007). ZnO nanoparticles are

environment-friendly, hence widely used in biological applications (Singh et al. 2009). CuO

is a narrow band gap, p-type semiconductor having diverse applications and industrial uses

(Phiwdang et al. 2013).

Stevia rebaudiana, called “candy leaf” is a perennial herb sweet in taste. It belongs to the

family Asteraceae and is indigenous to Brazil and Paraguay (Shivanna et al. 2012). It

produces zero-calorie diterpene glycosides; rebaudiosides and steviosides (Thiyagarajan and

Venkatachalam 2012). Stevia naturally protects from diabetes mellitus, hypertension and

obesity. It is propagated by tissue culture to produce elite plants (Yucesan et al. 2016). The

poor seed germination and low efficiency of stem cuttings actually paved the way for large-

scale in vitro propagation of Stevia (Rafiq et al. 2007). Different methods have been

developed to obtain higher amount of secondary metabolites from the Stevia leaf tissue.

Abiotic and biotic elicitors have potential of producing large quantities of sweetening

compounds and secondary metabolites by altering the metabolic cycle (Gupta et al. 2015;

64

Bayraktar et al.). However, the elicitors are useful up to a certain threshold level since they

become toxic at higher concentrations (Javed et al. 2016). The toxic effects of metallic oxide

nanoparticles as abiotic elicitors have been reported in many plants (Lee et al. 2008, 2010;

Lin and Xing 2008; Dimpka et al. 2012; Shaw and Hossain 2013; Javed et al. 2016). Both

ZnO and CuO nanoparticles have been found to create negative effects on the survival and

growth of organisms as well as the environment (Chang et al. 2012).

The objective of this study is to enhance the production of secondary metabolites of S.

rebaudiana by inoculating different concentrations of ZnO and CuO nanoparticles in

Murashige and Skoog (MS) medium during callogenesis. Previous studies have been

conducted on S. rebaudiana callus regarding different abiotic elicitors responsible for

enhancement of secondary metabolites (Hendawey and Abo El Fadl 2014; Gupta et al. 2015;

Khalil et al. 2015).


5.2.1 Callusing under ZnO and CuO nanoparticles stress

The seeds of Stevia rebaudiana are provided from Polisan Tarim, Istanbul, Turkey. For

germination, the seeds are surface-disinfected with 0.1% (w/v) mercuric (II) chloride (HgCl2)

and cultured on MS medium (Murashige and Skoog 1962) containing 3% (w/v) sucrose and

0.8% (w/v) agar. Later on, leaf explants (0.5 cm2) are excised from 30-days-old in vitro-

germinated seedlings, and cultured on MS medium supplemented with a combination of plant

growth regulators (PGRs); 0.5 mg/L kinetin (KIN) and 0.5 mg/L 2,4-dichlorophenoxyacetic

acid (2,4-D), and different concentrations of ZnO (34 nm in size) and CuO (47 nm in size)

nanoparticles (synthesized by co-precipitation method); 0.1-100 mg/L,. Hence, a total of nine

treatments are prepared, i.e., 8 having ZnO or CuO nanoparticles in different concentrations,

and one treatment devoid of ZnO or CuO nanoparticles as a control. The cultures are

maintained at 25±1°C under 16/8 (light/dark) photoperiod (50 μmol m−2

s−1

irradiance by

fluorescent lamps). The calli are harvested after 42 days of culture period and oven dried to

perform antioxidant assays.

5.2.2 Extract preparation for antioxidant assays

Stevia callus extracts are prepared by drying and then taking 0.1 g of fine powder of callus

produced under different ZnO and CuO concentrations. Powder is suspended in 500 μl

methanol and vortexed for 5 min. Then, sonication is done for 30 min followed by 15 min

centrifugation at 10,000 rpm. The supernatant is collected at the end to perform all anti-

oxidant activities. The antioxidant activities comprised of total phenolic content (TPC), total

65

flavonoid content (TFC), total antioxidant capacity (TAC), total reducing power (TRP) and

DPPH-free radical scavenging activity are performed according to the protocols described in

Chapter 2.

5.2.3 Statistical analysis

All experiments are conducted in completely randomized design in triplicates, and repeated

thrice. The data were statistically analysed using SPSS, Version 17.0 (SPSS Inc., Chicago,

IL, USA). The relationship between different parameters is assessed using Pearson’s

correlation coefficient (r). One-way ANOVA is used to check the significant mean difference

with Tukey’s HSD for post hoc analysis. A P< 0.05 is used to define significance.

5.3 Results

Callus induction response from leaf segments of Stevia rebaudiana and quantity of secondary

metabolites produced with or without exposure to ZnO and CuO nanoparticles stress is

investigated.

5.3.1 Physiology of Callus grown under ZnO and CuO nanoparticles stress

Callusing is performed using the method of Gupta et al. (2010) using leaf explants, and 0.5

mg/L KIN and 0.5 mg/L 2,4-D as plant growth regulators along with different concentrations

of ZnO and CuO nanoparticles. The results of treatments have been shown in table 5.1 and

5.2, and figures 5.1 and 5.2.

The best callus is obtained in 1 mg/L and 10 mg/L of ZnO nanoparticles treatment as its

amount is 2.5 cm like control, and is yellow and friable too. The fresh weight (FW) of callus

is highest (0.87 g) in the case of 0.01 mg/L of ZnO. There is no significant difference in the

dry weight (DW) of callus obtained at different treatments. The lowest amount (0.63 cm, 0.22

g FW and DW, respectively) and quality of callus (compact and very poor) is obtained in

case of 500 mg/L of ZnO nanoparticles treatment.

The best results (yellow and friable callus) in case of CuO nanoparticles treatment is obtained

when there is 1 mg/L of nanoparticles exposure rather than control. The amount (2 cm), and

FW (0.51 g) and DW (0.05 g) of callus is good in this treatment. The results are poor when

500 mg/L of CuO is added to MS basal medium, i.e., amount of callus is 0.33 cm, FW is 0.11

g, DW is 0.02 g and the callus texture is compact.

66



Conc. Of ZnO

NPs (mg/L)

FW (g) DW (g) Color Texture

Control 0.78±0.05b 0.06±0.01

b Yellow Friable

0.01 0.87±0.02a 0.08±0.01

a Yellow Compact

0.1 0.61±0.07e 0.06±0.03

b Yellow Compact

1 0.64±0.05d 0.06±0.05

b Yellow Friable

10 0.71±0.06c 0.06±0.03

b Yellow Friable

50 0.45±0.03f 0.04±0.06

d Yellow Compact

100 0.45±0.02f 0.05±0.04

c Yellow Compact

500 0.22±0.02g 0.02±0.02

e Yellow Compact

1000 0 0 - -




explants on MS medium supplemented with different concentrations of CuO nanoparticles.

Conc.Of CuO

NPs (mg/L)

FW (g) DW (g) Color Texture

Control 0.78±0.05a 0.06±0.02

a Yellow Friable

0.01 0.69±0.01b 0.06±0.01

a Green Compact

0.1 0.57±0.02b 0.05±0.05

b Green Compact

1 0.51±0.02c 0.05±0.03

b Yellow Friable

10 0.57±0.04b 0.05±0.03

b Green Compact

50 0.46±0.06d 0.04±0.01

c Yellow Compact

100 0.27±0.03e 0.03±0.01

d Yellow Compact

500 0.11±0.01f 0.02±0.02

e Yellow Compact

1000 0 0 - -



67

Figure 5.1: Callogenesis of Stevia grown in MS medium containing (a) 0 mg/L, (b) 0.01

mg/L, (c) 0.1 mg/L, (d) 1 mg/L, (e) 10 mg/L, (f) 50 mg/L, (g) 100 mg/L, (h) 500 mg/L and (i)

1000 mg/L concentration of ZnO nanoparticles.

Figure 5.2: Callogenesis of Stevia grown in MS medium containing (a) 0 mg/L, (b) 0.01

mg/L, (c) 0.1 mg/L, (d) 1 mg/L, (e) 10 mg/L, (f) 50 mg/L, (g) 100 mg/L, (h) 500 mg/L and (i)

1000 mg/L concentration of CuO nanoparticles.

68

5.3.2 Phytochemical Analysis of Callus under ZnO nanoparticles stress

According to Figure 5.3, the control treatment shows lowest amount of TPC (3.45 μg/mg of

DW), and the highest amount of TPC (5.65 μg/mg of DW) is obtained with the treatment

having 100 mg/L concentration of ZnO nanoparticles. Moreover, the TPC drops to 3.95

μg/mg of DW when MS medium is supplemented with 500 mg/L of ZnO nanoparticles.

Similarly, the highest amount of TFC (2.85 μg/mg of DW) is observed in the callus tissue

grown in the presence of 100 mg/L ZnO nanoparticles, while the lowest TFC (1.32 μg/mg of

DW) is obtained in 500 mg/L of treatment.

Regarding TAC and TRP, treatments containing 100 mg/L and 50 mg/L of ZnO nanoparticles

revealed highest amounts (9.69 μg/mg of DW and 10.83 μg/mg of DW respectively).

Whereas, the lowest amounts (5.44 μg/mg of DW and 8.75 μg/mg of DW) of TAC and TRP

are shown by the 500 mg/L of ZnO treatment. Both TAC and TRP increase with increasing

ZnO nanoparticles concentration until it reaches 500 mg/L of nanoparticles treatment.

According to Figure 5.4, the detoxification of DPPH free radical is found highest (85.91%) at

100 mg/L ZnO nanoparticles treatment, and lowest (58.43%) in treatment containing 500

mg/L ZnO nanoparticles. Hence, an ascending order of free-radical scavenging activity is

obtained from control to 50 mg/L ZnO nanoparticles, and suddenly drops in 500 mg/L ZnO

nanoparticles treatment.

Figure 5.3: Comparison of antioxidant activities at different concentrations of ZnO

nanoparticles. Total flavonoid content (TFC), total phenolic content (TPC), total antioxidant

capacity (TAC) and total reducing power (TRP) are shown by blue, red, green and purple

bars, respectively. Error bars are shown as standard deviation on each bar. Bars are

significantly different at confidence interval level of 95%.

a a a a a a a

b

0

c b

ab ab ab ab a

c

0

b b b b ab ab

a

c

0

ab ab ab ab ab a ab

b

0 0

2

4

6

8

10

12

Control 0.01 0.1 1 10 50 100 500 1000

An

tioxid

an

t act

ivit

ies

Concentration (mg/L)

TFC TPC TAC TRP

69

Figure 5.4: Comparison of % DPPH inhibition activity at different concentrations of ZnO

nanoparticles. Error bars are shown as standard deviation on each bar. Bars are significantly


5.3.3 Phytochemical Analysis of Callus under CuO nanoparticles stress

According to Figure 5.5, the highest TPC (5.88 μg/mg of DW) is found in calli produced on

MS medium supplemented with 10 mg/L of CuO nanoparticles while the lowest TPC (2.04

μg/mg of DW) is revealed by 500 mg/L CuO nanoparticles concentration. The TPC increases

with increasing nanoparticles concentration up to 10 mg/L, but after that, it starts to decline.

Whereas, the TPC is obtained at 5.26 μg/mg of DW and 4.88 μg/mg DW under 50 mg/L and

100 mg/L of CuO nanoparticles, respectively. The TFC is highest (2.23 μg/mg of DW) when

exposed to 100 mg/L of CuO nanoparticles and lowest (0.75 μg/mg of DW) in case of 500

mg/L of nanoparticles stress.

The highest TAC (8.84 μg/mg DW) and TRP (10.99 μg/mg DW) are revealed by the 10 mg/L

of CuO nanoparticles stress. Whereas, the lowest TAC (3.58 μg/mg of DW) and TRP (6.96

μg/mg of DW) are evaluated by 500 mg/L nanoparticles exposure. The DPPH inhibition is

highest (80.57%) and lowest (50.55%) by 10 mg/L and 500 mg/L CuO nanoparticles

exposure, respectively.

These findings clearly indicate that the antioxidant potential of callus tissues is significantly

enhanced by means of ZnO or CuO nanoparticles stress until it reaches up to 50 mg/L or 100

mg/L, respectively because the activities are declined thereafter. It can be concluded from

b b b ab ab ab

a

c

0

20

40

60

80

100

Control 0.01 0.1 1 10 50 100 500 1000

DP

PH

sca

ven

gin

g a

ctiv

ity (

%)


70

these experiments that the secondary metabolism of Stevia callus cultures scavenge more free

radicals when the concentration of nanoparticles increase up to a certain limit.

Figure 5.5: Comparison of antioxidant activities at different concentrations of CuO

nanoparticles. Total flavonoid content (TFC), total phenolic content (TPC), total antioxidant

capacity (TAC) and total reducing power (TRP) are shown by blue, red, green and purple

bars, respectively. Error bars are shown as standard deviation on each bar. Bars are



CuO nanoparticles. Error bars are shown as standard deviation on each bar. Bars are


a a a a a a a

b 0

b ab ab a a ab b

c

0

ab ab ab a a a ab

c

0

ab a a a a ab ab

b

0 0

2

4

6

8

10

12

Control 0.01 0.1 1 10 50 100 500 1000

An

tioxid

an

t act

ivit

ies


TFC TPC TAC TRP

ab ab a a a

ab b

c

0 0

20

40

60

80

100

Control 0.01 0.1 1 10 50 100 500 1000

Inh

ibit

ion

(%

)


71

5.4 Discussion

The natural antioxidants occurring in medicinal plants play a vital role in protection against

different types of environmental stresses. These antioxidants function as oxidation chain

breakers, free radical scavengers, and quenchers of single oxygen. Presence of both

nanoparticles, ZnO and CuO, exert stress on the callus induced from leaf explants of Stevia.

Though, higher concentrations prove toxic in both cases but ZnO stimulate callus induction

response and callus physiology at lower concentrations. However, CuO shows minor toxicity

at lower concentration. The results also show that in the presence of CuO nanoparticles,

callus texture is good and at some concentrations, green calli is produced. While in the

presence of ZnO, mostly yellow colored callus induction occurs from leaf explants.

In the present study, an oxidative stress induced by the ZnO and CuO nanoparticles create

toxic free radicals in S. rebaudiana. This stress increases by increasing the concentration of

ZnO or CuO nanoparticles due to the formation of reactive oxygen species, shown by the

enhanced antioxidant activities of the callus tissue as a response. However, after reaching a

certain threshold, different physiological parameters of callus and production of antioxidants

began to decline (Javed et al. 2016).

The role of ZnO and CuO nanoparticles as abiotic elicitors of callus physiology and

secondary metabolites production from the callus of Stevia rebaudiana is a new frontier in

abiotic stress elicitation. These nanoparticles impose oxidative stress to the callus tissues

grown by tissue culture, which trigger their metabolism and result in enhancement of all

antioxidant activities (Choi and Hu 2008). However, after reaching a certain threshold, the

growth of callus impairs and antioxidant activities are mitigated because the oxidative

stressers over-shadow the natural antioxidant ability and scavenging of free radicals (Dimpka

et al. 2014).

The callus of Stevia rebaudiana has certain valuable active metabolic constituents that are

involved in free radical scavenging. These chemicals are responsible for neutralization of

toxic free radicals, hence preventing excessive oxidation reactions. The current study reveals

influence of ZnO and CuO nanoparticles on the free radical scavenging activity and other

antioxidant activities of S. rebaudiana callus. This study suggests that the callus of S.

rebaudiana, provided either with ZnO and CuO nanoparticles, can be used as a source of

antioxidants. Consequently, a positive effect of these nanoparticles in the context of Stevia

physiology and antioxidant activities is observed. This study paves the way for conducting

72

further such cost-efficient and easy experiments using different nanoparticles and medicinal

plants in order to obtain elevated levels of antioxidants to be used in cure of various diseases.

73

CHAPTER 6

Application of ZnO Nanoparticles to Micropropagated Shoots of

Stevia rebaudiana Bertoni for Steviol Glycosides Production

Article Published:

Javed R, Usman M, Yucesan B, Zia M, Gurel E. (2017) Effect of Zinc Oxide (ZnO)

Nanoparticles on Physiology and Steviol Glycosides Production in Micropropagated Shoots

of Stevia rebaudiana Bertoni. Plant Physiology and Biochemistry. 110: 94-99.

http://dx.doi.org/10.1016/j.plaphy.2016.05.032

6.1 Introduction

Nanoparticles ranging in size from 1-100 nm possess specific physico-chemical properties

attributed to smaller size, large surface area and high reactivity compared to their bulk

counterparts (Yadav et al. 2013). The route of nanoparticles synthesis and their relative size

and structure plays pivotal role in exhibiting the biological properties of nanoparticles

(Kołodziejczak-Radzimska and Jesionowski 2014). The interaction of nanoparticles with the

biological system is of enormous importance, and nowadays researchers are trying to figure

out the potential effects of various kinds of nanoparticles in plants, animals and humans

(Boczkowski and Hoet 2010). Nanoparticles have numerous applications in agriculture

including synthesis of nano-pesticide or nano-fertilizer formulations, and their use as sensors

of soil conditions and for targeted delivery of genes in transformation (Aslani et al. 2014).

Stevia rebaudiana, belonging to the family Asteraceae, is a perennial herb native to Brazil

and Paraguay (Soejarto 2002). It is well-known for the production of steviol glycosides

specifically rebaudioside A, stevioside and rebaudioside C in its leaves that play a crucial role

in conferring anti-diabetic, anti-cancerous and anti-bacterial properties (Dey et al. 2013).

Stevia has been in-vitro propagated for many years, utilizing different protocols, in order to

obtain the enhanced quantity of pharmacologically valuable steviol glycoside products (Rafiq

et al. 2007). In recent years, the studies encompassing field of nanotechnology for

determination of effects of environmental stress on plant physiology have been progressing

rapidly (Bhattacharyya et al. 2015). Metallic oxide nanoparticles, specifically nano-scale zinc

oxide (ZnO), have gained paramount importance in this regard. However, the influence of

ZnO nanoparticles has been illustrated in only one medicinal plant, Fagopyrum esculentum

uptil now (Sooyeon et al. 2013).

http://dx.doi.org/10.1016/j.plaphy.2016.05.032

74

ZnO nanoparticles have largely been declared phytotoxic and their phytotoxity has been

manifested by the generation of reactive oxygen species (ROS), and formation of necrotic

lesions as well as yellow pigmentations on the leaves of different crop plants including

Lolium perenne, Glycine max, Cucumis sativus and Triticum aestivum (Lin and Xing 2008;

Lopez-Moreno et al. 2010; Kim et al. 2012). The effect of toxicity is dependent on the size of

nanoparticles, dissolution of metal ions, and their uptake and translocation in plant cells

(Franklin et al. 2007; Jiang et al. 2009). Recently, the influence of zinc (Zn) nanoparticles on

physiology and stevioside production of S. rebaudiana has been deciphered, and Zn

nanoparticles are to be found phytotoxic at a concentration of 400 mg/L and 1000 mg/L

(Desai et al. 2015). However, according to our knowledge, no study has been reported so far

on S. rebaudiana using the ZnO nanoparticles as abiotic stress elicitors. Consequently, the

aim of the present study is to observe the potential effects of synthesized and characterized

ZnO nanoparticles on the physiological characteristics of micro-propagated shoots, steviol

glycoside production, and anti-oxidant activity in leaves of S. rebaudiana.


6.2.1 Synthesis and Characterization of ZnO Nanoparticles

Synthesis of ZnO nanoparticles is performed by co-precipitation method (Mohan Kumar et

al. 2013) described in Chapter 2. ZnO nanoparticles are characterized using different

analytical techniques like X-ray diffraction (XRD), Fourier-transform infra-red (FTIR)

spectroscopy, UV-Visible spectrometry, Scanning electron microscopy (SEM) and Energy

dispersive X-ray (EDX) as described in Chapter 2.

6.2.2 Preparation of Medium containing ZnO Nanoparticles

The culture medium is prepared using Murashige and Skoog (MS) medium (Murashige and

Skoog 1962) and 3% (w/v) sucrose. A total of 6 media treatments were prepared having 0

mg/L, 0.1 mg/L, 1 mg/L, 10 mg/L, 100 mg/L and 1000 mg/L of ZnO nanoparticles. The pH

of media is adjusted to 5.7–5.8 and afterwards, 0.8% (w/v) plant agar is added for the

solidification of media. Later on, the culture media is autoclaved for 15 min at 121°C

temperature and 1.06 kg cm-2

pressure.

6.2.3 Growth Conditions of Plant Shoot Organogenesis

The seeds of S. rebaudiana were collected from Polisan Tarim, Istanbul, Turkey to perform

direct shoot organogenesis. After disinfection with 0.1% (w/v) mercuric (II) chloride (HgCl2),

seeds are cultured on plain MS medium. Finally, the axillary shoot nodes excised from 4

75

weeks-old seedlings are incubated in different media treatments, i.e., provided with various

concentrations of ZnO nanoparticles. The experimental set-up took place in growth room

having 16 h light/ 8 h dark photoperiod, provided by cool-white fluorescent light of 35 μmol

m-1

s-1

irradiance and 24 ± 1°C temperature at 55–60% relative humidity. The experiment is

conducted in triplicate. Total 15 nodal explants are used per treatment. After 4 weeks of

cultivation, various physiological parameters are noticed including mean percentage of

shooting, mean length of shoots, mean number of leaves, and fresh weight of the shoots

produced in vitro.

6.2.4 Extraction of Steviol Glycosides

Steviol glycoside analysis is performed using leaves from in vitro regenerated shoots grown

under ZnO nanoparticle stress. Shoots from all the treatments are washed with sterile distilled

water. After careful washing and soaking the plant material on filter paper, it is kept in oven

for drying at 60°C for 48 h. In order to carry out high performance liquid chromatography

(HPLC) analysis of samples, 20 mg sample from each treatment is added to 1 mL of 70%

(v/v) methanol in a micro-centrifuge tube. All samples are incubated in an ultrasonic bath at

55°C for 15 min and then centrifuged at 25°C and 12,000 rpm for 10 min. The supernatant

obtained is transferred to HPLC tubes after being filtered using 0.22 µm PTFE Millipore

syringe filters, and later on used for HPLC analysis. All samples are run in triplicates.

6.2.5 Analysis of Steviol Glycosides

Chromatography is performed with an autosampler (WPS-3000-SL Dionex Semi Prep

Autosampler) injecting 10 µL of each sample, a binary pump (LPG 3400SD Dionex) solvent

delivery system working at a flow rate of 0.8 mL min-1

, and a dual wavelength absorbance

detector operating at 210 and 350 nm (MWD-3100 Dionex UV-VIS Detector). The column,

Inertsil® ODS-3 (GL Sciences Inc., Japan) with 150 × 4.6 mm in length and 5 μm particle

size, is kept warm at 40°C in a column oven system (TCC-3000SD Dionex). Isocratic flow is

performed using acetonitrile and 1% (w/v) phosphoric acid buffer mixture (68:32) for 20 min

at the end.

6.2.6 Preparation of Extract for Anti-Oxidant Assays

Stevia leaf extracts are prepared by drying and then taking 0.1 g of fine powder of leaves

produced under different ZnO concentrations. Powder is dissolved in 500 μl methanol and

vortexed for 5 min. Then, the sonication is done for 30 min followed by 15 min

76

centrifugation at 10,000 rpm. The supernatant is collected at the end to perform all anti-

oxidant activities.

6.2.7 Antioxidant Assays

Methodology described in Chapter 2 is followed for determination of total phenolic content,

total flavonoid content, total antioxidant capacity, total reducing power and DPPH-free

radical scavenging activity.


The design of experiments is completely randomized, and later on, statistical analysis of data

is done using SPSS, Version 17.0 (SPSS Inc., Chicago, IL, USA). ANOVA was used for

determination of statistical difference, and the significance of difference between means ± SE

(standard error) values is calculated using Duncan’s multiple range tests performed at p<0.05.

6.3 Results

ZnO nanoparticles have been successfully synthesized by co-precipitation method and later

on, their characterization is carried out by different analytical methods.

6.3.1 Characterization of ZnO Nanoparticles

XRD Results

Crystal structure of ZnO nanoparticles is examined by powder XRD analysis. The peaks in

XRD spectrum confirmed the hexagonal wurtzite structure [Figure 6.1(a)]. The obtained

diffraction pattern is compared with PCPDFWIN card no. 891397. The characteristic peaks

with high intensities corresponding to the planes (100), (002), (101), (102), (110), (103),

(200), (112), (201) and (202) indicate that the product is of high purity. No peaks from other

phase of ZnO and impurities are observed; suggesting that high purity single phase of ZnO is

obtained. The particle size is determined as 34 nm from the peak broadening of diffraction

peaks using Scherrer’s formula (Cullity 1978). The lattice parameters (a and c) of ZnO

hexagonal structure and the lattice plane spacing d are related to the Miller indices (hkl) by an

equation:

2

2

2

22

2 3

41

c

l

a

khkh

d hkl

77

Using above equation along with the Bragg’s law (2dhklsinθ=nλ), we calculate the values of

“a” and “c”. The lattice constants calculated from the 100 and 002 peaks are a = 3.246 Å,

c = 5.205 Å and the ratio c/a = 1.603.

FTIR Results

Figure 6.1(b) shows the FTIR spectra of ZnO nanoparticles. In this spectra, the absorption

peak at 574 cm-1

is due to the O-H bending of the hydroxyl group, and broad absorption band

in the range of 3400–3450 cm-1

is assigned to the O H stretching vibration. The absorption at

857 cm-1

is due to the formation of tetrahedral coordination of Zn (Sharma et al. 2014). The

peak at 1025 cm-1

is due to the C O stretching vibration. Peaks in range 2850–2930 cm-1

are

due to the C H stretching vibration. The peaks observed at 1576 and peak 1420 cm-1

are due

to the asymmetrical and symmetrical stretching of the zinc carboxylate, respectively (Chithra

et al. 2015).

Figure 6.1: (a) X-ray diffraction pattern, (b) FTIR spectrum of the ZnO nanoparticles.

78

UV-Visible Spectroscopy Results

Figure 6.2 (inset) illustrates typical reflectance spectra of all the ZnO samples with band edge

between 375 400 nm. The band gap can be determined using the optical diffuse reflectance

spectroscopy (DRS). The reflectance R can be used to calculate the Kubelka−Munk function

(Kortum 1969) F(R)=(1−R)2/2R, and the gap size is obtained by linear extrapolation of the

leading edge of F2(R) function to zero (Tahir et al. 2009). Calculation of band edge value

from K-Munk Function of ZnO nanoparticles is shown in Fig. 6.2. The value of band edge

found is 3.229 eV and it is in good agreement with the reported value (Tahir et al. 2009).

Figure 6.2: UV-Visible spectrum of ZnO nanoparticles.

SEM Results

Morphology of the sample is investigated using field emission scanning electron microscope

(FESEM). Specimens are prepared by sticking ZnO nanoparticles to the carbon tape, and

blow away the excess of powder with compressed air. This specimen is sputter coated with a

thin Au-Pd layer of about 3 nm thick in vacuum to avoid charging. Typical SEM micrograph

79

for prepared ZnO nanoparticles is shown in Figure 6.3. The SEM micrograph clearly shows

irregular shaped morphologies of ZnO nanoparticles. The SEM observation shows the

presence of agglomerated nanoparticles with an average size of 40–100 nm.

Figure 6.3: FESEM image of ZnO nanoparticles (a) High Resolution image, (b) Low

Resolution image.

EDX Results

The EDX spectrum of ZnO nanoparticles is given in Figure 6.4. The EDX results show that

there are no other elemental impurities present in the prepared ZnO nanoparticles.

Figure 6.4: Energy dispersive X-ray profile of ZnO nanoparticles.

80

6.3.2 Determination of Physiological Parameters of Stevia rebaudiana

The results of the current study (Figure 6.5) show pertinent role played by increasing

concentration of ZnO nanoparticles in the growth of S. rebaudiana up to a certain threshold

level, i.e., 1 mg/L, but once this level is achieved, further increase of ZnO nanoparticles cause

toxicity in S. rebaudiana. Table 6.1 clearly indicates that the highest amount of shooting

(~90%) occurred in MS medium supplemented with 1.0 mg/L of ZnO nanoparticles.

However, the least shooting frequency (~31%) is found in MS medium having 1000 mg/L of

ZnO nanoparticles. Maximum shoot length (4.6 cm) is obtained with MS medium augmented

with 1.0 mg/L of ZnO nanoparticles, followed by MS containing 0.1 and 10 mg/L of ZnO

nanoparticles (4.5 cm) and MS control medium (4.1 cm). Based on our data, highest number

of nodes (5.4) and leaves (14.2) are produced by 1.0 mg/L ZnO nanoparticles treatment,

whereas the lowest number of nodes (2.0) and leaves (5.45) are formed in treatments

provided with 1000 mg/L of ZnO nanoparticles. Fresh weight of shoots is also measured to

be highest in MS medium fortified with 1.0 mg/L of ZnO nanoparticles (0.59g) and lowest

fresh weight (0.07g) is observed in 1000 mg/L of ZnO nanoparticles treatment.


nodal explants on MS medium supplemented with different concentrations of ZnO

nanoparticles.



Concentration

(mg/L)

Response

(%)

Mean shoot

length

(cm)

Mean no.

of nodes

per

explant

Mean no. of

leaves per

regenerated

shoot

FW of

shoots (g)

Control 85.7 4.1±0.2b

4.2±0.1c

10.0±0.1d

0.16±0.07e

0.1 85.4 4.5±0.1a

4.4±0.0b

13.6±0.1b

0.44±0.06b

1.0 89.6 4.6±0.1a

5.4±0.0a

14.2±0.2a

0.59±0.07a

10 85.5 4.5±0.1a

3.6±0.1d

13.8±0.2b

0.36±0.08c

100 50.5 3.6±0.1c

3.3±0.1e

12.3±0.1c

0.27±0.01d

1000 30.8 1.5±0.1d

2.0±0.0f

5.4±0.1d

0.07±0.04f

81

Figure 6.5: Effect of various concentrations of ZnO nanoparticles on the shoot formation in

Stevia rebaudiana. Length of shoots, number of nodes and number of leaves formed in

control medium (A), at a concentration of 0.1 mg/L (B), 1.0 mg/L (C), 10 mg/L (D), 100

mg/L (E), and 1000 mg/L (F).

6.3.3 Determination of Steviol Glycosides in Stevia rebaudiana

Figure 6.6 shows that S. rebaudiana produces more steviol glycosides (rebaudioside A and

stevioside) in the presence of up to 1 mg/L ZnO nanoparticles, thereafter, it results in causing

phytotoxicity. The amount of rebaudioside A enhances from 2.07% in control to 2.80% in

MS treatment supplemented with 0.1 mg/L ZnO nanoparticles. Furthermore, rebaudioside A

quantity obtained from shoots grown in MS having 1.0 and 10 mg/L ZnO nanoparticles is

found 3.65% and 3.11%, respectively. On increasing the ZnO nanoparticles concentration to

100 mg/L, a drastic decrease in rebaudioside A amount (1.02%) is observed. The decline in

rebaudioside A quantity occured more (0.14%) when shoots are allowed to grow in MS

medium containing 1000 mg/L ZnO nanoparticles. As anticipated from the data of

rebaudioside A content, HPLC spectra for quantity of stevioside reveals the similar pattern as

shown in Figure 6.6 having increased up to 1 mg/L ZnO nanoparticles followed by a sudden

decline, ultimately leading to zero stevioside amount in shoots obtained from MS medium

augmented with 1000 mg/L ZnO nanoparticles. The amount of stevioside is 0.73% obtained

in control group that is shown to rise to 1.12% and 1.17% in treatments having 0.1 and 1.0

mg/L ZnO nanoparticles, respectively. A sudden decline (0.99%) occurs by the effect of an

increase of ZnO nanoparticles concentration to 10 mg/L, lowering it to 0.35% in MS medium

supplied with 100 mg/L of ZnO nanoparticles. Moreover, stevioside is not detected at 1000

mg/L ZnO nanoparticles concentration.

82

Figure 6.6: Effect of ZnO nanoparticles at different concentrations ranging between 0 to

1000 mg/L on rebaudioside-A represented with blue bars and stevioside content with red

bars. Error bars have been shown as standard deviation on each bar. Bars are significantly


6.3.4 Determination of Anti-oxidant Activities of Stevia rebaudiana

It can be concluded from the Table 6.2 that a premier amount of total phenolic and flavonoid

content, total anti-oxidant capacity, and total reducing power is detected in stevia leaves that

are obtained from MS medium containing 1 mg/L ZnO nanoparticles, followed by the leaf

extracts formed from media plates having 10 mg/L nanoparticles. However, DPPH free

radical scavenging activity of Stevia shoot extracts is highest (74.8%) in shoots grown under

10 mg/L of ZnO nanoparticles stress, followed by 68.6% of activity obtained under 1 mg/L.

The lowest anti-oxidant activities are obtained from extracts containing 1000 mg/L of ZnO

nanoparticles employed in the MS medium.

d

c

a

b

e

f

c

a a b

d

0

1

2

3

4

0 0.1 1 10 100 1000

% o

f st

evio

l gly

cosi

des

(w

/w)

Conc. of ZnO (mg/L)

Amount of Reb A

Amount of Stevioside

83




different according to Duncan’s multiple range test at P<0.05. TFC: total flavonoid content,

TPC: total phenolic content, TAC: total antioxidant capacity, TRP: total reducing power, QE:

quercitine equivalent, GAE: gallic acid equivalent, AAE: ascorbic acid equivalent, DPPH:

2,2-diphenyl-1-picryl hydrazyl.

6.4 Discussion

Direct organogenesis method is used for tissue culture in order to grow Stevia shoots.

Previously, the soil and hydroponic system have remained a source of plant growth for

measurement of different growth parameters influenced by ZnO nanoparticles (Dimkpa et al.

2013; Watson et al. 2015). Moreover, the literature cited studies ZnO nanoparticles for their

phytotoxicity (mostly seed germination) only on crop plants (Boonyanitipong et al. 2011;

Prasad et al. 2012; Kouhi et al. 2014).

In the current study, ZnO nanoparticles applied to the MS shooting medium impose an

abiotic stress to the S. rebaudiana explants during their regeneration in to shoots. When the

concentration of ZnO nanoparticles is increased, it positively effects the plant growth as well

as production of steviol glycosides. Up to a certain concentration level, i.e., 1 mg/L, the

enhancement of steviol glycosides and increased shooting response of Stevia occur because

the ZnO nanoparticles behaves as an abiotic stress elicitor. This is clearly pointed out by the

powerful DPPH scavenging activity and other anti-oxidant activities of Stevia extracts. But

once a threshold level is achieved and still plant cells are prone to continued addition of ZnO

Concentration

(mg/L)

TFC

(µg QE/mg)

TPC

(µg GAE/mg)

TAC

(µg

AAE/mg)

TRP

(µg

AAE/mg)

% DPPH

Inhibition

Control 3.81±0.07d

4.52±0.03b

9.84±0.01c

8.28±0.02c

60.6

0.1 5.42±0.02b

4.54±0.02b

10.8±0.01b

8.63±0.02b

66.7

1.0 6.22±0.03a

4.67±0.03a

11.7±0.02a

10.3±0.01a

68.6

10 3.94±0.01c

4.24±0.05c

10.9±0.03b

10.3±0.01a

74.8

100 2.56±0.01e

3.01±0.01d

7.36±0.01d

9.99±0.04a

64.2

1000 1.36±0.01f

2.33±0.04e

4.62±0.05e

6.33±0.03d

59.5

84

nanoparticles, imbalance of plant growth regulators and the release and accumulation of free

radicals or reactive oxygen species (ROS) take place to a larger extent resulting in

degradation of few cells (Choi and Hu 2008). ZnO nanoparticles implicate an oxidative stress

by the release of metal ions or free radicals into MS culture medium (Gajewaska and

Sklodowska 2007; Dimkpa et al. 2012). It is the function of enzymatic and non-enzymatic

anti-oxidants naturally present in plants that help plant cells to cope with an oxidative stress

(Klaper et al. 2009), but at 1000 mg/L imbalance of anti-oxidative activities and oxidative

pressure occurs. Hence, the growth is impaired, steviol glycosides quantity reduced, and anti-

oxidant activities are mitigated as a consequence of oxidative damage caused by ZnO

nanoparticles at 1000 mg/L concentration. The ZnO nanoparticles phytotoxicity at 1000 mg/L

has also been manifested from the previously reported data (Wang et al. 2012; Watson et al.

2015; Zhao et al. 2013). Contrarily, it disagrees with Prasad et al. (2012) who reports positive

effects of >1000 mg/L of ZnO nanoparticles on seed germination of peanuts.

Steviol glycosides (SGs) production has been reported earlier by various researchers using

different abiotic stress elicitors like chlorocholine chloride (CCC), paclobutrazol (PBZ),

proline, polyethylene glycol (PEG), hydrogen peroxide (H2O2), 6-benzylaminopurine (BAP),

calcium chloride (CaCl2) and salicylic acid (Karimi et al. 2014; Gupta et al. 2015; Soufi et al.

2016). The current study is a new frontier of abiotic elicitors for the formation of SGs in the

leaves of Stevia rebaudiana.

In conclusion, this study demonstrates novel findings depicting the positive effect of ZnO

nanoparticles for in-vitro growth and steviol glycoside production in S. rebaudiana. In this

regard, a significantly enhanced quantity of steviol glycosides is obtained using up to 1 mg/L

concentration of ZnO nanoparticles in MS basal medium. Phytotoxic effects of ZnO

nanoparticles appear till threshold is achieved, and highest level of phtotoxicity is exhibited

by ZnO nanoparticles at 1000 mg/L concentration. This research opens up new avenues for

studying the fate of nanoparticles in medicinal plants in the context of their uptake,

translocation and alteration of metabolic pathways in a concentration-dependent manner.

85

CHAPTER 7

Application of CuO Nanoparticles to Micropropagated Shoots of

Stevia rebaudiana Bertoni for Steviol Glycosides Production

Article Submitted:

Javed R, Mohamed A, Yucesan B, Gurel E, Kausar R, Zia M. CuO Nanoparticles

Significantly Influence In-vitro Culture, Steviol Glycosides, and Antioxidant Activities of

Stevia rebaudiana (Bert.) Plant Cell Tissue Organ Culture (Under-Review).

7.1 Introduction

Nanoparticles having <100 nm size possess large surface area as compared to their bulk

counterparts. Such specific physical and chemical nature of nanoparticles result in their

higher reactivity (Yadav 2013). Apart from physio-chemical properties, nanoparticles also

play a key role in exhibiting specific biological properties to the living system

(Kołodziejczak-Radzimska and Jesionowski 2014). Nanoparticles have shown interaction

with the biological system involving different plants, animals and humans (Boczkowski and

Hoet 2010). Numerous applications of nanoparticles in agriculture have been elucidated

including biocide in plants, nano-fertilizer and nano-pesticide formulations, soil condition

sensors, and targeted gene delivery in transformation (Aslani et al. 2014; Perreault et al.

2014).

Stevia rebaudiana belongs to the family Asteraceae and is a perennial herb native to South

America (Soejarto 2002). Its metabolic pathways produce secondary metabolites in its leaves,

and are well-known for steviol glycosides production mainly rebaudioside A, stevioside and

rebaudioside C. The steviol glycosides play a pivotal role in conferring anti-diabetic, anti-

bacterial and anti-cancerous properties to Stevia (Dey et al. 2013). Different kinds of abiotic

and biotic stresses have been employed to S. rebaudiana during its in-vitro propagation in

order to enhance the steviol glycoside products of pharmacological importance (Rafiq et al.

2007). Oxidative nanoparticle stress elicitors such as metallic zinc oxide (ZnO) and copper

oxide (CuO) have gained enormous importance in recent years (Javed et al. 2016). The

reactivity and toxicity of metallic oxide nanoparticles depends on their size, surface,

structure, concentration, dissolution and exposure routes (Franklin et al. 2007; Jiang et al.

2009; Chang et al. 2012). CuO nanoparticles belong to the group of nanoparticles that are

used for both household and industrial purposes, so the toxicity of these nanoparticles should

86

be prevented (Chang et al. 2012). However, the CuO toxicity has largely been demonstrated

in aquatic organisms such as algae and zebrafish (Aruoja et al. 2009). CuO generated

intracellular oxidative stress involves the release of Cu ions (Cu+2

) causing toxicity after

exceeding the maximum physiological tolerance range of organisms, hence disturb the

balance between oxidation and anti-oxidation processes. CuO has largely been found

phytotoxic because of generation of reactive oxygen species (ROS) and necrotic lesions,

ultimately leading to cell death (Chang et al. 2012).

The effect of CuO nanoparticles on the growth, photosynthesis and oxidative response has

recently been studied in the crop plant, Oryza sativa (Da Costa and Sharma 2016). Zea mays

and Triticum aestivum have also been studied regarding the phytotoxic effect of CuO

nanoparticles. Furthermore, Lemna minor (Duckweed), an aquatic species was exposed to

CuO nanoparticles for the study of different enzymatic anti-oxidant activities and their level

of toxicity (Song et al. 2016). The CuO nanoparticles inhibitory effect has been noticed in

duckweed (Landoltia punctata) by comparing the influence of soluble Cu in bulk solution

and CuO nanoparticles (Shi et al. 2011). Another study was conducted examining the growth

and photosynthetic effects of CuO nanoparticles on an aquatic macrophyte, Lemna gibba L

(Perreault et al. 2014). Moreover, CuO nanoparticles effect on aquatic macrophyte, Elodea

nuttallii has been studied in the context of photosynthesis and oxidative stress (Regier et al.

2015). The negative influence of CuO nanoparticles on activated sludge in wastewater

treatment plants has also been determined (Hou et al. 2015).

Recently, the influence of copper oxide (CuO) nanoparticles on stevioside production and

enzymatic antioxidant activities of S. rebaudiana callus has been investigated (Hendawey et

al. 2015). Consequently, the aim of the present study is to observe the potential effects of

synthesized and characterized engineered CuO nanoparticles on the physiological

characteristics of micro-propagated shoots, steviol glycosides production, and non-enzymatic

anti-oxidant activities in leaves of S. rebaudiana.


7.2.1 Synthesis and Characterization of CuO Nanoparticles

CuO nanoparticles are synthesized by co-precipitation method of Ahamed et al. (2014) with

slight modifications as described in Chapter 3. Different analytical methods including X-ray

diffraction (XRD), Fourier-transform infra-red (FTIR) spectra, Scanning electron microscopy

87

(SEM) and Energy dispersive X-ray (EDX) spectra are performed for characterization of

CuO nanoparticles as described in Chapter 3.

7.2.2 Preparation of Medium having CuO Nanoparticles

MS medium comprising of total 6 treatments are prepared containing 0 mg/L, 0.1 mg/L, 1

mg/L, 10 mg/L, 100 mg/L and 1000 mg/L of CuO nanoparticles. The procedure described in

Chapter 6 is followed.

7.2.3 Growth Conditions of Shoot Organogenesis

Growth conditions of shoot organogenesis under CuO nanoparticles stress are optimized

according to the methodology described in Chapter 6.

7.2.4 Extraction and Analysis of Steviol Glycosides

Steviol glycosides are extracted from the leaves of in vitro regenerated shoots grown under

CuO nanoparticles stress according to procedure described in Chapter 6.

7.2.5 Preparation of Extract and Anti-Oxidant Assays

Methodology described in Chapter 6 is followed for the preparation of extracts and Chapter 2

is followed for performing antioxidant assays involving determination of total phenolic

content, total flavonoid content, total antioxidant capacity, total reducing power and DPPH-

free radical scavenging activity.


The design of experiments is completely randomized, and later on, statistical analysis of data

is done using SPSS, Version 17.0 (SPSS Inc., Chicago, IL, USA). ANOVA is used for

determination of statistical difference, and the significance of difference between means ± SE

(standard error) values is calculated using Duncan’s multiple range tests performed at p<

0.05.

7.3 Results

The chemically synthesized CuO nanoparticles are subjected to different characterization

methods.

88

7.3.1 Characterization of CuO Nanoparticles

XRD Results

The powder pattern is recorded with the use of Empyrean PANalytical X-ray diffractometer

with Bragg-Brentano geometry using Cu Kα radiation (λ = 1.54 Å). The step-scan covers the

angular range 20-80º with the step of 0.02º. Figure 6.1(a) shows the XRD pattern of CuO

nanoparticle. The diffraction data reveals that the material is composed of crystalline

monoclinic cubic cuprous oxides [Figure 7.1(a)]. The peak positions are in good agreement

with the PCPDFWIN data card 895899. The crystallite size determination is carried out using

the Scherrer’s equation (Cullity 1978).

B

kD

cos

Where, D is the crystallite size, k is a constant (~0.94 assuming that the particles are

spherical), λ is the wavelength of X-ray radiation, β is the line width at half maximum

intensity of peak and θB is the angle of diffraction. The particles size obtained from the XRD

data for CuO is 47 nm. The lattice parameter of CuO monoclinic structure and the plane

spacing d is related to the lattice constant and the Miller indices (hkl) (Cullity 1978).

ac

hl

c

l

b

k

a

h

d hkl

cos2sin

sin

112

2

2

22

2

2

22

Using above equation along with the Bragg’s law (2dhklsinθ=nλ), the values of lattice

parameters are calculated. Monoclinic CuO crystal have the lattice constants a = 4.69 Å, b =

3.418 Å and c = 5.122 Å and angle β = 99.57º.

FTIR Results

Figure 7.1(b) shows the FTIR spectrum of CuO nanoparticles. Various well-defined peaks are

observed at 535, 594, 869, 1052, 1411, 2094, 2847, 2920 and 3419 cm-1

. The appearance of

the peaks at 535, 594 corresponds to the characteristic stretching vibrations of Cu-O bonds in

the monoclinic crystal structure of CuO (Zheng and Liu 2007; Ethiraj and Kang 2012) and

869 cm-1

corresponds well to Cu-O-H vibration (Yu et al. 2012; Park and Kim 2004). The

bond at 1052 cm-1

is due to the C O stretching vibration (Zhao et al. 2012). Origin of two

well-defined absorption bands at 1411 cm-1

is due to the CH3 group and CH3 asymmetrical

stretching mode present on the surface of CuO nanostructures. The bands at 2847 and 2920

cm-1

are assigned to –CH2 and C-H stretching mode. The absorption peak around 2094 cm−1

89

is due to the existence of CO2 molecules in air, and the peak around 3419 cm-1

is assigned to

the O H stretching vibration.

Figure 7.1: (a) X-ray diffraction pattern, (b) FTIR spectrum of the CuO nanoparticles.

SEM Results

Morphology of the sample is investigated using field emission scanning electron microscope

(FESEM). Specimens are prepared by sticking CuO nanoparticles to the carbon tape, and

blow away the excess of powder with compressed air. This specimen is sputter coated with a

thin Au-Pd layer of about 3 nm thick in vacuum to avoid the charging. Typical SEM

5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 3 5 0 0 4 0 0 0

34

19

53

55

94

10

52

14

11

20

94

29

20

(b )

% T

ra

nsm

itta

nc

e

W a v e N u m b er (cm-1

)

2 0 3 0 4 0 5 0 6 0 7 0 8 0

(00

2)-

(a ) (31

1)

(22

0)(0

22

)

(20

2)

(11

2)

(02

0)

(11

1)-

(20

0)

(11

0)

In

ten

sit

y (

a.u

.)

A n g le 2 (d eg ree)

90

micrograph for prepared CuO nanoparticles is shown in Figure 7.2. The SEM micrograph

clearly shows irregular shaped morphologies of CuO nanoparticles. The SEM observation

shows the presence of agglomerated nanoparticles with an average size of 40–100 nm.

Figure 7.2: FESEM image of CuO nanoparticles (a) High Resolution image, (b) Low

Resolution image.

EDX Results

The EDX spectrum of CuO nanoparticles is given in Figure 7.3. The EDX results show that

there are no other elemental impurities present in the prepared CuO nanoparticles.

91

Figure 7.3: Energy dispersive X-ray profile of CuO nanoparticles.

7.3.2 Determination of Physiological Parameters of Stevia rebaudiana

The results of present study [Table 7.1, Figure 7.4)] shows that the growth of S. rebaudiana

shoots reaches to a maximum at a certain threshold level, i.e., 10 mg/L of CuO nanoparticles.

However, after attaining this threshold level, further increase results in toxicity to the plant.

Table 7.1 clearly indicates that the highest amount of shooting (~90%) occurred in MS

medium supplemented with 10 mg/L of CuO nanoparticles. In contrast, the lowest shooting

frequency (~41%) is obtained in MS medium containing 1000 mg/L of CuO nanoparticles.

Maximum shoot length (4.9 cm) is achieved with MS medium augmented with 10 mg/L of

CuO nanoparticles, followed by medium containing 1 mg/L (4.5 cm), 0.1 mg/L (4.3 cm) and

control (4.1 cm). Similarly, highest number of nodes (4.9) and leaves (16.1) are produced by

10 mg/L CuO nanoparticles treatment, while the lowest number of nodes (0.3) and leaves

(4.66) are formed in treatments provided with 1000 mg/L of CuO nanoparticles. Fresh weight

of shoots is also measured to be highest in MS medium fortified with 10 mg/L of CuO

nanoparticles (0.44 g) and lowest fresh weight (0.01 g) is found in 1000 mg/L of CuO

nanoparticles treatment.

92


nodal explants on MS medium supplemented with different concentrations of CuO

nanoparticles.


different according to Duncan’s multiple range test at confidence level of 95%.

Figure 7.4: Shoot organogenesis of Stevia in MS basal medium containing (a) no CuO (b)

0.1 mg/L CuO (c) 1 mg/L CuO (d) 10 mg/L CuO (e) 100 mg/L CuO (f) 1000 mg/L CuO

nanoparticles.

7.3.3 Determination of Steviol Glycosides in Stevia rebaudiana

Figure 7.5 shows the synthesis of more steviol glycosides (rebaudioside A and stevioside) in

S. rebaudiana in the presence of up to 10 mg/L of CuO nanoparticles, thereafter, it causes

phytotoxicity. The amount of rebaudioside A increased from 2.07% in control to 3.49% in

Conc.

(mg/L)

Response

(%)

Mean shoot

length

(cm)

Mean no. of

nodes per

explant

Mean no. of

leaves per

regenerated shoot

FW of

shoots (g)

0 84.7 4.1±0.1d

4.6±0.1a

13.1±0.3c

0.16±0.0d

0.1 85.4 4.3±0.1bc

4.7±0.1a

13.5±0.3c

0.30±0.0c

1.0 86.7 4.5±0.1b

4.8±0.1a

14.3±0.3b

0.39±0.0b

10 88.5 4.9±0.2a

4.9±0.1a

16.1±0.4a

0.44±0.0a

100 68.5 3.8±0.0d

2.3±0.0b

9.88±0.2d

0.14±0.0d

1000 40.8 1.0±0.0e

0.3±0.0c

4.66±0.0e

0.01±0.0e

93

MS supplemented with 0.1 mg/L of CuO nanoparticles. Furthermore, the enhanced quantity

of rebaudioside A obtained from shoots grown in MS having 1.0 mg/L and 10 mg/L of CuO

nanoparticles is found 3.64% and 4.17%, respectively. Increasing the CuO nanoparticles

concentration to 100 mg/L, a drastic decrease in rebaudioside A amount (3.49%) is observed.

The decline in rebaudioside A quantity occurs more (0.52%) when shoots are allowed to

grow in MS medium containing 1000 mg/L of CuO nanoparticles. Similarly, HPLC analysis

for quantity of stevioside reveals that the amount of stevioside is 0.73% obtained in control

group that is found to increase up to 1.17% and 1.19% in treatments having 0.1 mg/L and 1.0

mg/L of CuO nanoparticles, respectively. The stevioside amount was also found the highest

(2.06%) at 10 mg/L of CuO nanoparticles concentration. Thereafter, it decreases to 0.96% in

MS medium supplemented with 100 mg/L of CuO nanoparticles, and falls to a minimum

(0.13%) in 1000 mg/L of CuO nanoparticles.

Figure 7.5: Effect of CuO nanoparticles at different concentrations ranging between 0 and

1000 mg/L on rebaudioside A content represented with dark blue bars and stevioside content

with red bars. Error bars have been shown as standard deviation on each bar. Bars are


7.3.4 Determination of Antioxidant Activities in Stevia rebaudiana

Table 7.2 illustrates that a premier quantity of total phenolic content (6.22 µg GAE/mg), total

flavonoid content (7.49 µg QE/mg), total anti-oxidant capacity (11.9 µgAAE/mg), total

reducing power (11.5 µg AAE/mg) and % DPPH inhibition (74.8 %) is assessed from Stevia

C

ab ab

a

b

d C

ab ab

a

b

d

0

1

2

3

4

5

0 0.1 1 10 100 1000

Am

ou

nt

of

SGs

(%)

Concentrations of Nanoparticles (mg/L)

Rebaudioside A

Stevioside

94

leaves on 10 mg/L of CuO nanoparticles treatment. However, the lowest total phenolic

content (3.99 µg GAE/mg), total flavonoid content (2.11 µg QE/mg), total anti-oxidant

capacity (9.16 µgAAE/mg), total reducing power (10.3 µg AAE/mg) and % DPPH inhibition

(58.5 %) is found out from extracts containing 1000 mg/L CuO nanoparticles employed in

MS medium.


explants on MS medium supplemented with different concentrations of CuO nanoparticles.


different according to Duncan’s multiple range test at confidence level of 95%. TPC: total

phenolic content, GAE: gallic acid equivalent, TFC: total flavonoid content, QE: quercitine

equivalent, TAC: total antioxidant capacity, TRP: total reducing power, AAE: ascorbic acid

equivalent, DPPH: 2,2-diphenyl-1-picryl hydrazyl.

7.4 Discussion

Application of CuO nanoparticles in shoot induction medium stimulates the response upto

some extent, thereafter, all the parameters tend to decrease. CuO nanoparticle concentration

of 10 mg/L proved best as mean shoot length, number of nodes and leaves per plant and fresh

weight are maximum at this concentration. The results show that CuO nanoparticle

concentration up to 10 mg/L has a stimulatory effect and becomes toxic above that. The

potential harmful effects of CuO nanoparticles to organisms are poorly studied though CuO is

highly toxic. The extent of CuO nanoparticles toxicity is considered to be based on release of

metal ions from the corresponding nanoparticles. The generated metal ions may yield free

Conc.

(mg/L)

TPC

(µg

GAE/mg)

TFC

(µg

QE/mg)

TAC

(µg

AAE/mg)

TRP

(µg

AAE/mg)

% DPPH

Inhibition

0 4.5±0.0c

3.8±0.0d

10.4±0.0b

10.5±0.0c

60.6

0.1 4.6±0.0c

6.3±0.0b

10.9±0.0b

10.7±0.0b

66.7

1.0 5.6±0.0b

6.3±0.0b

11.5±0.0a

10.8±0.0b

68.6

10 6.2±0.0a

7.5±0.0a

11.9±0.0a

11.5±0.0a

74.8

100 4.1±0.0d

5.7±0.0c

9.52±0.0c

10.6±0.0bc

64.2

1000 3.9±0.0e

2.1±0.0e

9.16±0.0d

10.3±0.0d

58.5

95

radicals and cause intracellular oxidative stress. The oxidative stress might also be involved

in genotoxicity (Zafar et al., 2016). Although the toxicity and stimulatory mechansim of

nanoparticles is still not reported, however, it can be presumed that the nanoparticles itself or

their released ions generate modulatory effect inside the cell by interacting with

biomolecules, and even create an effect at transcription level.

Linked with better growth and increase in fresh weight, a trend in increase of SGs is observed

by an increase of CuO nanoparticle concentration in the media. Rebaudioside A and

stevioside continuously increase up to 10 mg/L of CuO treatment. Thereafter, these bioactive

compounds begin to decrease as CuO nanoparticles concentration is increased in the media.

Chlorocholine chloride and paclobutrazol have been reported to influence growth and SGs

production in Stevia rebaudiana (Karimi et al. 2014) and this is linked with enhanced

oxidative stress to some extent. Proline and polyethylene glycol (PEG) that induce stress on

regenerated plants are also found to stimulate enhanced production of SGs in callus and

suspension cultures of S. rebaudiana (Gupta et al. 2015).

The enzymatic and non-enzymatic antioxidants naturally present in plants actually helps them

cope with an oxidative stress of metal ions or free radicals (Klaper et al. 2009). CuO

nanoparticles exert oxidative stress on regenerated shoots of Stevia. As a result, total

antioxidant potential and reducing power potential tends to increase. The plantlets grown in

the presence of CuO nanoparticle concentration also show increase in DPPH-free radical

scavenging activity. To combat the oxidative stress, plants have internal mechanisms;

enzymatic and non-enzymatic. The non-enzymatic molecules, phenolics and flavonoids

increase as a result. However, these responses shut down when CuO concentration exceeds a

threshold level. Hence, accumulation of reactive oxygen species (ROS) cause depletion of

plant cells and their activities (Choi and Hu 2008). Based on this study, CuO nanoparticles

implicate an intracellular oxidative stress by the release of metal ions (Cu+2

) or free radicals

into MS culture medium (Gajewska and Skłodowska 2007; Dimpka et al. 2014), and as a

consequence of oxidative damage at 1000 mg/L, an impaired growth, reduced steviol

glycoside quantity, and mitigation of antioxidant activities is observed.

In conclusion, it is demonstrated from these findings that CuO nanoparticles confer positive

effects for in-vitro Stevia growth dynamics and steviol glycoside production. In this regard, a

significantly enhanced amount of secondary metabolites and antioxidant activities are

obtained at 10 mg/L of CuO nanoparticles concentration employed in MS basal medium.

96

Meanwhile, phytotoxic effects of CuO nanoparticles have also been observed, and the highest

level of phtotoxicity has been achieved by CuO nanoparticles at 1000 mg/L concentration.

This research opens up new avenues for the study of metabolic pathways in the context of an

interaction between different concentrations of nanoparticles and in vitro grown medicinal

plants. Metabolic engineering will be the next step to such kinds of studies.

97

CHAPTER 8

Application of PVP and PEG modulated CuO and ZnO

Nanoparticles to Micropropagated Shoots of Stevia rebaudiana

Bertoni for Steviol Glycosides Production

Article Submitted:

Javed R, Zia M, Yucesan B, Gurel E. Abiotic Stress of ZnO-PEG, ZnO-PVP, CuO-PEG and

CuO-PVP Nanoparticles Enhance Growth, Sweetener Compounds and Antioxidant Activities

in Shoots of Stevia rebaudiana Bertoni. IET Nanobiotechnology (Accepted).

8.1 Introduction

Nanoparticles are ultrafine particles, sized between 1 to 100 nm, that possess high surface

area and reactivity compared to their bulk counterparts. Metallic oxide nanoparticles possess

unusual stability at high temperature. Nanoparticles are surface-coated to provide high

stability and prevent aggregation. Zinc oxide (ZnO) and copper oxide (CuO) nanoparticles

linked with polymers contribute to the biological properties of nanoparticles (Javed et al.

2016a). Polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) are well-known coating

agents that bind to terminal hydroxyl or methoxy group (Liu et al. 2010). The surface area

and size of nanoparticles alters as a consequence of capping. The different methods of

capping of ZnO nanoparticles involve the use of organic compounds such as SiO2, Al2O3 and

metal ions as capping agents. In case of CuO nanoparticles, polyvinylpyrrolidone (PVP)-

capped and tripheny phosphine oxide (TPPO)-capped CuO nanoparticles have been

synthesized by Sharma et al. (2016), and chitosan-capped CuO nanoleaves have been

prepared by an ultrasound sonication method (Abiramanet al. 2017).

Stevia rebaudiana, belonging to Asteraceae (Sunflower family), is famous for its sweetener

compounds/ steviol glycosides (SGs) that are naturally produced in its leaves. In vitro

production of SGs in Stevia rebaudiana is effected by different mineral components, carbon

sources, plant growth regulators (PGRs) and different stress conditions. Few scientists have

reported that both rebaudioside A and stevioside were not produced by the callus or cell

suspension cultures, and they can only be produced in the whole plant (Bondarev et al. 2001).

The chemical stress induced by proline and polyethylene glycol (PEG) enhance the

production of SGs in callus and suspension culture of S. rebaudiana (Gupta et al. 2015).

Recently, the chilling stress induced by salicylic acid, hydrogen peroxide (H2O2), 6-


98

benzylaminopurine (BAP) and calcium chloride (CaCl2) resulted in a significantly higher

production of rebaudioside A and stevioside was identified (Soufi et al. 2016).

The elicitation of SGs can be performed by different nanoparticle molecules as a result of

optimization of specific protocols having coated or uncoated nanoparticles at different

concentrations. Later on, the process by which the highest content of SGs obtained can be

utilized for industrial and commercial scale applications of these compounds. The aims and

objectives of this study involve comparison of abiotic stress produced by ZnO, CuO, ZnO-

PEG, CuO-PEG, ZnO-PVP and CuO-PVP nanoparticles on the shoot organogenesis of Stevia

rebaudiana by elucidating the comparative physiology, SGs production and non-enzymatic

antioxidant activities.

8.2 Materials and methods

8.2.1 Shoots Development in Medium containing Nanoparticles

The seeds of Stevia rebaudiana are obtained from the private company, Polisan Tarim,

Istanbul, Turkey. After surface disinfection with 0.1 % (w/v) mercuric (II) chloride (HgCl2),

the seeds are cultured on MS medium (Murashige and Skoog 1962) having 3% (w/v) sucrose

and 0.8% (w/v) agar. PGRs are not added in MS medium containing shoot nodes for shoots

development. Different treatments are prepared having 1 mg/L and 10 mg/L concentrations

of ZnO (34 nm in size), ZnO-PEG (26 nm in size), ZnO-PVP (32 nm in size), CuO (47 nm in

size), CuO-PEG (27 nm in size) and CuO-PVP (27 nm in size) nanoparticles, all synthesized

by a chemical method of co-precipitation.

A total of 13 treatments were prepared; 1 treatment devoid of nanoparticles (lacking PGRs

for shoot organogenesis taken as control) and the others having 2 different concentrations of

6 types of nanoparticles for shoot organogenesis. All of cultures were maintained in growth

chamber under 16/8 h (light/dark) photoperiod (50 μmol m−2

s−1

irradiance by fluorescent

lamps) at 25 ± 1 °C. The shoot cultures are incubated for 4 weeks. The physiological

parameters involving percent shooting response, mean length of shoots, mean number of

nodes, mean number of leaves, FW of shoots, are measured. Later on, all cultures are dried to

form extracts for performing steviol glycosides (SGs) formation studies and antioxidant

activities.

8.2.2 Extract Preparation and Steviol Glycosides Analysis

Steviol glycosides are extracted from Stevia leaves grown under nanoparticles stress, and

analysed according to procedure described in Chapter 6.

99

8.2.3 Extract Preparation and Antioxidant Activities

Methodology described in Chapter 5 is followed for the preparation of extracts and

assessment of antioxidant assays involving determination of total phenolic content, total

flavonoid content, total antioxidant capacity, total reducing power and DPPH-free radical

scavenging activity.


The design of experiments is randomized and the statistical analysis of data is performed

using SPSS, Version 17.0 (SPSS Inc., Chicago, IL, USA). Statistical difference is determined

using ANOVA, and the significance of difference between means ± SE (standard error)

values are obtained using Duncan’s multiple range tests performed at p< 0.05.

8.3 Results

All nanoparticles produce effects on Stevia plant physiology, SGs production and antioxidant

activities.

8.3.1 Physiology Analysis of Stevia rebaudiana

Table 8.1 shows the comparative analysis of all physiological parameters, i.e., % shooting

response, mean shoot length, mean number of nodes, mean number of leaves and F.W of

shoots obtained from 1 mg/L and 10 mg/L concentration of nanoparticles stress induced

during the shoots development of Stevia rebaudiana. The results clearly reveal that all

parameters are higher in treatments of ZnO, ZnO-PEG and ZnO-PVP regarding 1 mg/L

concentration, whereas the best results obtained in case of 10 mg/L concentration of

nanoparticles are revealed by CuO, CuO-PEG and CuO-PVP. Moreover, the results of

capped nanoparticles (ZnO-PEG, CuO-PEG, ZnO-PVP, CuO-PVP) are higher than uncapped

nanoparticles (ZnO, CuO).

8.3.2 Steviol glycosides Analysis in Stevia rebaudiana

Figure 8.2 shows the SGs content obtained from leaves of Stevia rebaudiana under 1 mg/L of

nanoparticles concentration. Nanoparticles are applied at 1 mg/L to the MS medium

containing shoot nodes as explant for shoot organogenesis. The rebaudioside A is found to

be greater in amount than stevioside in case of all treatments. The SGs are greater in ZnO

nanoparticles as compared to the CuO nanoparticles. Overall, ZnO-PEG reveals highest SGs

followed by ZnO-PVP. Out of CuO nanoparticles, the highest rebaudioside A and stevioside

content is revealed by CuO-PVP and CuO-PEG, respectively. The lowest SGs content is

obtained by control (devoid of any nanoparticles). Figure 8.2 shows the effect of

100

nanoparticles at 10 mg/L of concentration on SGs production from leaves of Stevia

rebaudiana. CuO nanoparticles show greater SGs content as compared to ZnO nanoparticles.

CuO-PEG reveals highest content of SGs (rebaudioside A and stevioside) followed by CuO-

PVP. In case of ZnO, the ZnO-PEG and ZnO-PVP reveals highest content of SGs as

compared to the bare ZnO nanoparticles.


nodal explants on MS medium supplemented with 1 mg/L and 10 mg/L of different NPs.

±: standard error, the means with the same letter within the rows are not significantly


Physiological

parameters

Control ZnO ZnO-

PEG

ZnO-

PVP

CuO CuO-

PEG

CuO-

PVP

At 1 mg/L concentration of nanoparticles Response

(%)

84.7 87.0 90.5 92.2 85.8 87.4 88.7

Mean shoot

length (cm)

4.1±0.2a 4.3±0.1

c 4.4±0.1

d 4.4±0.2

d 4.2±0.1

b 4.3±0.1

c 4.3±0.1

c

Mean

number of

nodes

4.2±0.1a 4.4±0.2

c 4.6±0.1

e 4.7±0.1

f 4.3±0.1

b 4.5±0.2

d 4.5±0.2

d

Mean

number of

leaves

13.1±0.0a 14.6±0.1

bc 16.4±0.0

d 17.3±0.0

e 13.5±0.1

b 14.8±0.0

bc 15.3±0.0

c

F.W of

shoots

0.16±0.1a 0.26±0.2

e 0.33±0.1

f 0.46±0.1

g 0.19±0.1

b 0.22±0.1

c 0.24±0.2

d

At 10 mg/L concentration of nanoparticles Response

(%)

84.7 86.5 89.2 88.8 90.4 94.6 96.6

Mean shoot

length (cm)

4.1±0.1a 4.2±0.1

b 4.3±0.1

c 4.3±0.1

c 4.2±0.2

b 4.4±0.0

d 4.5±0.0

e

Mean

number of

nodes

4.2±0.2a 4.3±0.1

b 4.6±0.0

d 4.5±0.0

c 4.7±0.1

e 4.9±0.0

f 4.9±0.1

f

Mean

number of

leaves

13.1±0.1a 14.4±0.1

b 15.9±0.1

c 15.6±0.1

bc 15.4±0.1

bc 16.3±0.1

d 17.7±0.1

e

F.W of

shoots

0.16±0.2a 0.18±0.1

b 0.20±0.1

c 0.22±0.2

d 0.34±0.0

e 0.41±0.1

f 0.46±0.1

g

101

Figure 8.1: Amount of % SGs content at different nanoparticles of 1 mg/L concentration.

Rebaudioside-A represented with blue bars and stevioside with red bars. Error bars have been

shown as standard deviation on each bar. Bars are significantly different at confidence

interval level of 95%.

Figure 8.2: Amount of % SGs content at different nanoparticles of 10 mg/L concentration.

Rebaudioside-A represented with blue bars and stevioside with red bars. Error bars have been

shown as standard deviation on each bar. Bars are significantly different at confidence

interval level of 95%.

c

a

b

e

de d

f

c

a

b

e

bc d

f

0

1

2

3

4

5

6

ZnO ZnO-PEG ZnO-PVP CuO CuO-PEG CuO-PVP Control

Ste

vio

l Gly

cosi

de

s (%

)

Nanoparticles at 1 mg/L concentration

Rebaudioside A

Stevioside

e d

c bc

a b

f

d c bc b

a ab

e

0

0.5

1

1.5

2

2.5

3

3.5

4

ZnO ZnO-PEG ZnO-PVP CuO CuO-PEG CuO-PVP Control

Ste

vio

l Gly

cosi

de

s (%

)

Nanoparticles at 10 mg/L concentration

Rebaudioside A

Stevioside

102

8.3.3 Antioxidant Activities Analysis of Stevia rebaudiana

Table 8.2 shows the phytochemical results of 4 weeks old shoot leaves of Stevia rebaudiana

at 1 mg/L concentration and 10 mg/L concentration of nanoparticles. The data clearly

indicate that all antioxidant activities are higher in leaves grown in the presence of ZnO

nanoparticles as compared to the CuO nanoparticles at 1 mg/L concentration. Moreover, the

ZnO-PEG and ZnO-PVP or CuO-PEG and CuO-PVP reveals highest content of antioxidants

compared to ZnO or CuO due to capping. The results of 10 mg/L concentration are opposite

to the previous one because 10 mg/L concentration is found more suitable for shoots raised

under CuO nanoparticles than ZnO nanoparticles on comparative basis. However, the

antioxidants produced in the medium containing ZnO-PEG and ZnO-PVP or CuO-PEG and

CuO-PVP exploit highest antioxidant activities due to similar reason as described above.

Table 8.2 : Comparison of phytochemical assays in 4 weeks old shoots produced from nodal

explants on MS medium supplemented with 1 mg/L and 10 mg/L of different nanoparticles.

Control ZnO ZnO-PEG ZnO-

PVP

CuO CuO-

PEG

CuO-PVP

At 1 mg/L concentration of nanoparticles

TPC 2.3±0.1a 2.6±0.1

d 2.8±0.1

e 2.8±0.8

e 2.4±0.4

b 2.6±0.3

d 2.5±0.2

c

TFC 31.6±0.0a 38.2±0.2

c 80.1±0.6

g 60.2±0.5

f 32.4±0.2

b 55.4±0.5

d 59.3±0.1

e

TAC

1.2±0.1a 1.6±0.3

d 2.3±0.4

g 1.8±0.1

e 1.3±0.1

b 2.1±0.2

f 1.5±0.4

c

TRP

8.0±0.3a 12.1±0.2

d 14.3±0.2

f 13.2±0.3

e 8.5±0.1

b 9.9±0.7

c 9.75±0.9

bc

DPPH (%

inhibition

48.9 53.2 73.8 63.9 49.4 62.6 61.4

At 10 mg/L concentration of nanoparticles

TPC 2.3±0.1a 2.4±0.2

b 2.6±0.3

d 2.5±0.3

c 2.8±0.1

e 2.9±0.2

f 3.1±0.3

g

TFC 31.6±0.3a 35.8±0.5

b 44.6±0.7

d 40.4±0.3

c 55.2±0.2

e 63.7±0.2

f 87.5±0.1

g

TAC

1.2±0.2a 1.3±0.4

b 1.9±0.3

f 1.6±0.2

d 1.5±0.1

c 2.2±0.4

g 1.8±0.2

e

TRP

8.0±0.1a 8.42±0.6

b 8.76±0.5

c 9.85±0.3

d 10.5±0.3

e 11.4±0.7

f 11.4±0.4

f

DPPH (%

inhibition

48.9 51.5 65.6 58.7 64.9 68.5 67.5

±: standard error, the means with the same letter within the rows are not significantly

different according to Duncan’s multiple range test at P<0.05. FW: fresh weight, QE:

quercitine equivalent, GAE: gallic acid equivalent, AAE: ascorbic acid equivalent. TPC(µg

GAE/mg FW); TFC(µg QE/mg FW); TAC(µg AAE/mg FW); TRP(µg AAE/mg FW).

103

8.4 Discussion

The effect of ZnO nanoparticles was earlier studied on the medicinal plant, Fagopyrum

esculentum (Lee et al. 2013). In addition to this, comparative study of ZnO nanoparticles,

ZnO bulk particles and Zn+2 on Brassica napus was conducted regarding growth,

biochemical compounds, antioxidant enzyme activities and Zn bioaccumulation (Kouhi et al.

2015). Recently, effect of ZnO nanoparticles on Brassica nigra seedlings and stem explants

were reported in context of growth and antioxidant activities (Zafar et al. 2016). The effect of

CuO nanoparticles and dissolved Cu was studied in the aquatic macrophyte, Elodea nutellii

regarding Cu bioaccumulation, photosynthesis and oxidative stress (Regier et al. 2015).

Effect of CuO nanoparticles on Lemna minor regarding antioxidative activities was studied

(Song et al. 2016). Da Costa and Sharma (2016) studied the effect of CuO nanoparticles on

growth, photosynthesis and antioxidant activities of Oryza sativa. Different growth

parameters of Arabidopsis thaliana by the impact of CuO nanoparticles was studied (Nair

and Chung 2014; Wang et al. 2016). Genotoxic effects of ZnO and CuO nanoparticles on the

early growth of Fagopyrum esculentum was studied (Lee et al. 2013).

Previously,1 mg/L concentration was found best for ZnO nanoparticles in order to enhance

growth, SGs production and antioxidant activities in the leaves of Stevia rebaudiana (Javed et

al. 2016) which agrees with the current study. However, 10 mg/L concentration in case of

CuO resulted in highest growth parameters, SGs content and antioxidant activities.

Furthermore, the capped nanoparticles, i.e., ZnO-PEG, ZnO-PVP, CuO-PEG and CuO-PVP

elicited more SGs content formation as compared to their bare counterparts, i.e., ZnO and

CuO. The previous study by Javed et al. (2016) also determined higher biological activities in

PEG- or PVP-capped ZnO nanoparticles than the uncapped ZnO. Effect of Zn nanoparticles

on stevioside production of Stevia rebaudiana has also been reported (Desai et al. 2015).

Hendawey et al. (2015) reported the effect of Fe, Cu and Si nanoparticles on the callus of

Stevia rebaudiana with respect to stevioside content and enzymatic antioxidant activities.

In conclusion, the shoot culture of Stevia rebaudiana were raised under ZnO, CuO, ZnO-

PEG, CuO-PEG, ZnO-PVP and CuO-PVP nanoparticles stress. Two concentrations, i.e., 1

mg/L and 10 mg/L of all nanoparticles were used to grow Stevia leaves containing secondary

metabolites and sweetening compounds. ZnO nanoparticles revealed highest growth, SGs

content and phytochemical activities at 1 mg/L, and the highest growth, SGs content and

antioxidant activities of CuO nanoparticles was determined at 10 mg/L concentration of

nanoparticles. All assays declared capped ZnO and CuO nanoparticles (ZnO-PEG, CuO-

104

PEG, ZnO-PVP, CuO-PVP) to be the best for the growth, production of sweetener

compounds and non-enzymatic antioxidant activities as compared to the uncapped ones

(ZnO, CuO).

105

General Summary

Nanobiotechnology is an advanced area of research encompassing nanotechnology and

biotechnology. Nanotechnology has done wonders in different fields of engineering, physics,

chemistry, pharmacy, medicine and biology. Although scientists are working in this field of

research for the past few decades, nevertheless, a lot more work still needs to be done using

nanoparticles in various biotechnological applications. Nanobiotechnology is chosen for

research involving the synthesis and characterization of nanoparticles as well as biological

assays (medicine) and plant secondary metabolites production by tissue culture (plant

biotechnology).

First of all, uncapped and capped ZnO and CuO nanoparticles are synthesized using

surfactants/polymers as capping agents. The polymers used are polyethylene glycol (PEG)

and polyvinyl pyrrolidone (PVP). Then, the nanoparticles are characterized by X-ray

diffraction (XRD), fourier transform infra-red spectroscopy (FTIR), UV-Visible

spectrometry, scanning electron microscopy (SEM) and energy dispersive x-ray (EDX). It is

found by characterization that the size of nanoparticles get reduced when they are capped.

XRD pattern shows sample size and crystallinity. FTIR reveals different functional groups of

samples, UV-Visible spectroscopy measures the band gap energy, SEM elucidates

morphology of nanoparticles, and EDX depicts the purity of nanoparticles.

The antibacterial, antioxidant and antidiabetic activities of ZnO nanoparticles revealed a

significant increase in ZnO-PEG and ZnO-PVP nanoparticles as compared to the uncapped

ZnO nanoparticles. Similarly, the antibacterial, antioxidant, antitumor, cytotoxic and

antidiabetic activities are found significantly higher in CuO-PEG and CuO-PVP nanoparticles

as compared to the uncapped CuO nanoparticles. Gram-positive bacteria showed higher

antibacterial activity in comparison with Gram-negative bacteria in case of both ZnO and

CuO nanoparticles because the cell wall of Gram-positive bacteria is more permeable due to

the presence of only peptidoglycan as compared to the cell wall of Gram-negative bacteria

possessing peptidoglycan as well as lipolysaccharides. Regarding ZnO nanoparticles, highest

antibacterial activity is exhibited by ZnO-PVP, whereas the highest antioxidant and

antidiabetic activities are conferred by ZnO-PEG. Considering the CuO nanoparticles, the

highest antibacterial activity is obtained by CuO-PVP, the highest antioxidant and antitumor

activity by CuO-PEG, and the highest antidiabetic activity by CuO-PVP. The cytotoxic

activity is exhibited equally by all of CuO nanoparticles incase of 50 µg/ mL concentration

106

and this activity is obtained highest by CuO-PVP using either 25 µg/ mL or 12.5 µg/ mL of

nanoparticles concentration.

Feasible regeneration protocols for tissue culture of Stevia rebaudiana are optimized and

DPPH-free radical scavenging activity of different cultures of this antidiabetic medicinal

plant is determined. The highest DPPH-free radical scavenging activity is revealed by the

callus cultures, followed by Stevia shoots and plantlets, respectively. This difference is due to

the occurrence of different metabolic pathways in different plant parts obtained by

regeneration. The effect of ZnO and CuO nanoparticles on callus cultures of Stevia

rebaudiana is also calculated in context of their physiology and antioxidant activities. 1 mg/L

and 10 mg/L are declared the best ZnO and CuO nanoparticle concentrations, respectively

regarding various physiological parameters. The highest amount of total phenolic content

(TPC), total flavonoid content (TFC), total antioxidant capacity (TAC), and 1, 1-diphenyl-2-

picrylhydrazyl (DPPH)-free radical scavenging activity is obtained at 100 mg/L of ZnO

nanoparticles. However, the TPC, TAC, TRP and DPPH-free radical scavenging activity is

achieved highest at 10 mg/L concentration of CuO nanoparticles. Moreover, the highest TRP

regarding ZnO and the highest TFC regarding CuO were achieved at 50 mg/L and 100 mg/L,

respectively. This clearly indicates that CuO nanoparticles possess more toxicity to Stevia

callus as compared to the ZnO nanoparticles. Furthermore, it was revealed that the callus of

Stevia doesn’t produce steviol glycosides, both in the presence or absence of nanoparticles, as

estimated by the results of HPLC.

The positive effect of both ZnO and CuO nanoparticles is found during shoot organogenesis

of Stevia rebaudiana. In vitro growth of plants as well as its steviol glycosides (rebaudioside

A and stevioside) production and antioxidant activities enhance by the supplementation of

ZnO and CuO nanoparticles in to the MS growth medium having shoot nodes inoculated as

explants. The highest amount of physiological parameters, formation of steviol glycosides

and antioxidant activities are obtained at 1 mg/L and 10 mg/L of ZnO and CuO nanoparticles,

respectively. This rise is due to the oxidative stress of ZnO and CuO nanoparticles on Stevia

plant that triggered defence mechanism of the plant against this abiotic stress. However, after

reaching the maximum threshold level of ZnO (1 mg/L) and CuO (10 mg/L) nanoparticles,

further increment of both the nanoparticles results in phytotoxicity to Stevia. The maximum

level of phytoxicity is obtained at 1000 mg/L of ZnO and CuO nanoparticles that results in

repairing of growth and mitigation of secondary metabolite production of Stevia rebaudiana.

107

Hence, the positive as well as negative effect of ZnO and CuO nanoparticles is determined in

micropropagation (callogenesis and shoot organogenesis) of Stevia rebaudiana Bertoni.

The effect of different nanoparticles including capped nanoparticles (ZnO, CuO, ZnO-PEG,

CuO-PEG, ZnO-PVP, CuO-PVP) on the shoot organogenesis of Stevia rebaudiana is also

observed. Two different concentrations (1 mg/L and 10 mg/L) of all nanoparticles are

employed to the MS medium containing axillary shoot nodes as explants for shoot

organogenesis. ZnO, ZnO-PEG and ZnO-PVP shows the best effect at 1 mg/L concentration,

however, the highest effect of CuO, CuO-PEG and CuO-PVP is observed at 10 mg/L of

concentration. The most important finding is that the capped nanoparticles results in more

growth, steviol glycosides (SGs) production and antioxidant activities as compared to the

uncapped nanoparticles at similar concentration.

Summing up, the capped and uncapped ZnO and CuO nanoparticles have been synthesized

by chemical method of co-precipitation, and characterized by using different techniques

including XRD, FTIR, UV-Visible spectroscopy, SEM and EDX. The synthesized

nanoparticles have been analysed for biological activities as well as the production of

sweetener compounds, i.e., steviol glycosides (SGs) mainly Rebaudioside A (Reb A) and

Stevioside (ST) in in-vitro culture of Stevia rebaudiana. The results of biological assays

reveal that ZnO, ZnO-PEG and ZnO-PVP nanoparticles possess antibacterial, antioxidant and

antidiabetic potential, whereas CuO, CuO-PEG and CuO-PVP nanoparticles also exhibit

cytotoxic and antitumor potential alongwith the antibacterial, antioxidant and antidiabetic

activities. Furthermore, the in-vitro production of SGs (Reb A and ST), antioxidant activities

(TPC, TFC, TAC, TRP and DPPH-free radical scavenging activity) and growth parameters is

good at certain concentration of nanoparticles during callogenesis and shoot organogenesis of

Stevia plant. Capped nanoparticles (ZnO-PEG, CuO-PEG, ZnO-PVP, CuO-PVP) produce

best results as biological agents to be used as drugs or drug-carriers. Moreover, the SGs

production from an important antidiabetic plant, Stevia rebaudiana is higher in capped

nanoparticles (ZnO-PEG, CuO-PEG, ZnO-PVP, CuO-PVP) as compared to the uncapped

nanoparticles (ZnO, CuO).

108

Future Recommendations

Synthesis of other metallic/ metallic oxide nanoparticles should be performed and

capped with different capping agents. The effects of capping agents on the properties

of nanoparticles would be studied in this way. Furthermore, the green synthesis of

nanoparticles should be performed using extracts of medicinal plants as reducing

agents. The benefit of biogenic synthesis would be that they wouldn’t need to be

capped by capping/ polymerizing agents after synthesis because the green synthesized

nanoparticles get naturally capped by plant extracts and are also non-toxic unlike

chemically synthesized nanoparticles.

The biological studies shouldn’t be limited to in vitro experiments, rather need to be

explored in vivo, i. e., on experimental models and different human cell lines in order

to determine the ultimate effects produced by nanoparticles on the biological activities

of living organisms. Moreover, the bioactive compounds and secondary metabolites

of medicinal plants possess considerable importance on industrial and commercial

scale. Hence, different medicinal plants should be exploited for studying their

secondary metabolites production/ enhancement upon exposure of capped and

uncapped nanoparticles. Measurement of environmental toxicity of nanoparticles is of

considerable importance. Therefore, the toxicity levels of different nanoparticles

should be specified with respect to their production of secondary metabolites,

antioxidant activities and physiological parameters for different medicinal plants.

The study of metabolic pathways of medicinal plants elucidating different enzymes

and products produced as a result of oxidative stress of nanoparticles should be

carried out. In other words, the mechanisms of metabolic pathways resulting in the

production of desired secondary metabolites should be studied in detail. It will result

in the isolation of desired genes that are to be triggered for specific metabolite

production. Furthermore, the molecular studies involving gene expression and micro-

RNA expression are the steps towards metabolic engineering which should be done.

109

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