227
i
i
ii
PLANT MEDIATED SYNTHESIS, CHARACTERIZATION AND
BIOLOGICAL EVALUATION OF SILVER NANOPARTICLES USING
AQUEOUS EXTRACT(S) FROM LEAVES OF AGAVE AMERICANA,
MENTHA SPICATA AND MANGIFERA INDICA
FARAH SHIREEN ALI SHAH
A thesis submitted to the University of Peshawar in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in Biotechnology and
Microbiology
CENTRE FOR BIOTECHNOLOGY AND MICROBIOLOGY
UNIVERSITY OF PESHAWAR
Session 2012-2016
iii
PLANT MEDIATED SYNTHESIS, CHARACTERIZATION AND
BIOLOGICAL EVALUATION OF SILVER NANOPARTICLES USING
AQUEOUS EXTRACT(S) FROM LEAVES OF AGAVE AMERICANA,
MENTHA SPICATA AND MANGIFERA INDICA
This dissertation is submitted by Farah Shireen Ali Shah as partial fulfillment of
the requirements for the Degree of Doctor of Philosophy in Biotechnology and
Microbiology
Approved By:
1. ______________________
Prof. Dr. Bashir Ahmad
Research Supervisor
2. ______________________
External Examiner
3. _______________________
Director
Centre for Biotechnology and Microbiology
CENTRE FOR BIOTECHNOLOGY AND MICROBIOLOGY
UNIVERSITY OF PESHAWAR
iv
CERTIFICATE OF APPROVAL
This thesis titled “Plant Mediated Synthesis, Characterization and Biological
Evaluation of Silver Nanoparticles using Aqueous Extract(s) from leaves of
Agave americana, Mentha spicata and Mangifera indica” submitted by Farah
Shireen Ali Shah is hereby approved and recommended as partial fulfillment for
the award of Degree of Doctor of Philosophy in Biotechnology and Microbiology
1. Supervisor _________________________
2. External Examiner _________________________
3. Director _________________________
Centre for Biotechnology
& Microbiology, University of Peshawar
July 2016
v
AUTHOR’S DECLARATION
I solemnly declare that the research work presented in this thesis was carried out
in accordance with the requirements of the University of Peshawar’s regulations
for Research Degree Programs. The author has not submitted this work for any
other academic award. The work is original and author’s own data, while work
done in collaboration with or with the assistance of, others, is indicated as such.
The views expressed in this thesis, belongs to the author.
Date: ___________________ Signature: __________________
Farah Shireen Ali Shah
vi
Dedication
I wish to dedicate this work to my late father,
“Muhammad Ali Shah”, who taught me to value
myself and told me that I was the most precious thing
in his life.
vii
CONTENTS Tables i
Figures iii
Schemes vi
Pictures vi
Acknowledgments ix
Abstract x
C H A PTE R 1
IN TR O D U C TIO N & L ITE R A TU RE R E V IE W
1.1 General Introduction 1
1.2 Botanical Description 3
1.3 Agave americana (Plant) 3
1.3.1 Description 3
1.3.2 Distribution 5
1.3.3 Importance 5
1.4 Mentha spicata (Plant) 7
1.4.1 Description 7
1.4.2 Distribution 9
1.4.3 Importance 9
1.5 Mangifera indica (Plant) 11
1.5.1 Description 11
1.5.2 Distribution 13
1.5.3 Importance 13
1.6 Nanotechnology 15
1.6.1 Background 15
1.6.2 Current Status 16
1.7 Nanobiotechnology 17
1.7.1 Background 17
1.7.2 Current Status 17
1.8 Silver 19
1.8.1 Background 19
1.8.2 Current Status 19
1.9 Synthesis, Characterization and Optimization of Silver 21
viii
Nanoparticles
1.9.1 Physical Approach 21
1.9.1.1 Evaporation-Condensation Strategy 21
1.9.1.2 Laser Ablation Strategy 21
1.9.1.3 Arc Discharge Strategy 22
1.9.1.4 Direct Metal Sputtering Strategy 22
1.9.2 Chemical Approach 23
1.9.2.1 Chemical Reduction Strategy 23
1.9.2.2 Micro-Emulsion Strategy 24
1.9.2.3 Ultra-violet-Initiated Photo-Reduction Strategy 24
1.9.2.4 Microwave-Assisted Strategy 25
1.9.3 Green Approach 26
1.9.3.1 Microbe Mediated Strategy 26
1.9.3.2 Algae Mediated Strategy 28
1.9.3.3 Plant Mediated Strategy 28
1.10 Biological Efficacy of Silver Nanoparticles 30
1.10.1 Antimicrobial Activity 30
1.10.2 Anticancer Activity 31
1.10.3 Antioxidant Activity 32
1.10.4 Anti-Leshmanial Activity 32
1.10.5 Insecticidal Activity 33
1.11 Toxicity of Silver Nanoparticles 34
1.12 Aims and Objectives 35
C H A PTE R 2
METHODOLOGY
2.1 General Experimental Conditions 36
2.2 Plant Collection 36
2.3 Extraction 36
2.4 Phytochemical Screening 39
2.4.1 Mayer’s Test 39
2.4.2 Shinoda’s Test 39
2.4.3 Ferric Choride Test 40
2.4.4 Lead Acetate Test 40
2.4.5 Salkowski’s Test 41
2.4.6 Borntrager’s Test 41
2.4.7 Foam Test 41
ix
2.4.8 Benedict’s Test 41
2.4.9 Biuret’s Test 42
2.5 Synthesis of Silver Nanoparticles 43
2.6 Purification of Silver Nanoparticles 45
2.7 Characterization of Silver Nanoparticles 46
2.7.1 UV-VIS Spectroscopy 46
2.7.2 X-Ray Diffraction Measurements (XRD) 46
2.7.3 Scanning Electron Microscopy (SEM) 46
2.7.4 Energy-Dispersive X-Ray Spectroscopy (EDX) 47
2.7.5 Transmission Electron Microscopy (TEM) 47
2.7.6 Simultaneous Thermogravimetric And Differential Thermal
Analysis (TG-DTA)
47
2.8 Optimization of Silver Nanoparticles 48
2.8.1 pH Optimization 48
2.8.2 Temperature Optimization 48
2.9 Biological / Pharmacological investigation of silver
nanoparticles contrary to crude plant extracts
49
2.9.1 Antibacterial Assay 49
2.9.2 Minimum Inhibitory Concentration (MIC) Assay 51
2.9.3 Antifungal Assay 52
2.9.4 Anticancer Assay 54
2.9.5 Antioxidant Assay 56
2.9.6 Cytotoxic Assay 57
2.9.7 Phytotoxic Assay 59
2.9.8 Insecticidal Assay 60
2.9.9 Anti-Termite Assay 61
2.9.10 Enzyme Inhibition Assay 62
2.9.10.1 Acetylcholine-Esterase Inhibition 62
2.9.10.2 Urease Inhibition 63
2.9.11 Hemagglutination Assay 64
C H A PTE R 3 RESULTS & DISCUSSION
3.1 Phytochemical Screening 65
3.2 Characterization of Silver Nanoparticles 75
3.2.1 UV-VIS Spectroscopy 75
3.2.2 X-Ray Diffraction Measurements (XRD) 82
3.2.3 Scanning Electron Microscopy (SEM) 86
x
3.2.4 Energy-Dispersive X-Ray Spectroscopy (EDX) 96
3.2.5 Transmission Electron Microscopy (TEM) 100
3.2.6 Simultaneous Thermogravimetric And Differential Thermal
Analysis (TG-DTA)
103
3.3 Optimization of Silver Nanoparticles 110
3.3.1 pH Optimization 110
3.3.2 Temperature Optimization 110
3.4 Biological / Pharmacological investigation of silver
nanoparticles contrary to crude plant extracts
111
3.4.1 Antibacterial Assay 111
3.4.2 Minimum Inhibitory Concentration (MIC) Assay 120
3.4.3 Antifungal Assay 125
3.4.4 Anticancer Assay 131
3.4.5 Antioxidant Assay 136
3.4.6 Cytotoxic Assay 141
3.4.7 Phytotoxic Assay 146
3.4.8 Insecticidal Assay 151
3.4.9 Anti-Termite Assay 160
3.4.10 Enzyme Inhibition Assay 165
3.4.10.1 Acetylcholine esterase Inhibition 165
3.4.10.2 Urease Inhibition 169
3.4.11 Hemagglutination Assay 174
CONCLUSION 178
REFERENCES 180
PAPER PUBLISHED
TABLES
Table 3.1 Tabular depiction of phytochemical analysis of Agave americana,
xi
Mentha spicata and Mangifera indica (leaves)
Table 3.2 Tabular depiction of XRD values of Agave americana AgNPs
Table 3.3 Tabular depiction of XRD values of Agave americana aqueous
extract
Table 3.4 Tabular depiction of XRD values of Mangifera indica AgNPs
Table 3.5 Tabular depiction of XRD values of Mangifera indica aqueous
extract
Table 3.6 Tabular depiction of XRD values of Mentha spicata AgNPs
Table 3.7 Tabular depiction of XRD values of Mentha spicata aqueous
extract
Table 3.8 Tabular depiction of antibacterial assay by Agave americana
Table 3.9 Tabular depiction of antibacterial assay by Mangifera indica
Table 3.10 Tabular depiction of antibacterial assay by Mentha spicata
Table 3.11 Tabular depiction of MIC assay by Agave americana
Table 3.12 Tabular depiction of MIC assay by Mangifera indica
Table 3.13 Tabular depiction of MIC assay by Mentha spicata
Table 3.14 Tabular depiction of antifungal assay by Agave americana
Table 3.15 Tabular depiction of antifungal assay by Mangifera indica
Table 3.16 Tabular depiction of antifungal assay by Mentha spicata
Table 3.17 Tabular depiction of anticancer assay by Agave americana
Table 3.18 Tabular depiction of anticancer assay by Mangifera indica
Table 3.19 Tabular depiction of anticancer assay by Mentha spicata
Table 3.20 Tabular depiction of antioxidant assay by Agave americana
Table 3.21 Tabular depiction of antioxidant assay by Mangifera indica
Table 3.22 Tabular depiction of antioxidant assay by Mentha spicata
Table 3.23 Tabular depiction of cytotoxic assay by Agave americana
Table 3.24 Tabular depiction of cytotoxic assay by Mangifera indica
Table 3.25 Tabular depiction of cytotoxic assay by Mentha spicata
xii
Table 3.26 Tabular depiction of phytotoxic assay by Agave americana
Table 3.27 Tabular depiction of phytotoxic assay by Mangifera indica
Table 3.28 Tabular depiction of phytotoxic assay by Mentha spicata
Table 3.29 Tabular depiction of insecticidal assay by Agave americana
Table 3.30 Tabular depiction of insecticidal assay by Mangifera indica
Table 3.31 Tabular depiction of insecticidal assay by Mentha spicata
Table 3.32 Tabular depiction of anti-termite assay by Agave americana
Table 3.33 Tabular depiction of anti-termite assay by Mangifera indica
Table 3.34 Tabular depiction of anti-termite assay by Mentha spicata
Table 3.35 Tabular depiction of acetylcholine esterase inhibition by Agave
americana
Table 3.36 Tabular depiction of acetylcholine esterase inhibition by Mangifera
indica
Table 3.37 Tabular depiction of acetylcholine esterase inhibition by Mentha
spicata
Table 3.38 Tabular depiction of urease inhibition by Agave americana
Table 3.39 Tabular depiction of urease inhibition by Mangifera indica
Table 3.40 Tabular depiction of urease inhibition by Mentha spicata
Table 3.41 Tabular depiction of hemagglutination assay by Agave americana
Table 3.42 Tabular depiction of hemagglutination assay by Mangifera indica
Table 3.43 Tabular depiction of hemagglutination assay by Mangifera indica
FIGURES
Figure 3.1 Graphical depiction of absorbance values of Agave americana
AgNPs
xiii
Figure 3.2 Graphical depiction of transmittance values of Agave americana
AgNPs
Figure 3.3 Graphical depiction of absorbance values of Agave americana
aqueous extracts
Figure 3.4 Graphical depiction of absorbance values of Mangifera indica
AgNPs
Figure 3.5 Graphical depiction of transmittance values of Mangifera indica
AgNPs
Figure 3.6 Graphical depiction of absorbance values of Mangifera indica
aqueous extracts
Figure 3.7 Graphical depiction of absorbance values of Mentha spicata AgNPs
Figure 3.8 Graphical depiction of transmittance values of Mentha spicata
AgNPs
Figure 3.9 Graphical depiction of absorbance values of Mentha spicata
aqueous extracts
Figure 3.10 Graphical depiction of XRD values of Agave americana AgNPs
Figure 3.11 Graphical depiction of XRD values of Agave americana aqueous
extract
Figure 3.12 Graphical depiction of XRD values of Mangifera indica AgNPs
Figure 3.13 Graphical depiction of XRD values of Mangifera indica aqueous
extract
Figure 3.14 Graphical depiction of XRD values of Mentha spicata AgNPs
Figure 3.15 Graphical depiction of XRD values of Mentha spicata aqueous
extract
Figure 3.16 Graphical depiction of EDX values of Agave americana AgNPs
Figure 3.17 Graphical depiction of EDX values of Agave americana aqueous
extract
Figure 3.18 Graphical depiction of EDX values of Mangifera indica AgNPs
Figure 3.19 Graphical depiction of EDX values of Mangifera indica aqueous
extract
Figure 3.20 Graphical depiction of EDX values of Mentha spicata AgNPs
Figure 3.21 Graphical depiction of EDX values of Mentha spicata aqueous
extract
Figure 3.22 Graphical depiction of TD-DTA values of Agave americana AgNPs
xiv
Figure 3.23 Graphical depiction of TD-DTA values of Agave americana
aqueous extract
Figure 3.24 Graphical depiction of TD-DTA values of Mangifera indica AgNPs
Figure 3.25 Graphical depiction of TD-DTA values of Mangifera indica
aqueous extract
Figure 3.26 Graphical depiction of TD-DTA values of Mentha spicata AgNPs
Figure 3.27 Graphical depiction of TD-DTA values of Mentha spicata aqueous
extract
Figure 3.28 Graphical depiction antibacterial assay by Agave americana
Figure 3.29 Graphical depiction of antibacterial assay by Mangifera indica
Figure 3.30 Graphical depiction antibacterial assay by Mentha spicata
Figure 3.31 Graphical depiction of antifungal assay by Agave americana
Figure 3.32 Graphical depiction of antifungal assay by Mangifera indica
Figure 3.33 Graphical depiction of antifungal assay by Mentha spicata
Figure 3.34 Graphical depiction of anticancer assay by Agave americana
Figure 3.35 Graphical depiction of anticancer assay by Mangifera indica
Figure 3.36 Graphical depiction of anticancer assay by Mentha spicata
Figure 3.37 Graphical depiction of antioxidant assay by Agave americana
Figure 3.38 Graphical depiction of antioxidant assay by Mangifera indica
Figure 3.39 Graphical depiction of antioxidant assay by Mentha spicata
Figure 3.40 Graphical depiction of cytotoxic assay by Agave americana
Figure 3.41 Graphical depiction of cytotoxic assay by Mangifera indica
Figure 3.42 Tabular depiction of cytotoxic assay by Mentha spicata
Figure 3.43 Graphical depiction of phytotoxic assay by Agave americana
Figure 3.44 Graphical depiction of phytotoxic assay by Mangifera indica
Figure 3.45 Graphical depiction of phytotoxic assay by Mentha spicata
Figure 3.46 Graphical depiction of insecticidal assay by Agave americana at 12
hours exposure
Figure 3.47 Graphical depiction of insecticidal assay by Agave americana at 24
xv
hours exposure
Figure 3.48 Graphical depiction of insecticidal assay by Mangifera indica at 12
hours exposure
Figure 3.49 Graphical depiction of insecticidal assay by Mangifera indica at 24
hours exposure
Figure 3.50 Graphical depiction of insecticidal assay by Mentha spicata at 12
hours exposure
Figure 3.51 Graphical depiction of insecticidal assay by Mentha spicata at 24
hours exposure
Figure 3.52 Graphical depiction of anti-termite assay by Agave americana
Figure 3.53 Graphical depiction of anti-termite assay by Mangifera indica
Figure 3.54 Graphical depiction of anti-termite assay by Mentha spicata
Figure 3.55 Graphical depiction of acetylcholine esterase inhibition by Agave
americana
Figure 3.56 Graphical depiction of acetylcholine esterase inhibition by
Mangifera indica
Figure 3.57 Graphical depiction of acetylcholine esterase inhibition by Mentha
spicata
Figure 3.58 Graphical depiction of urease inhibition by Agave americana
Figure 3.59 Graphical depiction of urease inhibition by Mangifera indica
Figure 3.60 Graphical depiction of urease inhibition by Mentha spicata
SCHEMES
Scheme 2.1 Flowchart depicting episodes of research exploration
Scheme 2.2 Flowchart depicting steps involved in AgNPs synthesis
xvi
PICTURES
Picture 1.1 (a) Morphology of Agave americana plant
Picture 1.1(b) Zoom version of leaves of Agave americana plant
Picture 1.2 (a) Morphology of Mentha spicata plant
Picture 1.2 (b) Zoom version of leaf of Mentha spicata plant
Picture 1.3 (a) Morphology of Mangifera indica plant
Picture 1.3 (b) Zoom version of leaves of Mangifera indica plant
Picture 2.1 Crude ethanolic, methanolic, acetonic and aqueous fractions plus
green silver nanoparticles from leaves of Agave americana,
Mentha spicata and Mangifera indica
Picture 2.2 Blackish brown solution as productive plant mediated silver
nanoparticles
Picture 2.3 Dark colored purified fine AgNPs powder
Picture 3.1 Reddish pink color manifest positive flavonoids while reddish
purple indicate positive flavonone via Shinoda’s test
Picture 3.2 Green color manifest presence of phenolic compounds via ferric
chloride test while creamy white precipitate indicate positive
phenols via lead acetate test
Picture 3.3 Red color manifest positive steroids via Salkowski’s test
Picture 3.4 Reddish pink color manifest positive glycosidase via Borntrager’s
test
Picture 3.5 Froth production manifest positive saponins via Foam test
Picture 3.6 Green / Yellow color manifest positive carbohydrates via
Benedict’s test
Picture 3.7 Light pink color layer manifest positive proteins via Biuret’s test
Picture 3.8 SEM micrograph of Agave americana AgNPs at 150X
magnification
Picture 3.9 SEM micrograph of Agave americana AgNPs at 500X
magnification
Picture 3.10 SEM micrograph of Agave americana AgNPs at 1000X
magnification
Picture 3.11 SEM micrograph of Agave americana aqueous extract at 150X
xvii
magnification
Picture 3.12 SEM micrograph of Agave americana aqueous extract at 500X
magnification
Picture 3.13 SEM micrograph of Agave americana aqueous extract at 1000X
magnification
Picture 3.14 SEM micrograph of Mangifera indica AgNPs at 150X
magnification
Picture 3.15 SEM micrograph of Mangifera indica AgNPs at 500X
magnification
Picture 3.16 SEM micrograph of Mangifera indica AgNPs at 1000X
magnification
Picture 3.17 SEM micrograph of Mangifera indica aqueous extracts at 150X
magnification
Picture 3.18 SEM micrograph of Mangifera indica aqueous extracts at 500X
magnification
Picture 3.19 SEM micrograph of Mangifera indica aqueous extracts at 1000X
magnification
Picture 3.20 SEM micrograph of Mentha spicata AgNPs at 150X magnification
Picture 3.21 SEM micrograph of Mentha spicata AgNPs at 500X magnification
Picture 3.22 SEM micrograph of Mentha spicata AgNPs at 1000X magnification
Picture 3.23 SEM micrograph of Mentha spicata aqueous extracts at 150X
magnification
Picture 3.24 SEM micrograph of Mentha spicata aqueous extracts at 500X
magnification
Picture 3.25 SEM micrograph of Mentha spicata aqueous extracts at 1000X
magnification
Picture 3.26 TEM micrograph of Agave americana AgNPs
Picture 3.27 TEM micrograph of Mangifera indica AgNPs
Picture 3.28 TEM micrograph of Mentha spicata AgNPs
Picture 3.29 Zoomed TEM micrograph of fabricated green AgNPs
Picture 3.30 Pictorial depiction of antibacterial zone of inhibition formed by test
bacterial species in sensitivity response to green AgNPs and crude
leaves extracts
Picture 3.31 Pictorial depiction of MIC against test bacterial species
xviii
Picture 3.32 Pictorial depiction of antifungal assay against test fungal strains
Picture 3.33 Pictorial depiction of antioxidant activity with reference to DPPH
Picture 3.34 Pictorial depiction of phytotoxic activity against Lemna minor
Picture 3.35 Pictorial depiction of insecticidal activity against selected test
insect species
Picture 3.36 Pictorial depiction of anti-termite activity against Formosan
subterranean termite
Picture 3.37 Pictorial depiction of hemagglutination activity against ABO blood
group
1
ACKNOWLEDGEMENTS
All praises are for Almighty Allah, the most beneficent, the most merciful who
bestowed upon me with the sight to observe, the mind to think and the courage to work
more and more. Peace and blessing of Allah be upon the Holy Prophet (S.A.W) who
exhorted his follower to seek the knowledge from cradle to grave.
It is my privilege and honor to be a student of Prof. Dr. Bashir Ahmad, Centre for
Biotechnology and Microbiology (COBAM), University of Peshawar (UOP). I wish to
express my deepest gratitude for his expert guidance, appreciation and sincere advice,
marvelous and ongoing support during the period of this research work. His endless
encouragement and familiar deeds have been the major driving force throughout my
research career.
Words fail me to acknowledge the gratitude of my beloved mother (Melba Tan
Morilla), my brothers (Mansoor Ali Shah & Yousaf Ali Shah) and my spouse (Salman
Shehzada) for accepting and supporting my ambition. Without them I would have
never achieved this far.
I am extend my gratitude to Directorate Science & Technology (DOST) for their
financial support throught out the research work.
I am thankful to Dr. Javed Khan, PCSIR Laboratory, Peshawar, Dr. Ibrar Khan,
Assistant professor COBAM, UOP and Mr. Noshad, lab assistant COBAM, UOP for
being helpful during entire research period.
Last but not the least I am thankful to all my lab colleagues who succor and guided me
during my study at different occasions.
FARAH SHIREEN ALI SHAH
2
Abstract
Green silver nanoparticles were biosynthesized utilizing aqueous leaves extracts of Agave
americana, Mangifera indica and Mentha spicata plants due to presence of pre-eminent reducing
and stabilizing phytochemicals. These biocompatible nanostructures were purified, optimized
and characterized as mildly neutral, polycrystalline, monodispersed and thermally sensitive
compounds owing predominantly spherical shape and 30–150 nm diameter. Biological/
pharmacological analysis in comparison to crude ethanolic, methanolic, aqueous and acetone
leaves extracts evinced stupendous antibacterial activity against all pathogenic experimental
bacteria particularly E.coli and MRSA. Evaluated MIC values for AgNPs and crude leaves
extracts against experimental bacteria lies in the range of 40–320 µL. Green AgNPs remarkably
inhibited the growth of all test fungal mycelia while moderate fungal growth inhibition was
demonstrated by crude leaves extracts. Good anticancer activity was demonstrated by A.
americana AgNPs (69%) and acetone extracts (78%) while remaining AgNPs and extracts
moderately inhibited prostrate tumor proliferation. Robust antioxidant activity was demonstrated
by AgNPs and crude leaves extracts at highest sample concentration of 300 µL. In parallel,
significantly exalted cytotoxic, phytotoxic, anti-termite, insecticidal activity was exhibited by
bioinspired AgNPs and crude leaves extracts at highest sample concentration of 1000 µL.
Moderate enzyme inhibition was manifested against acetylcholine esterase and urease. Finally
absence of phyto-glutinins was evinced by negative hemagglutination reactions.
3
INTRODUCTION AND LITERATURE REVIEW
1.1 GENERAL INTRODUCTION
The term “Nanotechnology” refers to the state of the art of precise manipulations at the atomic
scale to construct potentially crucial products called “Nanoparticles” [1]. Nanoparticles are
regarded as fundamental building blocks which are prominently of small size with larger surface
area to volume ratio. Till date, nanoparticles produced such as silver (Ag), gold (Au), platinum
(Pt) and palladium (Pd) have been investigated adequately and being utilized in various areas of
medicine, electronics, catalysis, environmental and biotechnology [2 , 3]. Several organizations
have invested billions in the area owing to know its great potential. National Nanotechnology
Initiative Foundation, Japan for instance has funded $ 750 million, European Union (EU) $ 1.2
billion and United States of America (USA) $ 3.7 billion dollars to support the research in this
field [4].
Nanobiotechnology or nanobiology is an umbrella term that unifies physical and chemical
methods with biological principles to synthesize eco-friendly nanoparticles in economic ways
[5]. The trendy concept of nanobiotechnology has expanded in forms of nanoparticles, nanotools
and nanoscale phenomena. Biogenic nanoparticles serve as innovative foundation for novel
medicine which extends the prospects from symptomatic and restorative therapy to tissue
regeneration. Especially, silver nanoparticles have been used in many iatric and industrial sectors
[6].
Currently, many physical and chemical strategies are available for mass fabrication of silver
nanoparticles but many of these have undesired drawbacks such as high cost, use of toxic
4
solvents, synthetic additives and high energy requirements for maintaining constant high
pressure and temperatures [7].
A novel concept of green nanotechnology has evolved where nanoparticles are mass produced
using whole plant or plant parts. The basic concept originates from medicinal plants that
naturally produce effective therapeutic ingredients otherwise exhausting to be created in-vitro.
These ingredients can be directly isolated from their natural plant source and structurally
modified into formulations for many restorative therapies [8].
Asians, Romans, Greeks and Babylonians documented their own traditional therapeutic
approaches. These traditional theories instituted the foundation of modern practices [9].
Preference to herbal remedies over synthetic drugs is shown to be safer for human use because it
acts like vegetable and eliminates the risk of harmful side effects [10].
Our country, Pakistan is bestowed with potentially vital herbalism and approximately 120
different analeptic compounds are extracted from these medicinal herbs. These compounds are
exploited by the pharmaceutical industry to manufacture drugs for many challenging ailments
[11].
Hence, the synergism of these dynamic botanic constituents along with the silver ions will open
new perspective to produce eco-friendly nanoparticles in economic manner. This technology will
serve researchers across the globe due to its economically productive processing and prevalent
medicinal properties with minimal side effects.
5
1.2 BOTANICAL DESCRIPTION
The plants selected for research study are explained as follows:
1.3 AGAVE AMERICANA (PLANT)
1.3.1 Description
Kingdom: Plantae
Class: Angiosperms
Order: Asparagales
Family: Asparagaceae
Genus: Agave
Species: Agave americana
Agave americana is commonly known as maguey, century plant and American aloe in English
while Elwa in Urdu [12]. A. americana is monocotyledon and semelparous with life span of 10 -
30 years. The useful part is a modified stem. The rosette forming leaves are usually green but
most often glaucous having polychromatic borders, stretching about 1 m long, broad at the middle
and curving outwards. The apical spines are usually blackish-maroon and about 1 cm in size. The
marginal spines are facing downwards [13]. Morphology is depicted in (Picture 1.1 a , b).
6
(a) (b)
Picture 1.1: (a) Morphology of Agave americana plant
(b) Zoom version of leaves of Agave americana plant
7
1.3.2 Distribution
A. americana native to Mexico is a drought resistant and ornamental plant. Presently, it is
cultivated worldwide, and has naturalized in American, African, Mediterranean and Asian
regions including Pakistan [14].
1.3.3 Importance
A. americana is species of flowering plant in the family Agavaceae. The heart of flowering stem
accumulates a saccharine sap termed “aguamiel”. This sap is fermented to manufacture a milky
alcoholic beverage called “pulque” that possesses a pungent acidic flavor with viscous
consistency. A. americana is also source of one of the most notable distilled spirit, Tequila. This
type of hard liquor is inexpensive and consumed widely in European countries. Tequila is
manufactured by the mechanical heat extraction of plant sugars, which is carried out in ovens.
Pita, a durable leaf fiber of A. americana is used for making mats, coarse fabrics and ropes. It is
also used in “piteado” a form of leather embroidery. The major source for income in Mexico
comes from brewery and pita [15]. The heart of flower stalk is edible and nutritious as it contain
profuse amount of sugary matter. Commonly, it is baked or roasted and consumed like
Asparagus. Powdered seeds are used to make breads and cereals. It is also used as a thickening
agent in gravies and soups [16]. The sap “pina” of A. americana contains 16% fructans and 25%
inulin at leaf base. According to the reports, inulins extracted from A. americana are preferred
over that from Chicory by food industries owing to their low price and high water solubility.
They are used as low glycemic sweetners, food additives and fat substitute [17]. Gamma inulin is
used in the assembly of microspheres for the development of drug delivery vectors. It has been
tested successfully to be used as vaccine adjuvant [18]. It also acts as a prebiotics to maintain
8
healthy gut microflora and prevent colon cancers. Fructans from A. americana have been
investigated to function as hormone system modulator, blood cholesterol reducer, mineral
promoter particularly calcium and magnesium [19]. The plant extracts exhibit remarkable
antimicrobial potency against different pathogenic bacterial and fungal strains due to the
presence of a bioactive compound “triacontanol”. Approximately, 5 mg/ml leaf extract has
equivalent activity as compared to prime antibiotics such as streptomycin and griesfluvin. The
most susceptible species are Escherichia coli, Staphylococci, Pseudomonas and Alternaria
brassicae [20]. Along with antimicrobial potentials, crude leaf extracts also exhibit anti-
inflammatory, insecticidal, molluscacidal, antitumor, antidiabetic, antifouling, immuno-
regulatory and cardio-protective properties. Leaf extracts consists of vital enzymes called “angio-
tensin converting enzymes” that aids to medicate hypertension. Leaf extracts also consists of
various sapogenins such as “tigogenins” and “hecogenins” that are precursors for steroid
production [21]. Detergent industries exploit root and leaf extracts for the presence of active
compound “saponins”. The dense flesh of leaves is used for manufacturing facial petroleum jelly
while erect piercing thorns are used for needles and pins. Dried flowering stems are utilized as
razor strop and water proof thatch. In Mexico and Africa, the plant is employed to mark
territories as it dominates the land by ubiquitous proliferating nature. It is also used in
management of eroded and over-grazed lands. [22].
9
1.4 MENTHA SPICATA (PLANT)
1.4.1 Description
Kingdom: Plantae
Class: Angiosperms
Order: Lamiales
Family: Lamiaceae
Genus: Mentha
Species: Mentha spicata
Mentha spicata commonly known as Spear-Mint in English while Podina in Urdu [23].
It is green, acceptably fragrant and perennial herb. Stems are glabrous, quadrangular, apically
branched and about 30 – 60 cm in size. Leaves are serrated, glabrous, cuneate broadly, gland-
flecked underneath and tapering to the ends. Petioles are absent or approximately 2 – 6 × 0.5 –
1.5cm in size. It has an extended verticillasters are structured outlying or above. The bracts are
lanceolate and are usually short or can extend to the size of flowers. Calyx is also gland-
flecked, glabrous and about 1.5 – 2 mm in size. Restrictedly triangular eglandular hairs on teeth
are often present on calyx, which are equal. White or pastel pink colored corolla is present and
about 2.5 mm in size. Nutlets are brown [24]. Morphology is depicted in (Picture 1.2 a, b).
10
(a) (b)
Picture 1.2: (a) Morphology of Mentha spicata plant
(b) Zoom version of leaf of Mentha spicata plant
11
1.4.2 Distribution
M. spicata was initially discovered in 1843 AD, in the zones of Great Lakes, North America.
The herb prefers to proliferate in partial shady and organically moist areas. It is indigenous to
South-West Asia and Europe, but presently it is naturalized all over the planet [24].
1.4.3 Importance
Generally spearmint favors all temperate climates. It is often cultivated as ornamental plant in
pots due to its invasively expanding roots and revitalizing aroma. However, the leaves lose their
fragrant appeal as the plant borne flowers. They can be utilized in fresh, frozen and dried form.
Different sun or shade drying methods are applied for dehydrating purpose. The herb can also be
pickled in oil, alcohol, sugar and salt syrups for long term use [25]. Its potential compounds
abundantly present in plant oils include R-carvone, dihydrocarvone, limonene and 1, 8-cineol,
which provide it carminative and aromatic effects. While compounds such as menthone and
menthol are present in minimal amounts that are used in confectioneries, soaps, shampoos,
mouth washes and tooth pastes. The herb is also used as a cardinal element of Moroccan
Touareg tea, which imparts limpid subtle aroma to the refreshment. It is also used as a flavoring
agent in many summer beverages such as sweet ice-teas and potations like Mint Julep and Mojito
[25]. Studies proclaimed that tea of M. spicata leaves possess anti-androgenic effects that can be
exploited as a remedy for hirsutism in females without fluctuating total androstenolone and
testosterone levels in the body. However, experimental analysis on male rodents revealed that
administration of spearmint tea causes antipathetic reactions on their reproductive systems. It
also generates lipid peroxidation that leads to hepatic and renal dysfunctions. Hence its toxicity
is accountable in males [26]. From the previous research investigations, it has been testified that
at the dilution of 1/100 and 1/1000, the M. spicata extract exerts intense bactericidal against
12
many pathogenic Gram positive and Gram negative bacteria such as Bacillus subtilis,
Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus species. While at
dilution of 1/50,000, M. spicata extract shows 95% fungicidal activity against pathogenic fungi
such as Malassezia furfur, Trichophyton rubrum, and Trichosporon beigelii. M. spicata has also
been reported to have eminent antioxidant activity, which is employed by food industries to
restrain the formation of toxic substances and delay fat oxidation during processing of meat
products [27]. Spearmint extract also possess significant mosquitocidal and insecticidal activities
that are endorsed for many agricultural applications [28 , 29]. The spearmint extract can be
utilized to treat many gastric discomforts, insomnia and ageing due to its calming outcomes [30].
Despite having dynamic potentials, its mutagenic effects have not been described to date [31].
13
1.5 MANGIFERA INDICA(PLANT)
1.5.1 Description
Kingdom: Plantae
Class Angiosperm
Order: Sapindales
Family: Anacardiaceae
Genus: Mangifera
Species: Mangifera indica
Mangifera indica commonly known as Mango in English and Aam in Urdu.
The mango tree is glabrous and grows up to 15 m. Leaves are dark green, acuminate, coriaceous,
lanceolate, oblong and glossy on the exterior and about 11 – 24 cm in length and 4 – 8 cm in
breadth. Flowering panicles are longer than leaves and are erect, pubescent and conspicuous.
Calyx lobes are exteriorly pubescent and ovate. Corolla is oblong, imbricate and has three interior
nerves. Drupe is constricted, ovoid and about 3.5 – 20 cm in length. Mesocarp is fleshy while
endocarp is fibrous and stiff [32]. Morphology is depicted in (Picture 1.3 a, b).
14
(a) (b)
Picture 1.3: (a) Morphology of Mangifera indica plant
(b) Zoom version of leaves of Mangifera indica plant
15
1.5.2 Distribution
M. indica fruit is the national fruit of Philippines, Pakistan and India. It is local to India and
Burma but now it is tropically outspreading across the globe [33].
1.5.3 Importance
A therapeutically compelling flavonoid, mangiferin is obtained from the parts of the plant.
Estimated concentrations from old leaves, bark and young leaves are 94 g/kg, 107 g/kg and 172
g/kg respectively [34]. This phyto-active compound possesses multifarious preventive and
remedial properties such as antimicrobial, antiviral, antioxidant, anti-inflammatory, anti-
diabetic, anti-sclerotic, anticancer, anti-radical, radio-, cardio-, hepato- and neuro- protective
capacities. It also acts as frisky iron chelators to prevent Fenton- type reactions [35]. M. indica
plant has been used in Ayurvedic medicine for over a century by folk medicine practitioners to
treat acidity and digestive disorders along with other herbs particularly Asparagus racemosus
and Tinospora cordifolia [36]. Dried flowers are also utilized in herbalism to medicate chronic
dysentery, diarrhea and bladder disorders. While juice of fresh flowers consumed with curd is
also reported to treat diarrhea symptomatically [37]. Mango fruit possesses insoluble fibers that
function as prebiotics, which maintains healthy colon and gut flora by curbing constipation.
They also alkalize and energize the body. They also function as anticancer agent particularly
shielding against breast, colon, prostate cancer and leukemia. Fruits also own cardio-protective,
gastro-protective, immunomodulatory, and anticholesteremic potentials due to the presence of
flavonoids, fibers, ascorbic acid, pectins and indispensible enzymes [38]. Roasted ripe mango
juice also act as cough reliever. Research investigations revealed that consuming aqueous leaf
extracts or shade dried leaf powder twice a day assist to control diabetes in early onset of
affliction [39]. While aqueous bark extracts along with black salt is helpful to treat diarrhea. It
16
is also reported that leaves ash is applied on dermal burns to assist speedy recovery.
Decorticated kernel paste of M. indica revealed to assist in vaginitis and leucorrhea and may
also act as contraceptive. Tooth pastes produced from mango kernel is believed to strengthen
gums [40].
17
1.6 NANOTECHNOLOGY
1.6.1 Background
The concept of nanotechnology was pioneered by a prominent physicist, Richard Feynman in
1959 where he described the feasibility of configuration of matter at atomic scale as “There's
Plenty of Room at the Bottom”. The term “nanotechnology” was coined in 1974 by Norio
Taniguchi [41]. Further an American Engineer, Kim Eric Drexler in 1986 brought forward the
principles of nanotechnology via his book entitled as “Engines of Creation: The Coming Era of
Nanotechnology”. Persuaded by the Feynman’s objective, Drexler debated about the replication
of complex matter by atomic control as “nano-scale assembler”. In the same year, Drexler co-
sponsored an association, widely known as “The Foresight Institute” to promote and implement
nano-science in day to day technologies. His contributions laid the theoretical and experimental
foundations for the discipline nanotechnology. Motivated by Drexler’s enlightenments, in 1989
customization of atoms via scanning tunneling microscope (STM) took place positively. Later
atomic force microscope was also utilized for the same purpose [42]. In 1985 at William Marsh
Rice University, Robert Curl, Richard Smalley and Harry Kroto discovered a hollow carbon
molecule called Fullerene, for which they were awarded Nobel Prize in Chemistry in the year
1996. This C60 molecules was proposed to be employed in multitude of nano-electronic devices
exclusively as carbon nano-tubes, commonly known as bucky tubes or graphene tubes [43 , 44].
Enterprising progress in nano-tech products initiated in early 2000s, which commenced the
exploitation of silver nanoparticles as potential antimicrobial in pharmaceutical, textiles and food
industries. Later on in mid 2000s, nano-research was financed by United States National
Nanotechnology Initiative firm, through which projects of atomically operated materials were
designed, manufactured and serviced to benefit the community [45].
18
1.6.2 Current Status
With the advancements in science and technology, the enactment of nano-articles is tangible.
Since nano-tubes can be a promising packaging material, they are used by American automobile
companies and electronic industries to upgrade security of fuel and electricity networks. Among
others, cosmetics industry is also the topmost to implement theories of nanotechnology into
practice. By 2009, thousands of nanotechnology-based items were registered and merchandised
worldwide, including approximately 13% for cosmetic purposes. As per the research survey of
United States and Europe, between 2000 and 2010, nano-materials have grown in many medical
feilds such as dental, dermatology and pharmacy. The product consists of active formulations
and carriers that increase its competence [46]. Every year about 2 billion dollars are subsidized
on nanotechnology research and development. Of this fund, 40% is capitalized by United States
of America, rest is contributed by China, Japan, Singapore, Taiwan and European countries [47].
According to the study of Chuankrekkul, the diversity of nano-products trades globally, which
notably includes garments, electronics and health care products. Multiple metal nanoparticles
such as of silver, carbon, silica, titanium dioxide, zinc and gold are utilized for these purposes. It
is estimated that approximately 95% of silver nanoparticles are availed in product
manufacturing, followed by carbon nanoparticles (43%), silica nanoparticles (24%), titanium
dioxide (19%), zinc (18%) and gold (12%). The United States provides 52% of nanotech
consumer products internationally, while the rest is served by European and Asian companies
[48].
19
1.7 NANOBIOTECHNOLOGY
1.7.1 Background
In the course of 19th century, researchers struggled to begin with uniting engineering principles
of nano-tools with biological sciences. Although the aim was consistent, it called for a lot of
disagreements as erstwhile nano-materials were of inorganic origin and hydrophobic in nature
unlike the organic biotic systems. The field of “nanobiotechnology” or “nanobiology” ushered in
early 1990s, by scientific conventions and endurance, to blend physical and chemical properties
along with biological principles to create cost effective and environment friendly nanoparticles
[49]. These advancements enticed the scientists across the globe and many functional nano-
devices were fabricated according to hybrid principles of nanobiotechnology. For example,
nano-gadgets specifically ATP dependent organic motor and nano-electronic mechanical systems
were assembled by integration of synthetic and biological components [50].
1.7.2 Current Status
The novel domain of nano-biotechnology has been practice widely. It substantiates quality
pathways that are serviceable to humankind. In the field of medicine, inventions of nano-robots
have established mechanisms to treat fatal diseases such as cancer. This technology will help in
targeted treatment thus eliminating the risks of side effects that are caused by current chemo and
radiotherapies [51]. Nanobiotechnological procedures are modernizing the realm of medicine
from symptomatic remedies to sparking off cures. Organ culture has also been getting
materialized due to nano-biological strategies. Now it is possible to culture anthropoid uterus,
bladders, heart and limbs utilizing patient tissues. This strategy eliminates lethal health menace
due to distinct body rejections and absence of compatible donors [52]. The development of
fluorescent nano-sphere polymers can aid in diagnosis of pathological metabolites, tumors and
20
bacteria [53]. Self-assembling nano-tubes can generate and store biological data of living
organisms in the form of biochips that will enable optical computing process for future
proteomic programming. Lipids, proteins and synthetic membranes nanotechnology open new
dimensions for bioinformatics. This provides significant knowledge related to assembly, 3D-
folding, physiochemical properties and mutations of molecules that are fundamental for accurate
disease diagnosis and efficacious personalized cures [54 , 55]. The field of nanobiotechnology
also provides insights to many experimental tools such as optical tweezers, atomic force
microscopy for imaging, recombinant DNA technology and dual polarization interferometry for
self assembly and synthesis analysis, supercomputing, nanomechanics and multi-scale simulation
for computational analysis [56].
21
1.8 SILVER
1.8.1 Background
Silver (Ag) is a white and effulgent metallic element having intense thermal and electrical
conductivity along with reflectivity. It has been remarked for over a millennium for its
diversified utilization in commerce, costume, jewelry and utensils [57]. The reference goes back
to 400 B.C. as mentioned in the Book of Genesis, silver was extracted and refined from lead ores
from surface mining in Sardinia, Europe. The Roman economy was highly dependent on
business of silver bullion [58]. Estimated peak production of 200 tons per year was recorded by
Romans by 200 A.D. Many silver mines in Laureion were constructed during 438 B.C. [59]. Till
19th century, it was a main stock exchanger for Chinese Empire [60]. In early Bronze Age (60–
120 A.D.), a high temperature silver-lead cupellation technology was developed by pre- Inca
population of America [61].
Due to copper shortage during World War II, silver replaced its position in copious industrial
manufacturing such as engineering bus bars and bearings for aeronautics, connectors and
switches for electrical appliances, reflector for search light and other lightening systems, tin
solder and metal alloying particularly nickel alloying for metal quality enhancement.
Approximately 14,700 tons of metallic silver were acquired from United States Treasury to
process electromagnetic separation for Manhattan Project at Oak Ridge National Laboratory,
United States [62].
1.8.2 Current Status
Silver is used for several purposes in the world. It has been a ruling component of monetary
system till now. Silver alloys such as sterling silver (93% silver with 7.0% copper), britannia
silver (95% silver and 5.0% copper) and argentinum sterling silver (95.8% silver and balanced
22
amount of copper and germanium) are utilized in many silverwares and jewelry items due to hall
mark standard such as durability, resistance to corrosion and higher melting points (961.8oC).
These alloys can be polished further through flashing process by thin layer plating with 99.9%
fine silver, gold or rhodium to impart burnished appearance [63]. Silver as a reflective agent was
hired to construct analogous mirror panels, solar plasmonic batteries and photovoltaic panels
[64]. They are also utilized in water purifiers, textiles, electronics, glass coatings, microscopy,
and photography [65]. In the field of medicine, silver is well known for its antimicrobial potency
and therefore, it is widely applied as antiseptic in wound dressings as well as in medical
appliances such as catheters, nasogastric tubes, cardiac stent, etc. Silver is also applied in dental
amalgams to restore oral cavities [66].
Recently, silver nanoparticles (AgNPs) became appealing subject as this innovation shrinks the
mass of silver for a particular project. According to reports, they are successfully used to
manufacture conductive inks, super capacitors, radio frequency identification tags, electrodes
and light weight batteries. The versatility of nanotechnological applications is sought to be broad
as the products manufactured are cost effective and heavy duty [67].
23
1.9 SYNTHESIS, CHARACTERIZATION AND OPTIMIZATION OF
SILVER NANOPARTICLES
1.9.1 PHYSICAL APPROACH
Silver nanoparticles (AgNPs) were initially synthesized most predominantly by physical
approaches such as evaporation-condensation, laser ablation, arc discharge, and direct metal
sputtering methods, which are described below.
1.9.1.1 Evaporation-Condensation Strategy
Evaporation-condensation method has been proved to be the cynosure of physical approaches as
this advances to yield thermally stable AgNPs excluding the expense of time and energy. The
small ceramic heater instead of large tube furnace is used to facilitate evaporation of source
materials during AgNP synthesis. Further, the vapors are condensed by local heating body that
creates temperature gradient in the vicinity [68].
According to earlier studies, this method largely generates stable spherical nanoparticles that
range from 1.2 to 1.8 nm and 6.2 to 22 nm in diameter; and are resistant to agglomeration even at
higher temperatures [69]. These nanoparticles can be used for long-standing experiments such as
modeling nanoparticle estimation devices and inhalation toxicity studies [70].
1.9.1.2 Laser Ablation Strategy
AgNPs can also be mass produced by another physical approach called laser ablation. The
attributes of nanoparticles and efficacy of this technique depends on several criteria such as
provision of surfactants, nature of liquid medium, wavelength and volubility of laser streaks, and
24
ablation period (nanosecond, picosecond or femtosecond) [71 , 72]. Nanoparticles synthesized by
this technique are truly veritable as they are produced in chemical-free environment [73].
According to the earlier studies, exposure to laser beams at 800 nm for femtosecond generates
silver nanospheroids aqueously, ranging from 20 to 50 nm in diameter. However, in comparison
with femtosecond, the duration of nanosecond momentously increases the ablation efficacy in air
and water and gives less dispersed colloidal structures to nanoparticles [74 , 75].
1.9.1.3 Arc Discharge Strategy
Arc discharge method for the synthesis of AgNPs was first introduced by Tien et al. [76]. In this
method, silver wires (Gredmann, 1 mm in diameter and 99.99% pure) were used as electrodes
and immersed in deionized water in the absence of surfactants. AgNPs synthesized by this
method were 10 nm in diameter while the concentration of ionic silver recovered was estimated
as 11–19 ppm.
1.9.1.4 Direct Metal Sputtering Strategy
Direct metal sputtering method for the synthesis of AgNPs in an aqueous medium was first
introduced by Seigel et al. [77]. This method amalgamates physical deposition of metal ions
into glycerol, and generates round-shaped nanoparticles with estimated diameter of 2.4–3.5 nm.
It was also demonstrated that in diluted solutions, particle dispersion and size distribution remain
constant, thus yielding significant amount of nanoparticles even up to 1:20 glycerol-to-water
ratio.
25
1.9.2 CHEMICAL APPROACH
Chemically, AgNPs are synthesized predominantly by the use of many synthetic polymers and
polysaccharides through the multiple approaches such as chemical reduction, micro-emulsion
technique, UV-initiated photoreduction, and microwave-assisted synthesis, which are explained
as below.
1.9.2.1 Chemical Reduction Strategy
Chemical reduction is one of the most integrated and effective practice for the synthesis of
AgNPs in aqueous and nonaqueous solutions. Commonly procurable natural and synthetic
reducers are ascorbic acid, hydrogen ion, N,N-dimethyl-formamide, sodium borohydride, sodium
citrate, polyol, polyethylene glycol, and Tollen’s reagent, which reduce Ag+ ions to metallic Ago
particles. Certain surfactants and stabilizers, such as alcohols, amines, acids, thiols and polymers,
such as polyvinyl-pyrrolidone, polyethylene-glycol, polyvinyl-alcohol and para-
methoxyamphetamine are applied to preclude agglomeration, sedimentation, and loss of
functionalities [78 , 79, 80].
According to a recent study in 2013, Oliveira and his colleagues prepared dodecanethiol-capped
AgNPs, in which dodecanethiol acted as a competent stabilizing and solubilizing agent. These
refined nanoscopic particles were further analyzed for structure, distribution, and self-assembly
patterns [81, 82]. In additions, monotonously sorted and dissipated AgNPs were synthesized by
the co-processing of polyol and precursor injection technique. This synthesized nano-silver was
characterized as ≈17 nm in diameter and was balanced even at higher reaction temperatures [83].
At room temperature, AgNPs can also be chemically mass produced by simply reacting Ag+ ions
26
with poly-oxometalates, which acts as stabilizing and reducing entities. These nanoparticles
exhibit exclusive stable narrow distribution morphology in aqueous solutions [84].
1.9.2.2 Micro-emulsion Strategy
AgNPs can be prefabricated through duo-juncture reaction, which is established on spatial
isolation of reactants, that is, reducing agents and metal precursors into two immiscible phases.
Alkyl ammonium salt is used as a chemical mediator between the two solvent states, and stable
silver nano-clusters are generated at the interface [85].
Micro-emulsion technique uses noxious organic solvents that limit practice use by industries, but
later chemist Zhang and colleagues plied dodecane as oleaginous phase, which minimized
overall practice toxicity [86].
1.9.2.3 Ultra violet-initiated Photo-reduction Strategy
This is one of the most unambiguous and manageable chemical practice described for AgNP
synthesis. This couple irradiation along with potential stabilizing chemicals such as citrate,
collagen, polyacrylic acid, and polyvinyl pyrrolidone. According to a study, Ago was fabricated
by ultraviolet irradiation of AgNO3. The photoreduced bimodal nanoparticle diameter was quite
sizeable at exposure for 3 h, but advance exposure crumbled the AgNPs into preferred small size
owing to stable unimodal distributive patterns [87]. Laponite mineral clay suspension was used
as protective AgNP coatings to pre-empt clumping. Some studies elucidated the fabrication of
AgNP dendrites, nanospheres, and nanowires by photoreduction along with polyvinyl alcohol as
a fortifying agent [88].
27
1.9.2.4 Microwave-assisted Strategy
Microwave-assisted strategy is a promising synthetic pathway for the generation of AgNPs as
this practice eliminates the application of traditional oil bath; which results in minimal energy
dissipation and reaction period. The synthesized AgNPs were characterized as nanoscopic small
particles along with positive distribution and crystallization standards [89].
Typically utilized stabilizers include carboxymethyl cellulose, oligochitosan, starch, trisodium
citrate, polyethylene glycol, and formaldehyde [90, 91]. These stabilizers generate controllable
unvaried spherical AgNPs at room temperature and optimized pH of 9.0 [92]. The sizes of
AgNPs analyzed by transmission electron microscopy (TEM) were about 12 nm for starch, 15
nm for oligochitosan, and 62 nm for polyvinyl pyrrolidone as a reducing agent [93, 94].
28
1.9.3 GREEN APPROACH
According to several research works conducted, researchers have declared physical and chemical
practice pernicious and costly. To contend with the concerns, bio-based synthesis of AgNPs has
been proposed across the globe [95]. This method predominantly used biological systems such as
microbes, algae, and plants. Biological aspects such as class and genetics of organism, optimum
setup for enzyme function and cell proliferation, and application of favorable biocatalysts are
responsible for bulk production of economical and eco-friendly AgNPs [96]. Microbe, algae, and
plant-mediated syntheses of AgNPs are explained below.
1.9.3.1 Microbe Mediated Strategy
Microbial cultures such as Aeromonas, Bacillus, Corynebacterium, Enterococcus, Escherichia
coli, Fusarium, Klebsiella, Lactobacillus, and Pseudomonas species have been documented to
promote biosynthesis of AgNPs. According to the literature, supernatant cultures of Bacillus
subtilis and Bacillus licheniformis can potentially reduced Ag+ ions to Ag0 particles in aqueous
solutions. The yielded nanocrystals were characterized to be monodispersed and approximately
5–50 nm in diameter [97 , 98]. Particle aggregation was repressed by coupling microwave
irradiation technique to improve overall AgNP performance [99].
According to another study, AgNPs were intracellularly synthesized by Pseudomonas stutzeri
strain AG259, which, according to TEM analysis, were vacillating triangular, hexagonal, and
equilateral crystal topologies ranging from 35 to 46 nm in diameter. The nanocrystals were
precipitated in the periplasmic space of the cell by bacterial detoxification mechanism [100 ,
101].
29
Species of Lactobacillus present in the whey of buttermilk have been reported to biologically
produce AgNPs by reducing silver ions into AgNPs. Bioreduction of Ag+ ions takes place in
bacterial cell wall by the action of potential enzymes and saccharides. Lactobacillus casei was
reported to synthesize isolated spherical NPs of about 25–50 nm in diameter or clusters of 100
nm in diameter. Stability of the particles was governed by enzymes present in the cytoplasmic
membrane of the bacterial cell [102]. Studies revealed that the presence of nitro-reductase
enzyme in Aeromonas, Corynebacterium, E. coli, Enterobacter cloacae, and Klebsiella
pneumoniae cultures is responsible for the successful production of stable monodispersed AgNPs
in a speedy reaction time of about 5 min [103 , 104 , 105].
Moreover, fungal strains such as Aspergillus, Coriolus, Cladosporium, Fusarium, Penicillium,
and Phanerochaete sp. have been reported to extracellularly biosynthesize AgNPs due to the
presence of NADH-dependent nitro-reductase enzyme [106]. According to previous research
findings, Fusarium oxysporum and Fusarium acuminatum can prevalently produce AgNPs,
within 20 min, of different shapes from typical spherical to irregular triangular, pyramidal, and
hexagonal [107 , 108, 109]. Electron micrographs revealed that the diameter of stable
monodispersed AgNPs broadly ranges from 10–25 nm. Stability of the particles was rendered by
cellular proteins such as pH-dependent cytochrome c. Optimum pH recorded for protein was
≥12, as acidic pH denatures the proteins, thus affecting overall AgNP structure and function
[110].
As stated by early literature, real pathogenic fungi such as Aspergillus flavus, Aspergillus
fumigates and Cladosporium cladosporioides also produce spherical silver nanostructures
ranging from 5 to 25 nm in diameter according to scanning electron microscopy (SEM)
30
micrographs. The cellular components, such as organic acids, proteins, and polysaccharides,
were superintended to reduce silver ions into AgNPs [111 , 112].
Turkey tail mushroom, i.e., Coriolus versicolor or Polyporus versicolor, was also investigated to
comprise cardinal reducing elements such as glucose and thiol group of proteins that yield
monodispersed spherical AgNPs under alkaline pH, that is, ≥10 under reaction period of 1 h
[113].
1.9.3.2 Algae Mediated Strategy
There are hardly few documentations available corroborating algae-mediated synthesis of
AgNPs. In an earlier report, AgNPs were green synthesized by Chlorella vulgaris, Oscillatoria
willei, and Spirulina platensis at slightly acidic pH and room temperature [114, 115, 116].
Nanorods synthesized with the help of C. vulgaris were 45 nm in length and 16–24 nm in
breadth, while spheroids synthesized with the help of O. willei and S. platensis had a diameter of
100–200 nm and 7–16 nm, respectively. Reduction and stability to the AgNPs were conferred by
carboxyl moieties in Asp/Glu residues and hydroxyl moieties in Tyr residues [117].
1.9.3.3 Plant Mediated Strategy
In comparison with all other physical, chemical, and biological techniques involving microbes
and algae, plant-mediated synthesis has been proved to be eco-friendly and reasonable due to
ubiquitous availability, up-surged medicinal properties, accelerated upstream processing, and
decelerated downstream AgNP processing [118].
According to previous literature, leaf broths of Acalypha indica (Indian nettle) [119], Camellia
sinensis (green tea) [120], Capsicum annuum (chili pepper) [121], Cymbopogon flexuosus
(lemon grass) [122], Datura metel (metel or Devil’s trumpet) [122], Diospyros kaki (persimmon
31
or Sharon fruit) [123], Euphorbia hirta (asthma plant) [124], Eucalyptus citriodora (lemon
eucalyptus) [125], Ficus bengalensis (Indian banyan or figs) [125], Ginkgo biloba (maiden-hair
tree) [123], Garcinia mangostana (purple mangosteen) [126], Medicago sativa (Alfalfa) [122],
Magnolia kobus (Kobushi magnolia) [123], Ocimum sanctum (holy basil or tulsi) [126],
Pelargonium graveolens (geranium) [122], Pinus densiflora (red pine) [123] and Platanus
orientalis (oriental plane) [123] have been reported to synthesize AgNPs due to abundant
presence of potential phytocompounds such as polyphenols, polysaccharides, proteins, alkaloids,
alcoholic molecules, and flavonoids. These biomolecules act as decisive reducing and capping
agents that exchange Ag+ ions to Ago particles. SEM and TEM micrographs showed NPs of 10 to
500 nm in diameter, while shapes varied from incomplex spherical to complex rods, triangulars,
trapezoids, and prisms. AgNPs fabricated via green stratagem were found to degenerate at
temperatures ≥100oC and pH ≤ 2. The favorable conditions that assist in brisk reaction were
temperature 90o–95oC and slightly acidic to neutral pH 5.5–8 [127].
Research findings also reported that aqueous bark extracts of Cacumen platycladi (Platycladus
or Thujae), Cinnamon zeylanicum (cinnamon), Cochlospermum gossypium (gum kondagogu)
and Pinus eldarica (Afghan pine, Elder or Lone star Christmas tree) possessed eminent
biomolecules that are responsible to generate environment friendly and effective silver
nanostructures under optimum temperature of 90C within 30 min. Conformation of AgNPs
distorted from spherical to ellipsoidal at lower pH [128].
32
1.10 BIOLOGICAL EFFICACY OF SILVER NAOPARTICLES
Earlier studies suggest that plant-mediated synthesis synergizes the medicinal potency of plant
extracts and silver to curb many ailments. Greenly synthesized AgNPs offered new prospects
related to several industries specifically pharmacy, food and dairy, textile, water purification, and
engineering to tackle many adversities that the world is facing at present. Some of the reported
biological activities of nano-silver are explained below.
1.10.1 Antimicrobial Activity
Some studies revealed that AgNPs have the ability to impede replication of many pathogenic
bacterial and fungal species by destroying microbial membranes and impairing cellular
machinery. Synergism of formidable antibiotics such as ampicillin, clindamycin, erythromycin,
penicillin G, and vancomycin with AgNPs has practically unraveled antibiotic resistance
concerns [129]. In some groundwork investigations, the antimicrobial property of AgNPs was
compared with remarkable antibiotics such as kanamycin and tetracycline against well know
urinary tract infection causing microorganisms such as E. coli, Enterobacter aerogenes, E.
cloacae, Pseudomonas aeruginosa, Proteus morganii, K. pneumoniae, Staphylococcus aureus,
and Candida albicans [130]. The positive outcomes evidenced that low concentrations of
AgNPs, such as 20 mg/mL, could be microbicidal against all microorganisms by generating free
radicals. The minimum inhibitory concentration (MIC) recorded against E. coli and S. aureus
was 50 mg/mL and 12–96 µg/mL, respectively [131 , 132].
Dynamic antimicrobial ability of AgNPs can be employed for different therapeutic purposes such
as contriving curative creams, gels, and ointments to treat chronic wounds, burns, and other
cutaneous infections plus laminating medical equipment such as distinctively surgical utensils,
33
cardiac stents, prosthetic devices, and urinary catheters to restrict infection frequency. The
AgNPs can also beneficially assist in food and dairy storages and water purification to increase
shelf-life of perishable products and provide secure potable water. The bactericidal and
fungicidal property of AgNPs is also applied in textile and paint industries to curb bacterial and
fungal counts. A high rate of various epidemics has led many firms to implement AgNPs in
many household appliances to subdue the infective consequences [133].
1.10.2 Anticancer activity
Some studies showed that greenly synthesized AgNPs hold prodigious anticancer propensity by
evoking cytoprotective autophagy in cancer cells. Generalized outcomes on B16 mouse
melanoma cell cancers showed that AgNPs activate signaling mechanism Ptdlns 3K, which
results in antitumor activity by actuating autophagy [134]. Moderate-to-significant cytotoxic
potentials were demonstrated by marine algae such as Gelidiella and Ulva lactuca generated Ag
nanostructures against human HT–29, Hep–2, MCF–7, and Vero cell lines; this suggests
possibility of effective cancer therapies [135 , 136]. Aqueous leaf extracts of Melia dubia
(Malabar neem wood) and fruit extracts of Malus domestica (apple) also showed exceptional
anticancer potentials against human breast cancer cell line MCF–7 [137 , 138]. In 2013, Kaler
showed that at a concentration of 1 µg/mL, monodispersed AgNPs derived from Saccharomyces
boullardii display the potential to impede proliferation of cancer cell line MCF–7 by more than
70%, and this efficacy improves with the increase in AgNP concentration [139]. Overall results
revealed that biocompatible green AgNPs can offer new strategies to intercept malignancies.
34
1.10.3 Antioxidant activity
Multifarious studies exploring antioxidant properties showed that spherical AgNPs from aqueous
leaf extracts of Chenopodium murale (Australian spinach or Salt-green), Elephantopus scaber
(Elephant’s foot or Tutup bumi), and M. arvensis (Wild mint or field mint) possessed ≥50%
radical scavenging property at minimum concentration of 600 µg/mL. This seems to be
promising for many therapeutic applications with least side effects [140, 141, 142]. In contrast to
ascorbic acid and butylated hyroxytoluene, eco-friendly AgNPs from aqueous root extracts of
Helicteres isora also showed significant antioxidant activity [143]. Nano-silver from methanolic
extracts of stem and bark of Shorea roxburghii plant was also reported to possess eminent
antioxidant properties, which can be commercialized for therapeutic purposes [144].
1.10.4 Anti-leshmanial Activity
Leishmaniasis is one of the most incurable diseases worldwide as the available conventional
anti-leishmanial remedies are limited and have significant side effects. AgNPs from
Sargentodoxa cuneata plant proved to provide an alternative effective antileishmanial therapy
with least side effects [145]. It is reported that coupling ultraviolet and infrared radiation along
with plant-mediated AgNPs in the dark environment increases its anti-leishmanial potential by
generation of heat and reactive oxygen species [146 , 147]. In an in vitro study, the growth of
Leishmania tropica parasite was restrained 2- to 6-fold when exposed to radiation in dark, thus
controlling visceral progression of disease. These nanoparticles can aid in the development of
new medications to treat leishmaniasis [148].
35
1.10.5 Insecticidal activity
Insects are subtle disease initiator in various living organisms. They directly or indirectly act as a
vector. AgNPs have the pathetic influence on insects by mutilating their respiratory, gastric, and
lymphatic systems. It was remarked that AgNPs from Sargassum muticum (Japanese wire weed)
extracts possess insecticidal potency against Ariadne merione by altering conformation and
function of hemocytes, hemolymph, fat bodies, lumen, and gastric cecum [149]. AgNPs from
broth of Eclipta prostrate, Nelumbo nucifera, and Pergularia daemia were also found to posses
mosquitocidal and larvicidal potency against Anopheles subpictus and Culex quinquefasciatus
[150, 151, 152].
36
1.11 TOXICITY OF SILVER NANOPARTICLES
AgNPs have a great potential to be employed in medicine, engineering, optics, and electronics.
Interestingly, explicit toxicity issues, such as distinctively allergic reactions or organ
dysfunctions, have not been espied so far in case of AgNPs [153]. A dramatic derma condition
termed as argyrosis is observed when lacerations are healed with higher concentrations of silver.
Hence, the field of nanomedicine demands an extensive safety analysis of products before they
could be commercialized [154].
37
1.12 AIMS AND OBJECTIVES
The aims and objectives of this investigation were as follows:
1. To analyze the leaves of A. americana, M. spicata, and M. indica plants for alkaloids,
flavonoids, flavanones, phenolic compounds, steroids, glycosidases, saponins, carbohydrates,
and protein contents;
2. To prepare AgNPs from aqueous leaf extracts of A. americana, M. spicata, and M. indica
plants and then purify them via ultracentrifugation;
3. To characterize plant-mediated nano-silver via different spectroscopic techniques, such as
UV-Vis spectrophotometry, X-ray diffraction measurements, SEM, energy-dispersive X-ray
spectroscopy, TEM, simultaneous thermogravimetric, and differential thermal analysis;
4. To optimize AgNPs for their biocompatibility by monitoring parameters related to pH and
temperature;
5. To evaluate AgNPs and crude fractions of the leaf extracts for their antibacterial, antifungal,
anticancer, antioxidant, cytotoxic, phytotoxic, insecticidal, anti-termite, enzyme inhibition
(acetylcholine esterase and urease), and hemagglutination activities.
38
METHODOLOGY
2.1 GENERAL EXPERIMENTAL CONDITIONS
All research episodes i.e. AgNPs synthesis, purification, characterization, optimization and
comparative biological explorations were perfomed at Center of Biotechnology and
Microbiology (COBAM), University of Peshawar, Computerized Resource Lab (CRL),
Department of Physics University of Peshawar, Pakistan Counsil of Scientific and Industrial
Research (PCSIR), Peshawar and International Centre for Chemical and Biological Studies
(ICCBS), University of Karachi, Karachi. Commercial and analytical grade chemicals were plied
during the perusal. Experimental strategies involved in the current research exploration are
outlined in (Scheme 2.1).
2.2 PLANT COLLECTION
Leaves of A. americana, M. spicata and M. indica plants were obtained from disparate areas of
Peshawar District, Khyber Pukhtoon-Khuwa, Pakistan in March 2013 and were identified by
taxonomist, Ghulam Jelani, Botany Department, University of Peshawar, Pakistan.
2.3 EXTRACTION
The shade-dried leaves of A. americana, M. spicata, and M. indica plants were hewn into small
pieces. Then, the bits were triturated into the desired form of fine powder that ultimately
weighed 18 kg for A. americana, 15 kg for M. indica, and 12 kg for M. spicata. Further, the
triturated plant materials were submerged simultaneously in commercial grade ethanol,
methanol, and acetone for 14 days at room temperature. To obtain an aqueous extract, 25 g
39
triturated matter from each plant was seethed individually in 500 mL distilled water for half an
hour. Next, using Whatman filter paper, all solvent-soluble filtrates were collected and
concentrated at 40 °C under vacuum in a rotary evaporator (Sigma-Aldrich 4000, Germany).
Finally, the blackish green crude leaf extracts were procured that ultimately weighed 150 g each.
Crude aqueous ethanolic, methanolic and acetonic fractions are depicted in (Picture 2.1).
Picture 2.1: Crude ethanolic, methanolic, acetonic and aqueous fractions plus green silver
nanoparticles from leaves of Agave americana, Mentha spicata and Mangifera indica
40
Scheme 2.1: Flowchart depicti ng episodes of experimental strategy
Collection of Plant Material
Agave americana, Mentha spicata & Mangifera indica (Leaves)
PhytochemicalInvestigations
Alkaloids , Flavonoids, Flavonone, Phenolic Compounds, Steroids,
Glycosidases , Saponins,Carbohydrates,
&Protein
Synthesis of AgNPs
Purification
Characterization
UV-Vis Spectrophotometery
XRD, SEM, EDX, TEM, TG-DTA
Optimization
pH, Temperature
Biological/ Pharmacological Investigation
Antibacterial, Minimum inhibitory concentration (MIC), Antifungal,
Anticancer, Antioxidant, Cytotoxic, Phytotoxic, Insecticidal, Anti-
termite, Enzyme Inhibition (Acetylcholine esterase & Urease) &
Hemagglutination
Extraction (Aqueous,
Ethanol, Methanol, Acetone)
Biological / Pharmacological Investigation
Antibacterial, Minimum inhibitory concentration (MIC),
Antifungal, Anticancer, Antioxidant, Cytotoxic,
Phytotoxic, Insecticidal, Anti-termite, Enzyme Inhibition (Acetylcholine esterase &
Urease) & Hemagglutination
41
2.4 PHYTOCHEMICAL SCREENING
The contents of phytochemicals, such as alkaloids, flavonoids, flavanones, phenolic compounds,
steroids, glycosidases, saponins, carbohydrates, and protein in the leaves of A. americana, M.
spicata, and M. indica plants were determined by employing the methods described by Selvi et
al. [155] and Rahul et al [156].
2.4.1 MAYER’S TEST
2.4.1.1 Method
For alkaloid detection, 50 mg solvent-free extract was hydrolyzed with 1 mL of dilute
hydrochloric acid, and the mixture was filtered off using Whatman filter paper No.1. Then in a
test filtrate was reacted with few drops of Mayer’s regent (potassio-mercuric iodide solution), a
reaction in which a positive result was indicated by yielding a creamy white precipitate.
2.4.2 SHINODA’S TEST
2.4.2.1 Method
To detect flavonoids and flavanones, 50 mg solvent-free extract was carefully stirred with 1 mL
of alcohol and was filtered off using Whatman filter paper No.1. Then in a test tube, the
alcoholic test filtrate was reacted with few drops of concentrated hydrochloric acid and
fragments of magnesium ribbons or foil. A reddish pink color change of the magnesium ribbons
indicated the presence of flavonoids, whereas the reddish purple color showed the presence of
flavanones.
42
2.4.3 FERRIC CHORIDE TEST
2.4.3.1 Method
To determine the presence of phenols, 50 mg solvent-free extract was carefully dissolved in 5
mL distilled water followed by filtering with Whatman filter paper No.1. Further in a test tube,
the aqueous test filtrate was combined with 2 – 3 drops of neutral 5% ferric chloride solution.
The dusky green color change indicated a positive result.
2.4.4 LEAD ACETATE TEST
2.4.4.1 Method
To determine the availability of phenolic compounds, 50 mg solvent-free extract was dissolved
in 5 mL distilled water which was then filtered off using Whatman filter paper No.1. Next in a
test tube, the aqueous test sample was combined with 3 mL of 10% lead acetate solution. Bulky
white precipitate indicated positive results.
2.4.5 SALKOWSKI’S TEST
2.4.5.1 Method
For detection of steroids, 50 mg solvent-free extract was carefully stirred with chloroform which
was then filtered off using Whatman filter paper No.1. Further in a test tube, chloroform test
filtrate was uniformly mixed with concentrated sulfuric acid. Red color change denoted a
positive result.
43
2.4.6 BORNTRAGER’S TEST
2.4.6.1 Method
Glycosidases were identified by the following procedure: 50 mg solvent-free extract was
diligently dissolved in concentrated hydrochloric acid, which was then allowed to rest in a water
bath for two hours. Then, the hydrolysate was filtered off using Whatman filter paper No.1. In a
test tube then 2 mL test filtrate was reacted to 3 mL of chloroform with constant stirring. Then,
10% ammonia solution was added when a distant chloroform layer was formed. The pink color
change referred to a positive result.
2.4.7 FOAM TEST
2.4.7.1 Method
For detection of saponins, dry leaf powders of A. americana, M. spicata, and M. indica were
added to 5 mL distilled water which was then filtered off using Whatman filter paper No.1. Then
using a clean test tube, the aqueous test suspension was rigorously shaken for 15–20 min. The
appearance of a clear 2 cm layer of foam meant a positive result.
2.4.8 BENEDICT’S TEST
2.4.8.1 Method
For detection of reducing sugars or carbohydrates, 100 mg solvent-free extract was dissolved in
50 mL distilled water which was then filtered off using Whatman filter paper No.1. In a sterile
test tube then half volume of the test filtrate was combined with an equal volume of Benedict’s
reagent. The combination was then uniformly heated in a water bath for 2 min. The resultant
green, yellow, orange, or red color indicated the presence of reducing sugars.
44
2.4.9 BIURET’S TEST
2.4.9.1 Method
For detection of proteins, 100 mg solvent-free extracts was dissolved in 10 mL distilled water
which was then filtered off using Whatman filter paper No.1. Further in a test tube, 2 mL of the
test filtrate was treated with 1 drop of 2% copper sulfate solution and 1 mL of 95% ethanol. The
excessive amount of potassium hydroxide pellets was added to the reaction mixture. A positive
pink color change in the ethanolic layer indicated the presence of proteins.
45
2.5 SYNTHESIS OF SILVER NANOPARTICLES
Aliquots of 10 mL leaf broth of A. americana, M. spicata, and M. indica plants were blended
with 90 mL of AgNO3 (1 mM) solution at room temperature, which was then incubated for one
hour with incessant shaking at 75°C using a shaking water bath. At the end of the incubation
period, reduction of Ag+ ions to Ag0 nanoparticles was evidenced by the color change from
yellow to caliginous blackish brown as depicted in (Picture 2.2). Finally, the dark nanoparticle
solution was concentrated at 50°C under condition in a rotary evaporator (Sigma-Aldrich 4000,
Germany). Concentrated green AgNPs were further dehydrated at room temperature by evenly
strewing onto sterile petri-plates. Steps for AgNPs synthesis are depicted in (Scheme 2.2).
Picture 2.2: Blackish brown solution as productive plant mediated silver nanoparticles
46
Scheme 2.2: Flowchart depicting steps involved in AgNPs synthesis
Incubate in shaking water bath for 1 hour at 75oC
1mM AgNO3
solution (90ml)
Aqueous
leaves extracts
(10ml)
Blackish brown green AgNPs synthesized
47
2.6 PURIFICATION OF SILVER NANOPARTICLES
To purify the synthesized AgNPs from uncoordinated biological molecules, the method reported
by Forough et al. was followed [157]. According to the\is procedure, AgNPs were disseminated
in distilled water and then centrifuged at 12,000 rpm for 15 min. The supernatant was discarded,
and the purified AgNPs pellets were collected and spread for drying for use in the
characterization steps. Purified AgNPs are depicted in (Picture 2.3).
Picture 2.3: Dark colored purified fine AgNPs powder
48
2.7 CHARACTERIZATION OF SILVER NANOPARTICLES
To assess the physical, chemical, and biological attributes of the purified silver nanoparticles, the
following techniques were used.
2.7.1 UV-VIS SPECTROSCOPY
The optical characteristics of A. americana, M. spicata, and M. indica AgNPs were determined
at room temperature by using a UV-VIS spectrophotometer (Shimadzu UV-1601, Japan), which
was used at 10 nm resolution within the range 350–500 nm.
2.7.2 X-RAY DIFFRACTION MEASUREMENTS (XRD)
The metallic crystalline traits of the biocompatible AgNPs were estimated by a X-ray
diffractometer system (JDX-3532, China) equipped with Cu Ê (á) radiation of 1.54187 nm
wavelength, utilizing Ni gauze as a filter and a power supply of 30 kV/30 mA.
2.7.3 SCANNING ELECTRON MICROSCOPY (SEM)
Silver nanoparticles were morphologically evaluated by SEM (JEOL-JSM-5910, USA) model. A
thin coating of fabricated AgNPs was layered on the carbon-coated copper grid. The extra
solution was expunged off utilizing blotting paper. The AgNPs-coated copper grid was dried
under uninterrupted heat of mercury lamp for 5 min. Finally, AgNPs were observed via an
electron microscope at 150X, 500X, and 1000X magnification
49
2.7.4 ENERGY-DISPERSIVE X-RAY SPECTROSCOPY (EDX)
Elemental make-up of the green silver nanoparticles was probed by Energy-Dispersive X-Ray
Spectroscopy (INCA-200, UK). The assessment was conducted to substantiate that the stable
AgNPs solution contains solely silver.
2.7.5 TRANSMISSION ELECTRON MICROSCOPY (TEM)
Transmission electron microcopy of plant mediated nano-structures was performed by means of
TEM (Techni-G2-300kV, USA). The carbon-coated copper grid was surfaced with a minute
quantity of AgNPs suspension. The extra suspension was expunged off utilizing blotting paper.
The AgNPs that were on the surface of the grid were then placed under a mercury lamp for 5 min
to facilitate their drying.
2.7.6 SIMULTANEOUS THERMOGRAVIMETRIC AND DIFFERENTIAL
THERMAL ANALYSIS (TG-DTA)
Physical and chemical attributes of the green nano-silver were evaluated by Simultaneous
Thermo-gravimetric and Differential Thermal Analysis (Shimadzu DTG-60/DTG-60A, Japan)
model. The loss and gain of AgNPs mass was ascertained at various increasing temperatures.
50
2.8 OPTIMIZATION OF SILVER NANOPARTICLES
Bio-mediated silver nanoparticles were scrutinized at fluctuating pH values and temperatures to
assess the feasible ranges for superlative production and function of AgNPs.
2.8.1 pH OPTIMIZATION
The optimum pH for AgNPs was determined by following the procedure described by
Christopher et al. [158]. The suspensions of leaf-mediated AgNPs were inspected at a variable
pH range, i.e., 2.0–9.0. The reaction mixture pH was adjusted by adding 0.1 N hydrochloric acid
or 0.1 N sodium hydroxide solutions
2.8.2 TEMPERATURE OPTIMIZATION
The optimum temperature for aced preparation and function of silver nanoparticles was
determined by following the procedure described by Jiang et al. [159]. According to the
protocol, AgNPs solutions were incubated at a temperature range of 10–100°C using a constantly
shaking water bath for one hour. Then, the production and stability of AgNPs were observed.
51
2.9 BIOLOGICAL / PHARMACOLOGICAL INVESTIGATION OF SILVER
NANOPARTICLES CONTRARY TO CRUDE PLANT EXTRACTS
The biological and pharmacological properties of the AgNPs contrary to crude plant extracts
(aqueous, ethanol, methanol and acetone) were determined by following the standard protocols
for antibacterial, minimum inhibitory concentration (MIC), antifungal, anticancer, antioxidant,
cytotoxic, phytotoxic, insecticidal, anti-termite, enzyme inhibition (acetylcholine esterase and
urease) and hemagglutination activities.
2.9.1 ANTIBACTERIAL ASSAY
For over centuries, multifarious plant extracts have played a fundamental role in traditional folk
medicine for the therapy of many acute and chronic maladies. Potential phyto-constituents are
accountable for their curative properties [160]. Bearing in mind its clinical importance, the
antibacterial assays of biosynthesized AgNPs and crude extracts were conducted against
pathogenic bacteria to investigate the antibacterial efficacy of AgNPs versus crude fractions.
2.9.1.1 Test Organisms
Bacillus subtilus, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Methicillin
resistant Staphylococcus aureus (MRSA), Vancomycin resistant Staphylococcus aureus (VRSA)
and Streptomyces griseus.
52
2.9.1.2 Procedure
The antibacterial assay was carried out using the agar diffusion method [161]. Sterile nutrient
agar (Sigma-Aldrich, Germany) plates were prepared and uniformly inoculated with 24–hour
fresh bacterial cultures. Then, 6 mm wells were made in the solidified medium by a flame-
sterilized borer. Further, using sterile DMSO, a stock solution was prepared at a concentration of
3 mg/mL, from which 100 µL was transferred into each well. The robust antibiotic amoxicillin
was utilized as a positive control at a concentration 0.5 mg/mL in DMSO, while sterile DMSO
was employed as a negative control. The petri plates were kept undisturbed for a couple of hours
in an aseptic environment of laminar flow for preferable diffusion of the test sample into the
media. Finally, all petri-plates were incubated overnight at 37°C, and the results were evaluated
after 24 h of incubation on the basis of the presence of clear zones of inhibition. Percent
inhibition was computed by formula illustrated below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝑮𝒓𝒐𝒘𝒕𝒉 𝑰𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏 =𝒁𝒐𝒏𝒆 𝒐𝒇 𝑰𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 (𝒎𝒎)
𝒁𝒐𝒏𝒆 𝒐𝒇 𝑰𝒏𝒉𝒊𝒃𝒊𝒕𝒐𝒏 𝒐𝒇 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 (𝒎𝒎) × 𝟏𝟎𝟎
53
2.9.2 MINIMUM INHIBITORY CONCENTRATION (MIC) ASSAY
Minimum inhibitory concentrations (MICs) were estimated as the minimum concentration of the
antimicrobial agents to suppress the replication of bacteria during 24 h of incubation period
[162].
2.9.2.1 Test Organisms
Bacillus subtilus, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Meticillin
resistant Staphylococcus aureus (MRSA), Vancomycin resistant Staphylococcus aureus (VRSA)
and Streptomyces griseus.
2.9.2.2 Procedure
AgNPs and plant extracts were investigated for possible MICs at volumes of 10, 20, 40, 80, 160,
320, and 640 µL against the selected bacterial species by using the preceding protocol of Banso
et al. [163]. Autoclaved nutrient broth (Sigma-Aldrich, Germany) was prepared in autoclaved
test tubes. The test samples were pipetted into the medium, and then inoculated with fresh
bacterial culture. All the test tubes were then incubated at 37°C for 24 h. After the incubation
period, the results were assessed on the basis turbidity.
54
2.9.3 ANTIFUNGAL ASSAY
In the current century, fungal infections are recognized as a prominent threat to humankind due
to developing resistance of fungal strains towards multiple recommended medications. On the
other hand, certain medicinal plants have exhibited outstanding broad-spectrum antifungal
properties owing to the presence of potent phytocompounds. Thus, the biosynthesized AgNPs
and crude fractions were screened for possible antifungal properties against some pathogenic
fungal strains.
2.9.3.1 Test Organisms
Aspergillus niger, Aspergillus parasitica, Fusarium oxysporum, Penecillium and Verticillium
2.9.3.2 Procedure
An antifungal assay was conducted prior to the implementation of the tube dilution method
[161]. According to the assay results, we prepared a stock solution, using sterile DMSO,
containing 24 mg/mL of AgNPs and crude plant extracts. Further, aseptic Sabouraud dextrose
agar (Sigma-Aldrich, Germany) was prepared in immaculate aseptic test tubes to support fungal
proliferation. Then, from the stock solution, 67.6 µL test sample was pipetted into the medium,
and the mixture was allowed to cool in slanting orientation to solidify in the aseptic environment
of a laminar flow hood. In the next step, the prepared slants were inoculated with fresh fungal
spores by using a flame-sterilized inoculating loop. Miconazole and pure sterile DMSO were
used as a positive and a negative control, respectively. Finally, all test tubes were incubated for a
week at 28°C, and the results were assessed at the end of the incubation period by measuring the
55
linear mycelial growth on the medium. Percent inhibition was computed by formula illustrated
below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝒈𝒓𝒐𝒘𝒕𝒉 𝑰𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏 = 𝟏𝟎𝟎 −𝑳𝒊𝒏𝒆𝒂𝒓 𝒈𝒓𝒐𝒘𝒕𝒉 𝒊𝒏 𝒕𝒆𝒔𝒕 𝒔𝒂𝒎𝒑𝒍𝒆(𝒎𝒎)
𝑳𝒊𝒏𝒆𝒂𝒓 𝒈𝒓𝒐𝒘𝒕𝒉 𝒊𝒏 𝒄𝒐𝒏𝒕𝒓𝒐𝒍 (𝒎𝒎)× 𝟏𝟎𝟎
56
2.9.4 ANTICANCER ASSAY
Natural phyto-ingredients are a key source for drug production. The well-known cancer drugs,
such as camptothecin, doxorubicin, etoposide, and paclitaxel possess active phytochemicals,
especially alkaloid derivatives. Unlike chemo-, radio-, hormonal, and surgical therapies, these
natural compounds can aid to treat cancer in an effective and risk-free manner. Thus, the
anticancer efficacy of AgNPs and crude leaf fractions was evaluated to investigate natural
anticancer agents.
2.9.4.1 Test Cancer Cell line
Human prostate cancer cell line (PC-3)
2.9.4.2 Procedure
The anticancer assays of the crude extracts and AgNPs from the leaves of A. americana, M.
spicata, and M. indica plants were conducted following an earlier protocol for MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assays described by
Mosmann [164]. First, breast cancer cells were cultured in an essential medium supplemented
with 15% fetal bovine serum and 1% penicillin (Invitrogen) using autoclaved petri plates and
was then incubated overnight at 37°C with relative humidity and 5% CO2. Next, the proliferated
cells were harvested using 0.25% trypsin/EDTA solution. Furthermore, the harvested cancer
cells were sub-cultured onto an aseptic 96-well microtiter plate. In the next step, a stock solution
of 10 mg/mL was prepared in 50% methanol. From the stock solution, a working solution of 1.0
mg/mL was prepared by adding cell culture broth. The working solution was then added to
cultured MCF–7 cells (1 × 104 cells/well) on microtiter plate for 24 h. After 24 h of incubation,
57
10 µL MTT reagent (Invitrogen, USA) was added to each well ,and the solution was re-
incubated at the for 3 h same temperature. Finally, the media containing MTT reagent was
withdrawn, and sterile 200 µL DMSO was added into each well followed by incubation for
additional 20 min. Absorbance at 550 nm was recorded using a Synergy microplate reader
(BioTek, USA). The standard drug doxorubicin was used as a positive control, and trials were
conducted in triplicate. Optical density was enumerated and percent cancer cytotoxicity was
enumerated by formula illustrated below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝒄𝒂𝒏𝒄𝒆𝒓 𝒄𝒚𝒕𝒐𝒕𝒐𝒙𝒊𝒄𝒊𝒕𝒚 = 𝟏𝟎𝟎 –𝑶𝑫 𝒐𝒇 𝒕𝒆𝒔𝒕 𝒘𝒆𝒍𝒍
𝑶𝑫 𝒐𝒇 𝒄𝒐𝒏𝒕𝒓𝒐𝒍 𝒘𝒆𝒍𝒍 × 𝟏𝟎
58
2.9.5 ANTIOXIDANT ASSAY
According to several studies, it is estimated that approximately two-thirds of earth’s flora possess
medicinal properties, especially antioxidant potential. This antioxidant property has been used to
treat numerous human conditions, particularly inflammations, cancers, and cardiovascular
disorders by reducing cellular oxidative stress. Thus, the possible antioxidant potential of
biocompatible AgNPs and crude leaf extracts of A. americana, M. spicata, and M. indica was
investigated in our study.
2.9.5.1 Materials
DPPH (1, 1-diphenyl-2- 8 picrylhydrazyl), methanol and UV-Vis spectrophotometer
2.9.5.2 Procedure
The antioxidant assay was conducted according to a preceding protocol for 1, 1-diphenyl-2-8
picrylhydrazyl (DPPH) radical scavenging appraisal [161]. According to this method, the
reaction mixture was prepared by blending different strengths of the stock solutions, i.e., 100,
200, 300 µg/mL, and 1 mL of 1 mM DPPH in methanol. The reaction mixture was then allowed
to rest for 30 min at room temperature. Finally, the percentage of radical reduction was estimated
by absorbance at 517 nm, where ascorbic acid was used as a positive control and DPPH was used
as a reference compound. Experiment was run in triplicate and percent absorbance was computed
by formula illustrated below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 =𝑪𝒐𝒏𝒕𝒓𝒐𝒍 𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 − 𝑬𝒙𝒕𝒓𝒂𝒄𝒕 𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆
𝑪𝒐𝒏𝒕𝒓𝒐𝒍 𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 × 𝟏𝟎𝟎
59
2.9.6 CYTOTOXIC ASSAY
Medicinal plants are known for a long time to contain natural compounds of therapeutical
interest. These natural compounds possess unique pharmacological benefits along with some
concomitant side effects.
2.9.6.1 Test Organism
Artemia salina (Shrimp eggs)
2.9.6.2 Procedure
The cytotoxic potential of AgNPs and crude leaf extracts was evaluated by brine shrimp
(Artemia salina) lethality assay [165]. The brine shrimps were subjected to variable sample
dilutions of green AgNPs and crude fractions obtained from the leaves of A. americana, M.
spicata, and M. indica plants to determine their cell toxicity. To facilitate hatching, brine shrimp
eggs were placed in a saline environment, assembled in rectangular plastic salver with an
approximate diameter of 22 × 32 cm. The salver was partitioned unevenly by a perforating
devise into large dark and small normal light chambers. Eggs (50 mg) were introduced into the
dark chamber and were allowed to hatch and mature at room temperature for two days. Then, the
nauplii were collected from the lighter end of the chamber using a Pasteur pipette. A further
stock solution of 10 mg/mL was prepared using methanol from which 10, 100, and 1000 µL
dilutions were transferred into sterile flasks. The organic solvent was allowed to vaporize by
placing it in a laminar flow hood for 30 min. Next, 1 mL brine sea water was added to it, and ten
shrimp larvae were placed in the flasks by a Pasteur pipette to adjust the final volume of the sea
water up to 5 mL. All the flasks were incubated for 24 h at 28°C. Etoposide drug was used as a
60
positive control, while methanol was utilized as a negative control. Shrimp lethality was
estimated by observations through a magnifying glass. Percent lethality was computed by
formula illustrated below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝒔𝒉𝒓𝒊𝒎𝒑 𝒍𝒆𝒕𝒉𝒂𝒍𝒊𝒕𝒚 =𝑵𝒐. 𝒐𝒇 𝒅𝒆𝒂𝒅
𝑻𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝒔𝒉𝒓𝒊𝒎𝒑𝒔× 𝟏𝟎𝟎
61
2.9.7 PHYTOTOXIC ASSAY
Natural phytotoxic products are considered effective, biodegradable, and eco-friendly herbicides
and phyto-stimulants. The plant secondary metabolites when used as herbicides act as
allelochemicals against many problematic weeds. To assess their phytotoxic potency, crude
extracts and green AgNPs from leaves of A. americana, M. spicata, and M. indica plants were
screened against the free-floating aquatic plant Lemna minor (L. minor) at variable sample
dilutions.
2.9.7.1 Test Organism
Lemna minor (Duckweeds)
2.9.7.2 Procedure
Green synthesized silver nanoparticles and crude aqueous, methanolic, ethanolic, and acetonic
fractions were investigated for possible phytotoxic activity by using Lemna bioassay protocols
that were reported earlier [161]. According to the protocol, 30 mg/mL stock solutions were
prepared using analytical grade methanol, from which 10, 100, and 1000 µg/mL dilutions were
transferred into sterile petri plates. All the petri plates were left undisturbed for 30 min at room
temperature to evaporate the organic solvent. Further, to support growth of Lemna minor, E-
medium (20 mL) was added to the petri plates containing the evaporated test sample. Finally,
sixteen healthy L. minor plants were put into the experimental petri plates which were then
incubated in an incubator for one week at 28°C. The results were computed at the end of
incubation. Percent growth regulation was computed by formula illustrated below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝒈𝒓𝒐𝒘𝒕𝒉 𝒓𝒆𝒈𝒖𝒍𝒂𝒕𝒊𝒐𝒏 =𝑬𝒙𝒑𝒆𝒓𝒊𝒎𝒆𝒏𝒕𝒂𝒍 𝑻𝒆𝒔𝒕 𝑺𝒂𝒎𝒑𝒍𝒆
𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅× 𝟏𝟎𝟎
62
2.9.8 INSECTICIDAL ASSAY
Phyto-constituents also possess insecticidal potential, and can be used as natural insecticides.
They are affordable, less to non-toxic, and environment-friendly. Considering the importance of
these advantages, the possible insecticidal potential of green silver nanostructures and crude leaf
extracts was screened against four insect species.
2.9.8.1 Test Organisms
Callosobruchus maculates (Cow pea Beetle), Tribolium castaneum (Red flour Beetle),
Cryptolestes pusillus (Flat grain Beetle) and Oryzaephilus surinamensis (Saw toothed grain
Beetle)
2.9.8.2 Procedure
The insecticidal assay was conducted before the contact toxicity evaluation [165]. According to
the protocol, 9 cm filter paper was snipped into the shape of petri plates. Then, a stock solution
of AgNPs and crude leaf extracts was prepared at a concentration of 3 mg/mL using methanol as
an organic solvent. Further, using autoclaved micropipette tips, stock solution was poured on the
filter paper lining placed in the petri plate. The plates were left undisturbed for 30 min at room
temperature to vaporize the methanolic content. Then, 12 healthy insect species were carefully
placed into each petri plate separately using a sterile brush. Finally, all test petri plates were
incubated in a growth chamber at 28°C with 50% relative humidity. Observations were
performed at 12 and 24 h, and the results were estimated by calculating number of survived
insects. Permethrin was used as a positive control. Percent lethality was enumerated by formula
illustrated below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝒊𝒏𝒔𝒆𝒄𝒕 𝒍𝒆𝒕𝒉𝒂𝒍𝒊𝒕𝒚 =𝑵𝒐. 𝒐𝒇 𝒅𝒆𝒂𝒅 𝒊𝒏𝒔𝒆𝒄𝒕𝒔
𝑻𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝒊𝒏𝒔𝒆𝒄𝒕𝒔× 𝟏𝟎𝟎
63
2.9.9 ANTI-TERMITE ASSAY
Termite infestation has been traditionally controlled by a chemical suspension of the pesticide
known as Termiticide, which pose negative effects on animal health and the environment. The
utilization of phyto-ingredients as anti-termite agents can efficiently aid in achieving desirable
outcomes while avoiding these adverse effects in a cost-effective and eco-friendly way.
2.9.9.1 Test Organisms
Coptotermes formosanus (Formosan subterranean termite)
2.9.9.2 Procedure
A termiticide assay was also conducted before the contact toxicity bioassay [165]. Clean and
autoclaved pieces of filter paper were incised in the shape of a petri plate. Stock solution at the
concentration of 2 mg/mL was prepared using analytical grade methanol. The filter paper was
adjusted properly to the base of petri plate, stock solution was poured uniformly onto it, and was
then left undisturbed at room temperature to allow vaporization. After solvent evaporation, 12
healthy termites were placed into the test petri plates, all of which were then incubated at 28°C
using desiccators to maintain constant relative humidity. Fipronil was used as a positive control.
Lethality was estimated through observations at 12 and 24 h and calculating the number of dead
termites. Percent lethality was enumerated by formula illustrated below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝒕𝒆𝒓𝒎𝒊𝒕𝒆 𝒍𝒆𝒕𝒉𝒂𝒍𝒊𝒕𝒚 =𝑵𝒐. 𝒐𝒇 𝒅𝒆𝒂𝒅 𝒕𝒆𝒓𝒎𝒊𝒕𝒆𝒔
𝑻𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝒕𝒆𝒓𝒎𝒊𝒕𝒆𝒔× 𝟏𝟎𝟎
64
2.9.10 ENZYME INHIBITION ASSAY
Enzymatic actions account for the onset of many serious diseases; therefore, enzymes are chief
targets for drugs in the prevention and treatment of a number of disorders. A multitude of
commercially available therapeutic options are based on the actions of enzyme inhibitors that
suppress the effect of enzymes mediating disease phenotypes. In our study, crude leaf extracts
and silver nanoparticles were screened for possible acetylcholine esterase and urease inhibitory
activities to add a new dimension to the field of medicine.
2.9.10.1 ACETYLCHOLINE-ESTERASE INHIBITION
2.9.10.1.1 Test Enzyme
Acetylcholine esterase from Electrophorus electricus (Electric eel)
2.9.10.1.2 Procedure
Acetylcholine esterase inhibitory activity was probed by following the protocol described by
Ingkaninan et al. [166]. According to the protocol, a stock solution of 10 µg/mL was prepared in
analytical grade methanol. Then, a reaction mixture of 100 µL AChE, 275 µL Tris-HCl buffer,
and 500 µL Ellman's reagent (DNTB) was prepared in a clean and aseptic cuvette. To this
reaction mixture, 100 µL test sample was added and allowed to rest at room temperature for 30
min. Absorbance was observed at 405 nm, and the rate of inhibition was computed. The standard
drug Galanthamine was used as a positive control. The experiment was performed in triplicate at
pH 8.0. Percent enzyme inhibition was enumerated by formula illustrated below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝑬𝒏𝒛𝒚𝒎𝒆 𝑰𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏 = 𝟏𝟎𝟎 − 𝑬𝒙𝒕𝒓𝒂𝒄𝒕 𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆
𝑪𝒐𝒏𝒕𝒓𝒐𝒍 𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 × 𝟏𝟎𝟎
65
2.9.10.2 UREASE INHIBITION
2.9.10.2.1 Test Enzyme
Urease from Canavalia ensiformis (Jack bean)
2.9.10.2.2 Procedure
Urease inhibition assay was performed following the indophenol protocol described by Akhtar et
al. [167]. A reaction mixture consisting of 25 µL Jack bean urease, 55 µL buffer, 100 mM urea,
and 5 µL test sample (0.5 mM) was prepared and incubated at 30°C for 15 min. Afterwards,
aliquots from the mixture were transferred to a 96-well microtiter plate and re-incubated at the
same temperature for 15 min. Further, to each well, 50 µL phenol reagent (1% w/v phenol and
0.005% w/v sodium nitroprusside) and 70 µL alkali reagent (0.5% w/v sodium hydroxide and
0.1% sodium hypochloride) were added. After 50 min of incubation, absorbance was recorded at
630 nm using a microtiter plate reader (SpectraMax Plus 384 Molecular Device, USA). Thiourea
was utilized as a reference compound. The experiment was carried out in triplicate at pH 8.0.
Percent enzyme inhibition was enumerated by formula illustrated below;
𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝑬𝒏𝒛𝒚𝒎𝒆 𝑰𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏 = 𝟏𝟎𝟎 –𝑶𝑫 𝒐𝒇 𝒕𝒆𝒔𝒕 𝒘𝒆𝒍𝒍
𝑶𝑫 𝒐𝒇 𝒄𝒐𝒏𝒕𝒓𝒐𝒍 𝒘𝒆𝒍𝒍 × 𝟏𝟎𝟎
66
2.9.11 HEMAGGLUTINATION ASSAY
Medicinal plants extracts and AgNPs were analyzed for the content of possible phyto-lectins
which are exploited in many blood typing reagents due to their agglutination potential towards
specific blood groups. Owing to their low cost, easy purification methods, and harmlessness to
human health, plant lectins have many advantages over animal preparations.
2.9.11.1 Test Blood Samples
All ABO groups (A+, A-, B+, B-, AB+, AB-, O+, O-)
2.9.11.2 Procedure
The hemagglutination assays of the produced AgNPs and crude leaf extracts were carried out
according to Shahzada et al. [161]. Stock solution (1 mg/mL) was prepared using sterile DMSO
at the protean strengths of 1:2, 1:4, 1:8, and 1:16. On the day of the experiment, fresh blood
samples from healthy volunteers were collected and centrifuged at 2500 rpm for 15 min. Then,
utilizing phosphate buffer, 2% RBC suspension was prepared using pelleted RBCs obtained by
centrifugation. Next, from each sample strength, 1 mL test sample was put into a sterilized test
tube, followed by the addition of 1 mL of the RBC suspension. The reaction mixture was
incubated in an incubator at 37°C for 30 min. Finally, the blood agglutination reactions occurring
in response to the addition of test samples were observed, and the appearance of smooth button
formation indicated a positive result.
67
RESULTS AND DISCUSSION
Green biocompatible AgNPs were fabricated with A. americana, M. spicata, and M. indica
leaves. To detect the presence of bioactive phytoconstituents, responsible for Ag+–Ago reduction
in an economic and ecofriendly way, the plants were first phytochemically analyzed. Elements of
these plant act as vigorous bioreducers and capping ligands to afford monodispersed, stable
AgNPs. These NPs were further purified, characterized, and optimized using previously
documented protocols. In addition, the biological/pharmacological properties of AgNPs were
evaluated and compared with the crude ethanolic, methanolic, acetonic, and aqueous leaf extract
of the selected plants.
3.1 PHYTOCHEMICAL SCREENING
Phytochemical analysis revealed that A. americana, M. spicata, and M. indica leaves possessed
an exorbitant quantity of biologically active elements, termed as polyphenols; these elements
reduce AgNO3 to AgNPs. The A. americana leaf extracts not only had high quantities of
flavonoids, flavanones, phenolic compounds, saponins, and steroids but also had a high protein-
carbohydrate ratio. However, they did not have alkaloid content and had low amounts of
glycosidase. Furthermore, the M. indica leaf extracts contained significant amounts of
flavonoids, flavanones, phenolic compounds, glycosidase, steroids, proteins, and carbohydrates.
Alkaloids were not detected, but moderate amount of saponins was detected in the extracts.
Adequate amounts of flavonoids, flavanones, phenolic compounds, and glycosidases and less-to-
moderate amounts of saponins and steroids were detected in the M. indica leaf extracts; however,
alkaloids, proteins, and carbohydrates were absent. Results are summarized and depicted in
(Table 3.1) and (Pictures 3.1 - 3.7).
68
Previous studies have revealed that aerial parts of A. americana possess various potential
phytoconstituents, mainly phenolic compounds, which are responsible for the overall biological
competence of the plant. The total phenolic content and flavonoid content in the leaves were
10.541–39.35 mg/100 g and 43.35–304.8 mg/100 g, respectively [168]. A potential flavanone,
agamanone, was isolated and structurally elucidated to be 5,7-dihydroxy–6,5′-dimethoxy–3′,4′-
methylene-dioxy flavanone, which contributes to the antimicrobial, antioxidant, lipid
peroxidation inhibition, and hemolytic activities. However, no bioactive compounds from A.
americana roots have been reported to date [169 , 170]. Further, M. spicata leaves and oils
possess profuse amounts of phenolic compounds, i.e., the total phenolic content was estimated to
be 8.43 mg/100 g [171]. Spear mint leaves also possess other elements such as carbohydrates
14.46 ±0.15%, proteins 1.75 ±1.0%, fats 3.20 ±0.003%, and fibers 2.1 ±0.03%. All these
ingredients can be exploited for their possible bactericidal, fungicidal, radical scavenging,
chemotherapeutic activities [172]. On evaluating M. indica leaves, the presence of
anthraquinone, cardiac glycosidases, flavonoids, tannins, steroids, saponins, and reducing sugars
was detected. Traces of calcium oxalate and absence of alkaloids were also reported [173 , 174].
Similarly, methanolic extracts of mango peels and flesh were also analyzed for bioactive
compounds. Mass spectroscopy and HPLC revealed that the functional compounds were
quercetin family, fatty acids, and tannin derivatives that account for the nutritional value of the
fruit [175].
69
Table 3.1: Tabular depiction of phytochemical analysis of Agave americana, Mentha spicata
and Mangifera indica (leaves)
Name of
Phytochemicals
Presence of compounds
Agave americana
(Leaves)
Mentha spicata
(Leaves)
Mangifera indica
(Leaves)
Alkaloids - - -
Flavonoids +++ +++ +++
Flavonones +++ +++ +++
Phenolic
compounds +++ + +++
Steroids +++ + +++
Glycosidases + +++ +++
Saponins +++ + ++
Carbohydrates + - ++
Proteins +++ - +++
Note: (+) sign manifest less quantity of bioactive phytochemical
(++) sign manifest moderate quantity of bioactive phytochemical
(+++) sign manifest high quantity of bioactive phytochemical
(-) sign manifest absence of bioactive phytochemical
70
(a) (b)
Picture 3.1 (a, b): Reddish pink color manifest positive flavonoids while reddish purple indicate
positive flavonone via Shinoda’s test
71
(a) (b)
Picture 3.2 (a,b): Green color manifest presence of phenolic compounds via ferric chloride test
while creamy white precipitate indicate positive phenols via lead acetate test
72
Picture 3.3: Red color manifest positive steroids via Salkowski’s test
73
Picture 3.4: Reddish pink color manifest positive glycosidase via Borntrager’s test
74
Picture 3.5: Froth production manifest positive saponins via Foam test
75
Picture 3.6: Green / Yellow color manifest positive carbohydrates via Benedict’s test
76
Picture 3.7: Light pink color layer manifest positive proteins via Biuret’s test
77
3.2 CHARACTERIZATION OF SILVER NANOPARTICLES
Purified AgNPs from A. americana, M. spicata, and M. indica leaves were characterized using
several spectrophotometric techniques and the results were compared with aqueous leaf extracts
of selected plants as follows.
3.2.1 UV-VIS SPECTROSCOPY
According to the UV-VIS spectroscopy, the λmax for the plant-mediated AgNPs ranged between
350 and 500 nm; however, the aqueous extract in the same range showed irregular absorbance
patterns. The λmax for A. americana and M. indica AgNPs was arrayed at 430 nm, revealing the
highest absorbance of 0.72 and 0.81 and lowest transmittance of 53.71 and 47.21, respectively.
The λmax for M. spicata AgNPs was found to be at 410 nm, revealing the highest absorbance of
0.68 and lowest transmittance of 56.78. Spectroscopic upshots are summarized in (Figure 3.1 –
3.9), thus accrediting it as bio-inspired green nano-silver.
Earlier studies reported the surface plasmon resonance peak of AgNPs from aqueous leaf
extracts of Couroupita guianensis tree and Curcuma longa herb in the range of 420 – 440 nm
[176 , 177]. Moreover, some reports have suggested that ethanolic plant preparations for
production of Ago particles exhibit a maximum absorbance crest at 410 nm [178].
78
Figure 3.1: Graphical depiction of absorbance values of Agave americana AgNPs
Figure 3.2: Graphical depiction of transmittance values of Agave americana AgNPs
0.052 0.055 0.057
0.058
0.118
0.22
0.72
0.124
0.12 0.118 0.116 0.1120.071
0.0680
0.2
0.4
0.6
0.8
1
370 380 390 400 410 420 430 440 450 460 470 480 490 500
Ab
sorb
ance
Wavelength (nm)
Absorbance
79
Figure 3.3: Graphical depiction of absorbance values of Agave americana aqueous extracts
0.043
0.0510.062
0.108
0.119
0.211
0.215
0.216
0.245
0.257
0.3
0.304
0.321
0.346
0.359
0.378
0
0.2
0.4
0.6
0.8
1
350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500
Ab
sorb
ance
Wavelength (nm)
Absorbance
80
Figure 3.4: Graphical depiction of absorbance values of Mangifera indica AgNPs
Figure 3.5: Graphical depiction of transmittance values of Mangifera indica AgNPs
0.069 0.071
0.076
0.08
0.083
0.121
0.156
0.221
0.81
0.231 0.224
0.217
0.193
0.1810.164
0
0.2
0.4
0.6
0.8
1
350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500
Ab
sorb
ance
Wavelength (nm)
Absorbance
86.4
85.89
81.48 79.35 78.56 75.45
69.21
47.2153.9
64.36
74.8780.21
81.26
83.41
84.86
0
10
20
30
40
50
60
70
80
90
100
350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500
Tran
smis
sio
n (%
)
Wavelength (nm)
Transmission
81
Figure 3.6: Graphical depiction of absorbance values of Mangifera indica aqueous extracts
0.072
0.075
0.091
0.125
0.143
0.145
0.187
0.228
0.256
0.249
0.268
0.325
0.294
0.402
0.416
0.421
0
0.2
0.4
0.6
0.8
1
350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500
Ab
sorb
ance
Wavelength (nm)
Absorbance
82
Figure 3.7: Graphical depiction of absorbance values of Mentha spicata AgNPs
Figure 3.8: Graphical depiction of transmittance values of Mentha spicata AgNPs
0.0540.056 0.0720.080.114
0.12
0.68
0.1460.124 0.122
0.105
0.0890.0880.079
0.071
0.066
0
0.2
0.4
0.6
0.8
1
350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500
Ab
sorb
ance
Wavelength (nm)
Absorbance
82.53
82.21
81.91
80.54
78.29
75.7
56.78
60.73
68.89
70.41
72.35
74.65
75.34
78.67
81.483.26
0
10
20
30
40
50
60
70
80
90
100
350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500
Tra
nsm
issi
on
(%)
Wavelength (nm)
Transmission
83
Figure 3.9: Graphical depiction of absorbance values of Mentha spicata aqueous extracts
0.049
0.053
0.0540.062
0.107
0.138
0.129
0.169
0.211
0.213
0.292
0.303
0.309
0.329
0.341
0.322
0
0.2
0.4
0.6
0.8
1
350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500
Ab
sorb
ance
Wavelength (nm)
Absorbance
84
3.2.2 X-RAY DIFFRACTION MEASUREMENTS (XRD)
Crystallographic analysis of green AgNPs corroborated that all the three contrived NPs were
polycrystalline in texture and their whole XRD spectrum for 2θ readings ranged from 10° to 80°.
The intense peaks for A. americana at 2θ angles were 5.166, 3.906, 2.032, 1.448, and 1.438,
which lie in the range of 17°–65°, whereas the d-values for M. indica and M. spicata AgNPs
ranged from a short peak of 1.354 to extended peak of 6.831 at 12°–69°C, respectively. In
addition, sample purity was testified by the absence of impurity peaks. The analysis was
compared with the aqueous leaf extracts of selected plants, demonstrating many irregular d-
values in the same 2θ range. Crystal lattice was calculated using the Debye–Scherrer’s equation,
and the estimated average crystal size was found to be 28 nm, 35 nm and 43 nm for A.
americana, M. indica and M. spicata, which was further confirmed by TEM analysis. XRD
upshots are summarized in (Table 3.2 – 3.7) and (Figure 3.10 – 3.15).
Previous research indicates that green-synthesized AgNPs provide sharp crystallographic peaks
in the plane of 10°–80° [179]. Leaves-mediated AgNPs of Eucalyptus chapmaniana and
Paederia foetida manifested X-ray diffraction angles from acute 26.459° to wide 73.628°,
respectively, thereby identifying it as crystalline orthorhombic AgNPs that are approximately 24
nm in size [180, 181].
85
Figure 3.10: Graphical depiction of XRD values of Agave americana AgNPs
Figure 3.11: Graphical depiction of XRD values of Agave americana aqueous extract
86
Figure 3.12: Graphical depiction of XRD values of Mangifera indica AgNPs
Figure 3.13: Graphical depiction of XRD values of Mangifera indica aqueous extract
87
Figure 3.14: Graphical depiction of XRD values of Mentha spicata AgNPs
Figure 3.15: Graphical depiction of XRD values of Mentha spicata aqueous extract
88
3.2.3 SCANNING ELECTRON MICROSCOPY (SEM)
Conformation, surface topologies, and stability of AgNPs were examined at 150×, 500×, and
1000× magnification using SEM. Electron micrographs revealed that plant extracts act as
capping ligands and reducing agents to provide relatively stable and monodispersed
nanospheroids. This analysis was compared with aqueous leaf extracts, which displayed
asymmetrical crisp images. SEM upshots are depicted in (Pictures 3.8 – 3.25).
AgNPs from aqueous leaf extracts of Anthoceros, Cycas, Ficus carica, Helianthus annus, Oryza
sativa, Urtica dioica, Paederia foetida, Sorghum bicolor, and Zea mays have varied
conformations, majorly spherical, cuboidal, spheroid, rectangular, and triangular. The particle
assembly, stability, and depressiveness largely depends on the experimental conditions and
precursor used as bioreductant [182 , 183].
89
Picture 3.8: SEM micrograph of Agave americana AgNPs at 150X magnification
Picture 3.9: SEM micrograph of Agave americana AgNPs at 500X magnification
90
Picture 3.10: SEM micrograph of Agave americana AgNPs at 1000X magnification
Picture 3.11: SEM micrograph of Agave americana aqueous extract at 150X magnification
91
Picture 3.12: SEM micrograph of Agave americana aqueous extract at 500X magnification
Picture 3.13: SEM micrograph of Agave americana aqueous extract at 1000X magnification
92
Picture 3.14: SEM micrograph of Mangifera indica AgNPs at 150X magnification
Picture 3.15: SEM micrograph of Mangifera indica AgNPs at 500X magnification
93
Picture 3.16: SEM micrograph of Mangifera indica AgNPs at 1000X magnification
Picture 3.17: SEM micrograph of Mangifera indica aqueous extracts at 150X magnification
94
Picture 3.18: SEM micrograph of Mangifera indica aqueous extracts at 500X magnification
Picture 3.19: SEM micrograph of Mangifera indica aqueous extracts at 1000X magnification
95
Picture 3.20: SEM micrograph of Mentha spicata AgNPs at 150X magnification
Picture 3.21: SEM micrograph of Mentha spicata AgNPs at 500X magnification
96
Picture 3.22: SEM micrograph of Mentha spicata AgNPs at 1000X magnification
Picture 3.23: SEM micrograph of Mentha spicata aqueous extracts at 150X magnification
97
Picture 3.24: SEM micrograph of Mentha spicata aqueous extracts at 500X magnification
Picture 3.25: SEM micrograph of Mentha spicata aqueous extracts at 1000X magnification
98
3.2.4 ENERGY-DISPERSIVE X-RAY SPECTROSCOPY (EDX)
Comparative elemental evaluation of biosynthesized AgNPs and aqueous leaf extracts was
conducted using energy dispersive X-ray spectroscopy, revealing that plant-mediated AgNPs
from A. americana, M. spicata, and M. indica leaves possess 34.91%, 9.96%, and 9.93% weight
of silver along with other biological elements such as carbon, oxygen, magnesium, silicon,
sulfur, chlorine, potassium, and calcium. The absence of silver element in the aqueous extract
confirmed the presence of synthesized AgNPs. Spectroscopic results are summarized in (Figure
3.16 – 3.21).
According to Gopinath et al., the EDX spectrum for AgNPs from Allium cepa, Acalypha indica,
Cinnamomum camphora, Jatropha curcas, Cinnamon zeylanicum, Murraya koenigii, Mimosa
pudica, and Ocimum tenuiflorum had pellucid silver peak, testifying the accurate production of
biogenic AgNPs. The results of this study supports the EDX spectrums found for the early
fabricated AgNPs [184, 185 ].
99
Figure 3.16: Graphical depiction of EDX values of Agave americana AgNPs
Figure 3.17: Graphical depiction of EDX values of Agave americana aqueous extract
100
Figure 3.18: Graphical depiction of EDX values of Mangifera indica AgNPs
Figure 3.19: Graphical depiction of EDX values of Mangifera indica aqueous extract
101
Figure 3.20: Graphical depiction of EDX values of Mentha spicata AgNPs
Figure 3.21: Graphical depiction of EDX values of Mentha spicata aqueous extract
102
3.2.5 TRANSMISSION ELECTRON MICROSCOPY (TEM)
The diameter and detailed two-dimensional conformations of AgNPs were analyzed using TEM.
The TEM micrographs confirmed that the most particles had a diameter ranging from 30 to 150
nm. However, some had 15–20 nm diameters. The morphology of the bioinspired nanostructures
was spherical, spheroids, rods, ellipsoidal, or barely triangular. Micrographic upshots are
depicted in (Pictures 3.26 - 3.29).
The TEM results of the green synthesized AgNPs concorded with those reported by Heydari et
al., Singh et al. and Garg [186, 187, 188]. The TEM micrograph showed the average diameter of
AgNPs lying in the ranges of 10−100 nm with assorted structures.
103
Picture 3.26: TEM micrograph of Agave americana AgNPs
Picture 3.27: TEM micrograph of Mangifera indica AgNPs
104
Picture 3.28: TEM micrograph of Mentha spicata AgNPs
Picture 3.29: Zoomed TEM micrograph of fabricated green AgNPs
105
3.2.6 SIMULTANEOUS THERMOGRAVIMETRIC AND DIFFERENTIAL THERMAL
ANALYSIS (TG-DTA)
The thermal stability and mass loss of aqueous leaf extracts and AgNPs were monitored using
TG-DTA in the temperature range of 0°–800°C. The aqueous leaf extracts were thermally stable
up to 100°C, and the rise in temperature indicated loss in mass and activity of the extract.
Although AgNPs from selected plants were thermally stable ≤200°C, the loss in particle aptitude
and mass was observed by a gradual increase in temperature (200–800oC), thus affirming it as
thermally sensitive compound. TG-DTA results are summarized in (Figure 3.22 – 3.27).
An early analysis revealed that AgNPs act as a semiconductor of heat and electricity because
biologically manufactured AgNPs remain stable at higher temperatures till 300°C. Further, a
steady weight dissipation was observed between 150°–600°C [189].
106
Figure 3.22: Graphical depiction of TD-DTA values of Agave americana AgNPs
107
Figure 3.23: Graphical depiction of TD-DTA values of Agave americana aqueous extract
108
Figure 3.24: Graphical depiction of TD-DTA values of Mangifera indica AgNPs
109
Figure 3.25: Graphical depiction of TD-DTA values of Mangifera indica aqueous extract
110
Figure 3.26: Graphical depiction of TD-DTA values of Mentha spicata AgNPs
111
Figure 3.27: Graphical depiction of TD-DTA values of Mentha spicata aqueous extract
112
3.3 OPTIMIZATION OF SILVER NANOPARTICLES
The optimum pH and temperature of green-synthesized AgNPs were evaluated to assist time-
and labor-effective mass production of AgNPs. The observations are explained as follows.
3.3.1 pH OPTIMIZATION
The AgNPs were analyzed in a wide pH range of 2.0–9.0, and the optimum pH that supports the
robust and true biogenic synthesis of AgNPs was found to be in the range of 6.0–9.0,
demonstrating that mildly acidic to mildly alkaline are the most favorable conditions. Early
studies on olive-mediated AgNPs indicated that the particle size of AgNPs was larger at acidic
pH than at pH 8. Fairly alkaline condition enhances the stabilizing and reducing capacity of plant
extract, allowing a rapid rate of reaction [190].
3.3.2 TEMPERATURE OPTIMIZATION
The optimum temperature was in the range of 10°–100°C, and the results indicated that the
optimum temperature magnitude for green synthesis was in the range of 70°–90°C. Any
fluctuation in the optimum pH or temperature directly affects the rate of reaction and whole
biological virtue of AgNPs. A conducive incubation temperature may yield a significant amount
of AgNPs within half an hour of the reaction. At room temperature, the AgNP synthesis is slow
and yields irregular conformation, but an increase in temperature may increase the rate of
reaction. At ≥70°C, a preferable temperature, nanospherical AgNPs were produced in lesser
reaction time, i.e., within maximum 45 min [190].
113
3.4 BIOLOGICAL / PHARMACOLOGICAL INVESTIGATION OF SILVER
NANOPARTICLES CONTRARY TO CRUDE PLANT EXTRACTS
The outcomes of the comparison between the biological/pharmacological properties of
biosynthesized AgNPs and the crude ethanolic, methanolic, aqueous, and acetonic leaf extracts
of A. americana, M. spicata, and M. indica are given below.
3.4.1 ANTIBACTERIAL ASSAY
AgNPs from A. americana leaves exhibited significant bacteriostatic and bactericidal potentials
against nosocomial pathogenic bacteria, MRSA (96%) and E. coli (95%). In addition, they
possessed good-to-moderate activity against VRSA (76%), P. mirabilis (72%), B. subtilis (64%),
P. aeruginosa (48%), and S. griseus (45%). In comparison to AgNPs, the crude aqueous extracts
of A. americana exhibited significant zone of inhibition against E. coli (91%), whereas the
methanolic crude extract exhibited good-to-excellent antibacterial efficacy against P. aeruginosa
(85%), MRSA (81%), and VRSA (77%). The crude ethanolic and acetonic leaf extracts of A.
americana exhibited good antibacterial potentials against all the test organisms, except P.
mirabilis, which was resistant to it. Furthermore, the crude ethanolic, methanolic, and acetonic
leave extracts of M. indica exhibited significant activity against S. griseus (90%, 80%, and 90%,
respectively), whereas the aqueous leaf extracts were moderately active against all the test
bacteria. In contrast, AgNPs from M. indica leaves manifested good activity against all the test
bacteria. Green AgNPs from M. spicata leaves exhibited excellent activity against E. coli (83%)
only, whereas the replication of rest of test organisms was moderately inhibited. Similarly, the
crude acetonic leaf extracts of M. spicata manifested significant activity against P. mirabilis
(80%), whereas the rest of the test bacterial species were moderately sensitive towards it, except
114
B. subtilis, which was resistant to all the fractions. All the results are summarized in (Table 3.8 –
3.10) and (Figure 3.28 - 3.30) and zones of inhibition in response to bacteria growth suppression
are depicted in (Pictures 3.30).
An earlier study has reported that the ethanolic and chloroformic extracts of A. americana L.
possess moderate antibacterial potency against E. coli, P. aeruginosa, and B. subtilis [191]. The
three biologically active compounds isolated from the aerial parts of A. americana impart the
yearned antibacterial efficacy, and they were designated as tetratriacontanol, tetratriacontyl
hexadecanoate, and 5-hydroxy-7-methoxy-2-tritriacontyl-4H-1-benzopyran-4-one [192]. In
addition, leaf extract of M. indica and its biofabricated AgNPs have excellent antibacterial
efficacy against many gram positive and gram negative bacteria, particularly B. subtilis, E. coli,
S. typhi, Streptococcus agalactiae, S. aureus, Pseudomonas fragi, and Proteus vulgaris [193 ,
194]. Similarly, essential oils from M. spicata leaves exhibit bactericidal activity against Listeria
monocytogenes [195].
115
Table 3.8: Tabular depiction of antibacterial assay by Agave americana
Bacterial Species
Standard
(mm)
Growth inhibition by Agave americana (Leaves)
AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract Z
on
e o
f In
hib
itio
n
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Bacillus subtilus 25 16 64 15 60 15 60 8 32 9 36
Escherichia coli 23 22 95 0 0 0 0 21 91 0 0
Pseudomonas aeruginosa
27 13 48 21 77 23 85 10 37 14 52
Proteus mirabilis 25 18 72 0 0 0 0 9 36 0 0
MRSA 26 25 96 20 76 21 81 0 0 16 62
VRSA 26 20 76 20 77 20 77 10 38 19 73
Streptomyces griseus 20 9 45 14 70 12 60 12 60 11 55
116
Figure 3.28: Graphical depiction antibacterial assay by Agave americana
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
6460
60
32
36
95
0 0
91
0
72
0 0
36
0
48
77
85
37
52
96
7681
0
62
76 7777
38
73
45
7060 60
55
Per
cen
t In
hib
itio
n
Agave americana
Bacillus subtilus
E.coli
Proteus mirabilus
Pseudomonas aeruginosa
MRSA
VRSA
Streptomyces griseus
117
Table 3.9: Tabular depiction of antibacterial assay by Mangifera indica
Bacterial Species
Standard
(mm)
Growth inhibition by Mangifera indica (Leaves)
AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract Z
on
e o
f In
hib
itio
n
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Bacillus subtilus 25 19 76 14 25 13 52 14 56 16 64
Escherichia coli 23 12 52 11 48 10 43 0 0 0 0
Pseudomonas aeruginosa
27 20 74 21 77 20 74 19 70 13 48
Proteus mirabilis 25 15 60 0 0 0 0 3 12 0 0
MRSA 26 18 69 22 85 18 69 15 58 18 69
VRSA 26 11 42 17 65 10 38 13 50 11 42
Streptomyces griseus 20 14 70 18 90 16 80 11 55 18 90
118
Figure 3.29: Graphical depiction of antibacterial assay by Mangifera indica
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
76
5652
56
64
5248
43
0 0
60
0 0
12
0
7477
7470
48
69
85
69
57
69
42
65
38
5042
70
90
80
55
90
Per
cen
t In
hib
itio
n
Mangifera indica
Bacillus subtilus
E.coli
Proteus mirabilus
Pseudomonas aeruginosa
MRSA
VRSA
Streptomyces griseus
119
Table 3.10: Tabular depiction of antibacterial assay by Mentha spicata
Bacterial Species
Standard
(mm)
Growth inhibition by Agave americana (Leaves)
AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract Z
on
e o
f In
hib
itio
n
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Zo
ne
of
Inh
ibit
ion
(mm
)
% I
nh
ibit
ion
Bacillus subtilus 25 5 20 0 0 0 0 0 0 0 0
Escherichia coli 23 19 83 11 48 0 0 16 70 0 0
Pseudomonas
aeruginosa 27 15 55 12 44 19 70 15 55 20 74
Proteus mirabilis 25 14 56 0 0 9 36 12 48 20 80
MRSA 26 15 58 18 69 13 50 0 0 0 0
VRSA 26 12 46 13 50 19 73 10 38 11 42
Streptomyces griseus 20 9 45 9 45 5 25 3 15 11 55
120
Figure 3.30: Graphical depiction antibacterial assay by Mentha spicata
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
20
0 0 0 0
83
48
0
70
0
56
0
36
48
80
55
44
70
55
74
58
69
50
0 0
4650
73
384245
45
25
15
55
Per
cen
t In
hib
itio
n
Mentha spicata
Bacillus subtilus
E.coli
Proteus mirabilus
Pseudomonas aeruginosa
MRSA
VRSA
Streptomyces griseus
121
(a) (b)
(c) (d)
Picture 3.30 (a,b,c,d): Pictorial depiction of antibacterial zone of inhibition formed by test
bacterial species in sensitivity response to green AgNPs and crude leaves extracts
122
3.4.2 MINIMUM INHIBITORY CONCENTRATION (MIC) ASSAY
According to the MIC assay, a minimum volume 40, 80, and 160 µL of biogenic AgNPs from A.
americana, M. spicata, and M. indica leaves can restrain proliferation of the test bacterial
species. However, the crude ethanolic, methanolic, and acetonic leaf extracts of A. americana
and M. spicata had negative medium turbidity at a concentration of 40 µL toward S. griseus
only. Furthermore, the ethanolic leaf extract of M. indica could curb MRSA proliferation at a
volumr of 40 µL. The remaining bacterial species showed relative MIC in the range of 80–320
µL. Results are summarized in (Table 3.11 – 3.13) and depicted in (Pictures 3.31).
The antibacterial MIC100 range for Bacillus cereus, E. coli, P. aeruginosa, P. mirabilis, S. typhi,
Shigella flexneri, S. aureus, and Streptococcus spp. ranged from 10 to 180 mg/mL [194]. The M.
spicata leaves and oil extracts have a MIC100 value of 32 mg/mL against P. aeruginosa [195].
123
Table 3.11: Tabular depiction of MIC assay by Agave americana
Bacterial Species
MIC values for Agave americana
(10, 20, 40, 80, 160, 320 & 640µl)
AgNPs Ethanolic Extract
Methanolic Extract
Aqueous Extract
Acetone Extract
Bacillus subtilus 80 80 80 160 160
Escherichia coli 40 160 160 40 160
Pseudomonas aeruginosa 160 80 80 160 160
Proteus mirabilis 80 320 320 160 320
MRSA 40 40 40 160 80
VRSA 80 80 80 320 80
Streptomyces griseus 80 40 40 160 40
124
Table 3.12: Tabular depiction of MIC assay by Mangifera indica
Bacterial Species
MIC values for Mangifera indica
(10, 20, 40, 80, 160, 320 & 640µl)
AgNPs Ethanolic Extract
Methanolic Extract
Aqueous Extract
Acetone Extract
Bacillus subtilus 80 160 160 160 80
Escherichia coli 80 160 160 320 320
Pseudomonas aeruginosa 80 80 80 80 160
Proteus mirabilis 80 320 320 320 320
MRSA 80 40 80 80 80
VRSA 160 160 320 160 160
Streptomyces griseus 160 160 320 320 160
125
Table 3.13: Tabular depiction of MIC assay by Mentha spicata
Bacterial Species
MIC values for Mentha spicata
(10, 20, 40, 80, 160, 320 & 640µl)
AgNPs Ethanolic Extract
Methanolic Extract
Aqueous Extract
Acetone Extract
Bacillus subtilus 160 320 320 320 320
Escherichia coli 80 160 160 80 160
Pseudomonas aeruginosa 160 160 80 160 80
Proteus mirabilis 160 320 320 160 80
MRSA 160 160 160 320 320
VRSA 160 160 80 320 160
Streptomyces griseus 40 40 40 160 40
126
(a)
(b)
Picture 3.31 (a,b): Pictorial depiction of MIC against test bacterial species
127
3.4.3 ANTIFUNGAL ASSAY
According to the antifungal evaluation, green-synthesized AgNPs from A. americana leaves
broth had a remarkable activity against Fusarium oxysporum (89%) and Verticillium (82%). It
has a good activity against Aspergillus niger (70%) and moderate activity against Penicillium
(55%). All the crude leaf extracts of A. americana exhibited moderate activity against the test
fungal strains, except for the aqueous extracts, which had significant activity against F.
oxysporum (85%). Aspergillus parasiticus was resistant toward all extracts except for the
acetonic extracts, which showed relatively good activity of 70%. AgNPs from M. indica leaves
exhibited good inhibition against A. niger (80%); however, Penicillium (51%) and F. oxysporum
(41%) were moderately inhibited. In addition, the ethanolic and acetonic extracts restrained the
proliferation of A. niger mycelia up to 74% and 35%, respectively. The aqueous extracts also
inhibited the growth of A. niger up to 61% along with Verticillium (37%), demonstrating good-
to-moderate activity. The methanolic extracts were incapable of restraining all the test fungi. In
comparison to AgNPs of A. americana and M. indica, AgNPs from M. spicata leaf broth
exhibited less activity against Verticillium (12%), whereas the crude extracts revealed moderate
activity against the tested fungi, except the acetonic extracts, which were inactive. Results are
summarized in (Table 3.14 – 3.16) and are graphically depicted in (Figure 3.31 – 3.33).
Inhibition of fungal mycelia in response to AgNPs and crude fractions are depicted in (Picture
3.32).
Earlier studies indicated that steroidal saponins, C–27, isolated from A. americana leaves
exhibited exclusive antifungal potentials against human opportunistic pathogens, primarily
Aspergillus, Cryptococcus, and Candida species [196]. Crude ethyl acetate, methanolic, and n-
butanol extracts of A. americana leaves were used to manage epidemics of Alternaria blight in
128
Brassica crops [197]. Leaf extracts of M. indica, containing active flavonoids, terpenoids, and
tannins, have a computed percent mycelial inhibition of 60%–90% against Alternaria,
Aspergillus, Macrophomina, and Penicillium species [198 , 199]. In addition, M. spicata leaves
prevent food spoilage by inhibiting growth of food borne molds, particularly, A. flavus [200 ,
201].
129
Table 3.14: Tabular depiction of antifungal assay by Agave americana
Fungal species
Standard
(mg/ml)
Percent growth inhibition by Agave americana
(Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
Verticillium
100
82
0
39
68
33
Fusarium oxysporum
100
89
54
0
85
0
Aspergillus niger
100
70
40
66
60
61
Aspergillus parasiticus
100
0
0
0
0
70
Penecillium
100
55
0
0
0
0
Figure 3.31: Graphical depiction of antifungal assay by Agave Americana
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
82
0
39
68
33
89
54
0
85
0
70
40
6660 61
0 0 0 0
70
55
0 0 0 0
Per
cen
t In
hib
itio
n
Agave americana
Verticillium
Fusarium oxysporum
Aspergillus niger
Aspergillus parasiticus
Penecillium
130
Table 3.15: Tabular depiction of antifungal assay by Mangifera indica
Fungal species
Standard
(mg/ml)
Percent growth inhibition by Mangifera indica
(Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
Verticillium
100
0
0
0
37
0
Fusarium oxysporum
100
41
0
0
0
0
Aspergillus niger
100
80
74
0
61
35
Aspergillus parasiticus
100
0
0
0
0
0
Penecillium
100
51
0
0
0
0
Figure 3.32: Graphical depiction of antifungal assay by Mangifera indica
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
0 0 0
37
0
41
0 0 0 0
8074
0
61
35
0 0 0 0 0
51
0 0 0 0
Per
cen
t In
hib
itio
n
Mangifera indica
Verticillium
Fusarium oxysporum
Aspergillus niger
Aspergillus parasiticus
Penecillium
131
Table 3.16: Tabular depiction of antifungal assay by Mentha spicata
Fungal species
Standard
(mg/ml)
Percent growth inhibition by Mentha spicata
(Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
Verticillium
100
12
19
45
64
0
Fusarium oxysporum
100
0
50
0
57
0
Aspergillus niger
100
0
38
53
43
0
Aspergillus parasiticus
100
0
0
0
0
0
Penecillium
100
32
0
0
0
0
Figure 3.33: Graphical depiction of antifungal assay by Mentha spicata
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
1219
45
64
00
50
0
57
00
38
53
43
00 0 0 0 0
32
0 0 0 0
Per
cen
t In
hib
itio
n
Mentha spicata
Verticillium
Fusarium oxysporum
Aspergillus niger
Aspergillus parasiticus
Penecillium
132
Picture 3.32: Pictorial depiction of antifungal assay against test fungal strains
133
3.4.4 ANTICANCER ASSAY
According to the anticancer evaluation against prostate cancer cell line (PC-3), it was manifested
that crude acetone extract of A. americana dynamically inhibited the proliferation of cancer cells
owing percent inhibition of 78% and IC50 value 10.96 µg/mL. Good inhibitory metastasis, 69%
was also evinced by AgNPs from leaves of A. americana owing IC50 values of 14.02 µg/mL.
Rest of crude ethanolic, methanolic and aqueous fractions remained inactive. Following A.
americana, green AgNPs, ethanolic and acetone extracts from leaves of M. spicata plant evinced
moderated anticancer aptitude i.e. 42%, 48% and 55% respectively. IC50 values estimated for
AgNPs, ethanolic and acetone extracts were 225.14 µg/mL. Moderate inhibitory competence was
also manifested by biogenic AgNPs from leaves of M. indica that is 44%. While similar to
aqueous and methanolic crude extracts of M. spicata, all crude fractions of M. indica remained
inactive against PC-3 cell line by revealing low inhibitory potentials. Results are summarized in
(Table 3.17 – 3.19) and depicted in (Figure 3.34 – 3.36).
Preliminary investigations revealed that ethanolic extract of A. americana possess effective
antitumor potency against human ovarian teratocarcinoma cell line (PA-1). IC50 values was
documented as 0.01 µg/mL [202]. Human breast adenocarcinoma cell lines (MDA-MB-231) and
(MCF-7) proliferation was moderately inhibited by leaves and kernel extracts of M. indica and
induced cytotoxicity IC50 values were documented as 15-30 µg/mL [203]. Ediriweera et al.
reported antiproliferation aptitude of M. zeylanica bark extracts against breast and ovarian cancer
cell lines by n-hexane and choloroform extract owing IC50 values upto 86.6=92.9 µg/mL [204].
Similarly, aqueous and methanolic extracts of M. spicata herb was previously evaluated against
eight varied human carcinoma cell lines (A-549, COLO-205, HCT-116, MCF-7, NCI-H322, PC-
3, THP-1 and U-87MG). From the experiment it was interpreted that methanolic extract
134
significantly inhibited COLO-205, MCF-7, NCI-H322 and THP-1 cell lines while aqueous
extract was active against HCT-116 and PC-3 [205]. In comparison to crude fractions of selected
plants, AgNPs biofabricated earlier also displayed worthwhile anticancer potency. It was
observed to agressively target hepatocellular carcinomas and colorectal cancers in mice by
inducing apoptosis [206 , 207].
135
Table 3.17: Tabular depiction of anticancer assay by Agave americana
Cancer
cell line
Control
(mg/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
PC-3 cell
line
81.97 69 15 36 25 78
Figure 3.34: Graphical depiction of anticancer assay by Agave americana
0
10
20
30
40
50
60
70
80
90
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
81.97
69
15
36
25
78
Per
cen
t In
hib
itio
n
Agave americana
PC-3 Cell line
136
Table 3.18: Tabular depiction of anticancer assay by Mangifera indica
Cancer
cell line
Control
(mg/ml)
Percent inhibition by Mangifera indica (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
PC-3 cell
line
81.97 44 37 28 39 23
Figure 3.35: Graphical depiction of anticancer assay by Mangifera indica
0
10
20
30
40
50
60
70
80
90
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
81.97
44
37
28
39
23
Per
cen
t In
hib
itio
n
Mangifera indica
PC-3 Cell line
137
Table 3.19: Tabular depiction of anticancer assay by Mentha spicata
Cancer
cell line
Control
(mg/ml)
Percent inhibition by Mentha spicata (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
PC-3 cell
line
81.97 42 48 25 15 55
Figure 3.36: Graphical depiction of anticancer assay by Mentha spicata
0
10
20
30
40
50
60
70
80
90
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
81.97
42
48
25
15
55
Per
cen
t In
hib
itio
n
Mentha spicata
PC-3 Cell line
138
3.4.5 ANTIOXIDANT ASSAY
In the antioxidant assay, AgNPs from A. americana, M. indica, and M. spicata demonstrated
moderate-to-good activity at various sample dilutions. In contrast to green AgNPs, aqueous leaf
extract of A. americana and ethanolic and broth quintessence of M. spicata demonstrated
excellent activity at the highest dilution of 300 µL (82%, 82%, and 83%, respectively).
Moreover, the methanolic extract of M. indica demonstrated good activity at 300 µL (76%). The
other crude extracts also demonstrated moderate-to-good percent absorbance at variable
dilutions. Therefore, at the highest dilution of 300 µL, radical scavenging potentials of AgNPs
and crude extracts increase considerable antioxidant activity. Results are summarized in (Table
3.20 – 3.22) and are depicted in (Figure 3.37 – 3.39) and (Picture 3.33).
Previous antioxidant studies confirmed that phenolic constituents from A. americana leaves
possess powerful radical scavenging activity [208]. Apart from leaves, the flowers also possess
significant antioxidant potentials due to increased contents of antioxidant flavonol (1210.4 µg/g
dry extract). At a concentration of 30.2 mg, its radical scavenging activity is comparable to
vitamin C [209]. Furthermore, M. indica leaf extracts potentially scavenge hydroxyl and
hypochlorous acids [210]. Leaf extracts of M. spicata also efficiently inhibited superoxides. The
total antioxidant activity presented by 20 µg/mL of ethyl acetate extract was 95%, by 30 µg/mL
of aqueous extract was 84%, and by 20 µg/mL of chloroform and n-hexane extract was 50%
[211].
139
Table 3.20: Tabular depiction of antioxidant assay by Agave americana
No. of
dilutions
(µl)
Control
(µg/ml)
Percent absorbance by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100 100 52 22 68 71 56
200 100 68 48 72 72 65
300 100 73 53 77 82 73
Figure 3.37: Graphical depiction of antioxidant assay by Agave americana
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
52
22
6871
56
68
48
72 72
65
73
53
7782
73
Per
cen
t A
bso
rba
nce
Agave americana
100µl
200µl
300µl
140
Table 3.21: Tabular depiction of antioxidant assay by Mangifera indica
No. of
dilutions
(µl)
Control
(µg/ml)
Percent absorbance by Mangifera indica (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100 100 43 15 16 44 43
200 100 62 46 46 48 48
300 100 64 50 76 50 56
Figure 3.38: Graphical depiction of antioxidant assay by Mangifera indica
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
43
15 16
44 43
62
46 46 48 48
64
50
76
5056
Per
cen
t A
bso
rba
nce
Mangifera indica
100µl
200µl
300µl
141
Table 3.22: Tabular depiction of antioxidant assay by Mentha spicata
No. of
dilutions
(µl)
Control
(µg/ml)
Percent absorbance by Mentha spicata (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100 100 40 68 71 71 46
200 100 73 72 75 77 54
300 100 79 82 78 83 55
Figure 3.39: Graphical depiction of antioxidant assay by Mentha spicata
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
40
6871 71
46
73 7275 77
54
7982
7883
55
Per
cen
t A
bso
rba
nce
Mentha spicata
100µl
200µl
300µl
142
Picture 3.33: Pictorial depiction of antioxidant activity with reference to DPPH
143
3.4.6 CYTOTOXIC ASSAY
According to the cytotoxic evaluation, it was manifested that crude ethanolic, methanolic and
aqueous fractions of A. americana exhibited exemplary 100% cytotoxic potentials at highest
concentration of 1000 µL. LD50 recorded were 17.20 µg/mL for ethanolic extract, 5.76 µg/mL
for methanolic extract and 10.52 µg/mL for aqueous extracts. While good cytotoxic activity was
exhibited by green AgNPs (77%) and acetone extracts (67%) from leaves of A. americana plant
at highest lethal concentration of 1000 µL. LD50 recorded was 0.312 µg/mL for AgNPs and
35.65 µg/mL for acetone extracts respectively. At lower sample concentration of 100 µL,
ethanolic, methanolic and aqueous extracts leaves extracts demonstrated aced activity, 80%, 86%
and 97%, while biosynthesized silver nanoparticles revealed good lethal potency, 73% and
acetone extract exhibited moderate inhibition, 50%. At least sample concentration of 10 µL,
moderate cytotoxicity was manifested by all extracts except for AgNPs, 63% and methanolic
extracts, 60%. In comparison to biofabricated AgNPs and crude extracts from A. americana,
green AgNPs from leaf extracts of M. indica appraised good activity, 73% and 77% at 100 and
1000 µL lethal concentration and LD50 recorded was 42.76 µg/mL. Crude aqueous fraction
demonstrated good activity, 60% at highest 1000 µL sample concentration and LD50 recorded
for it was estimated as 2225.63 µg/mL. At highest 1000 µL concentration, acetone and
methanolic extract revealed moderate activity, 50% and 43%. LD50 recorded was 1249.40
µg/mL and 7968.36 µg/mL. Crude ethanolic extract from leaves of M. indica demonstrated low
to no activity at all lethal concentrations. Crude methanolic extracts from leaves of M. spicata
also manifested excellent cytotoxicity at highest 1000 µL concentration and LD50 recorded was
161.85 µg/mL respectively. Moderated activity was revealed by crude ethanolic and acetone
extract, 57% and LD50 recorded was 625.17 µg/mL and 540.75 µg/mL. Biogenic silver
144
nanoparticles and aqueous extracts revealed low to negative cytotoxicity at all concentrations.
Results are summarized in (Table 3.23 – 3.25) and depicted in (Figure 3.40 – 3.42).
Early studies testified that silver nanoparticles possess cytotoxic effects on Artemia salina cysts
by positively promoting genetic damage, apoptosis, aggregation in viscera and cease hatching of
nauplii [212]. Green spherical AgNPs about 33 - 40nm in size from Sargassum ilicifolium plant
documented acute cytotoxicity against shrimp larvae manifesting LD50 value of 10 µg/mL
[213]. Leaves of A. americana were also asserted to possess cell toxicity manifesting LD50 value
of 923.10 µg/mL [202]. Similarly moderate toxicity was also demonstrated by aqueous leaf and
bark extracts of M. indica manifesting LD50 value of 5.05 µg/mL [214]. Leaf extracts of M.
spicata were also screened for cell toxicity, which testified low toxicity manifesting LD50 value
of 1701 µg/mL [215]. The concentration was further experimented on animal models which
revealed negative morphological, histopathological, hematological and biochemical alterations
[216].
145
Table 3.23: Tabular depiction of cytotoxic assay by Agave americana
No. of
dilutions
(µl)
Control
(mg/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
10 100 11 63 18 40 12 60 16 47 16 47
100 100 8 73 6 80 4 86 1 97 15 50
1000 100 7 77 0 100 0 100 0 100 10 67
Figure 3.40: Graphical depiction of cytotoxic assay by Agave Americana
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
63
40
60
47 47
7380
8697
50
77
100 100 100
67
Per
cen
t In
hib
itio
n
Agave americana
10µl
100µl
1000µl
146
Table 3.24: Tabular depiction of cytotoxic assay by Mangifera indica
No. of
dilutions
(ss)
Control
(mg/ml)
Percent inhibition by Mangifera indica (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
10 100 22 27 26 13 22 27 26 13 29 3
100 100 8 73 24 20 21 30 22 27 29 3
1000 100 7 77 23 23 17 43 12 60 15 50
Figure 3.41: Graphical depiction of cytotoxic assay by Mangifera indica
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
27
13
27
13
3
100
73
20
30 27
3
77
23
43
60
50
Per
cen
t In
hib
itio
n
Mangifera indica
10µl
100µl
1000µl
147
Table 3.25: Tabular depiction of cytotoxic assay by Mentha spicata
No. of
dilutions
(µl)
Control
(mg/ml)
Percent inhibition by Mentha spicata (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
No.
of
surv
ivors
% I
nh
ibit
ion
10 100 26 13 23 23 27 10 24 20 23 27
100 100 24 20 21 30 19 37 21 30 20 33
1000 100 21 30 13 57 5 83 20 33 13 57
Figure 3.42: Tabular depiction of cytotoxic assay by Mentha spicata
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
13
23
10
2027
20
3037
30 33
100
30
57
83
33
57
Per
cen
t In
hib
itio
n
Mentha spicata
10µl
100µl
1000µl
148
3.4.7 PHYTOTOXIC ASSAY
The phytotoxic assay indicated that AgNPs from leaves of selected plants possessed minimal
percent inhibition at minimum test sample concentration of 10 µL. The percent inhibition
amplifies when the sample concentration increases three-folds, i.e., 1000 µL. Therefore, at 1000
µL, A. americana exhibited good activity (63%), M. indica manifested excellent activity (88%),
and M. spicata exhibited moderate activity (50%). In contrast to AgNPs, crude leaf extracts also
manifested outstanding-to-good activity, particularly ethanolic, aqueous, and acetonic extracts of
M. indica, i.e., 81%, 69%, and 63%, respectively, and acetonic and methanolic extracts of A.
Americana, i.e., 63% and 50%, respectively. Finally, the crude leaf extracts of M. spicata
exhibited low activity at all sample concentrations. Results are summarized in (Table 3.26 –
3.28) and depicted in (Figure 3.43 – 3.45) and (Pictures 3.34).
149
Table 3.26: Tabular depiction of phytotoxic assay by Agave americana
No. of
dilutions
(µl)
Control
(mg/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
10 100 2 13 0 0 2 13 2 13 3 19
100 100 5 31 3 19 4 25 3 19 6 38
1000 100 10 63 5 31 8 50 6 38 10 63
Figure 3.43: Graphical depiction of phytotoxic assay by Agave americana
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
13
0
13 1319
31
1925
19
38
63
31
50
38
63
Per
cen
t In
hib
itio
n
Agave americana
10µl
100µl
1000µl
150
Table 3.27: Tabular depiction of phytotoxic assay by Mangifera indica
No. of
dilutions
(µl)
Control
(mg/ml)
Percent inhibition by Mangifera indica (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
10 100 4 25 4 25 1 6 3 19 3 19
100 100 8 50 9 56 3 19 6 38 5 31
1000 100 14 88 13 81 5 31 11 69 10 63
Figure 3.44: Graphical depiction of phytotoxic assay by Mangifera indica
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
25 25
6
19 19
5056
19
3831
8881
31
6963
Per
cen
t In
hib
itio
n
Mangifera indica
10µl
100µl
1000µl
151
Table 3.28: Tabular depiction of phytotoxic assay by Mentha spicata
No. of
dilutions
(µl)
Control
(mg/ml)
Percent inhibition by Mentha spicata (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
No.
of
dea
d
%
Inh
ibit
ion
10 100 2 13 2 13 0 0 2 13 2 13
100 100 4 25 4 25 0 0 3 19 2 13
1000 100 8 50 6 38 0 0 6 38 4 25
Figure 3.45: Graphical depiction of phytotoxic assay by Mentha spicata
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
13 13
0
13 13
25 25
0
1913
50
38
0
38
25
Per
cen
t In
hib
itio
n
Mentha spicata
10µl
100µl
1000µl
152
(a)
(b)
Picture 3.34 (a,b): Pictorial depiction of phytotoxic activity against Lemna minor
153
3.4.8 INSECTICIDAL ASSAY
The insecticidal assays revealed that at 12 h of incubation, AgNPs from aqueous leaf extracts of
A. americana showed moderate insecticidal activity against C. maculates (50%), whereas the
remaining test insects species were less sensitive toward it. In addition, AgNPs from M. indica
possessed less-to-moderate activity against C. pusillus (25%), C. maculates (42%), and O.
surinamensis (50%), whereas T. castaneum was resistant to it at the initial 12 h exposure. For the
same initial exposure time, AgNPs from M. spicata possessed less activity against C. maculates
(17%) and O. surinamensis (17%), whereas the remaining two insect species were resistant to it.
In comparison to AgNPs, at 12 h primary exposure, the crude fractions of A. americana, M.
indica, and M. spicata possessed similar less-to-moderate activity against the experimental insect
species. However, according to the 24 h observation, AgNPs and crude ethanolic extracts from
A. americana demonstrated significant activity against C. maculates (83%) and good activity
against O. surinamensis, i.e., 66% and 75%, respectively. Furthermore, the aqueous and
methanolic leaf extracts demonstrated good activity against C. maculates, i.e., 66% and 67%,
whereas the acetone extracts were inactive against T. castaneum and moderately active against
the remaining test species. The AgNPs and crude leaf extracts from M. indica demonstrated good
activity against all the test insects, except for the aqueous and acetonic extracts, which were
biologically inactive. The crude methanolic extract of M. spicata was moderately active against
T. castaneum (50%), C. maculates (42%), and C. pusillus (33%). Further, the green M. spicata
AgNPs demonstrated moderate activity against O. surinamensis (42%) and C. maculates (33%).
The aqueous extract was totally inactive, but acetonic extracts demonstrated minimum inhibition
against T. castaneum (17%) and O. surinamensis (8%). Therefore, doubling the sample exposure
154
period doubles its insecticidal potentials. Results are summarized in (Table 3.29 – 3.31) and
depicted in (Figure 3.46 – 3.51) and (Pictures 3.35).
Earlier studies on the insecticidal activity of M. spicata leaves reported that at variable extract
concentrations of 25, 50, 100, 150, 200, 250, 300, and 500 μg/mL, M. spicata leaves
demonstrated excellent insecticidal potentials against green peach aphids [Myzus persicae
(Sulzer)] [217]’/. At 600 mg/mL, the bark and leaf extracts of M. indica demonstrated significant
activity against dengue vectors, i.e., Asian tiger mosquito (Aedes albopictus) and yellow fever
mosquito (Aedes aegypti) [218].
155
Table 3.29: Tabular depiction of insecticidal assay by Agave americana N
am
e o
f O
rg
an
ism
Co
ntr
ol
(mg
/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous Extract
Acetone Extract
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
Call
oso
bru
ch
us
ma
cu
late
s
100
6
50
10
83
6
50
10
83
1
8
4
67
4
33
8
66
3
25
7
58
Tri
boli
um
ca
sta
neu
m
100
2
17
5
42
4
33
7
58
4
33
4
33
0
0
3
25
0
0
0
0
Cry
pto
lest
es
pu
sill
us
100
3
25
6
50
1
8
4
33
0
0
2
17
0
0
1
8
2
17
5
42
Ory
zaep
hil
us
suri
nam
en
sis
100
3
25
8
66
4
33
9
75
2
17
3
25
1
8
3
25
2
17
5
42
156
Figure 3.46: Graphical depiction of insecticidal assay by Agave americana at 12 hours exposure
Figure 3.47: Graphical depiction of insecticidal assay by Agave americana at 24 hours exposure
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
50 50
8
3325
17
33 33
0 0
25
80 0
1725
33
178
17
Perc
en
t In
hib
itio
n (
12
hou
rs)
Agave americana
Callosobruchus
maculates
Tribolium castaneum
Cryptolestes pusillus
Oryzaephilus
surinamensis
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
83 83
67 6658
42
58
3325
0
50
33
17
8
42
66
75
25 25
42
Perc
en
t In
hib
itio
n (
24
hou
rs)
Agave americana
Callosobruchus
maculates
Tribolium castaneum
Cryptolestes pusillus
Oryzaephilus
surinamensis
157
Table 3.30: Tabular depiction of insecticidal assay by Mangifera indica N
am
e o
f O
rg
an
ism
Co
ntr
ol
(mg
/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous Extract
Acetone Extract
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
Call
oso
bru
ch
us
ma
cu
late
s
100
5
42
9
75
6
50
9
75
4
33
7
58
2
17
4
33
3
25
7
58
Tri
boli
um
ca
sta
neu
m
100
0
0
5
42
2
17
6
50
0
0
3
25
0
0
0
0
0
0
0
0
Cry
pto
lest
es
pu
sill
us
100
3
25
7
58
4
33
5
42
1
8
4
33
0
0
3
25
0
0
0
0
Ory
zaep
hil
us
suri
nam
en
sis
100
6
50
8
67
4
33
9
75
3
25
4
33
3
25
6
50
0
0
2
17
158
Figure 3.48: Graphical depiction of insecticidal assay by Mangifera indica at 12 hours exposure
Figure 3.49: Graphical depiction of insecticidal assay by Mangifera indica at 24 hours exposure
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
4250
33
1725
0
17
0 0 0
2533
80 0
50
3325 25
0
Perc
en
t In
hib
itio
n (
12
hou
rs)
Mangifera indica
Callosobruchus
maculates
Tribolium castaneum
Cryptolestes pusillus
Oryzaephilus
surinamensis
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
75 75
58
33
58
4250
25
0 0
58
4233
25
0
6775
33
50
17
Perc
en
t In
hib
itio
n (
24
hou
rs)
Mangifera indica
Callosobruchus
maculates
Tribolium castaneum
Cryptolestes pusillus
Oryzaephilus
surinamensis
159
Table 3.31: Tabular depiction of insecticidal assay by Mentha spicata N
am
e o
f O
rg
an
ism
Co
ntr
ol
(mg
/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous Extract
Acetone Extract
12 hours
24 hours
12 hours
24 hours
12 hours
24 hours
12 hours
24 hours
12 hours
24 hours
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
Call
oso
bru
ch
us
ma
cu
late
s
100
2
17
4
33
0
0
3
25
2
17
5
42
0
0
0
0
0
0
0
0
Tri
boli
um
ca
sta
neu
m
100
0
0
0
0
0
0
0
0
3
25
6
50
0
0
0
0
1
8
2
17
Cry
pto
lest
es
pu
sill
us
100
0
0
3
25
3
25
4
33
0
0
4
33
0
0
0
0
0
0
0
0
Ory
zaep
hil
us
suri
nam
en
sis
100
2
17
5
42
2
17
4
33
1
8
3
25
0
0
0
0
0
0
1
8
160
Figure 3.50: Graphical depiction of insecticidal assay by Mentha spicata at 12 hours exposure
Figure 3.51: Graphical depiction of insecticidal assay by Mentha spicata at 24 hours exposure
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
17
0
17
0 00 0
25
08
0
25
0 0 0
17 17
80 0
Perc
en
t In
hib
itio
n (
12
hou
rs)
Mentha spicata
Callosobruchus
maculates
Tribolium castaneum
Cryptolestes pusillus
Oryzaephilus
surinamensis
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
3325
42
0 00 0
50
0
1725
33 33
0 0
4233
25
08
Perc
en
t In
hib
itio
n (
24
hou
rs)
Mentha spicata
Callosobruchus
maculates
Tribolium castaneum
Cryptolestes pusillus
Oryzaephilus
surinamensis
161
(a) (b)
(c) (d)
Picture 3.35 (a,b,c,d): Pictorial depiction of insecticidal activity against selected test insect
species
162
3.4.9 ANTI-TERMITE ASSAY
From the antitermite assay, it was confirmed that AgNPs from A. americana, M. indica, and M.
spicata leaf extracts had less activity at 12 h exposure. However, at 24 h exposure, good activity
was observed against Formosan subterranean termite, i.e., 75%, 67%, and 50%, respectively.
Analogous to AgNPs, crude leaf extracts also manifested less activity 12 h exposure, but termite
sensitivity increased at 24 h exposure, i.e., the A. americana ethanolic extracts (50%),
methanolic extracts (42%), and aqueous extracts (42%) possessed moderate activity. The M.
indica ethanolic extract (67%) demonstrated good activity, whereas the all crude extracts of M.
spicata demonstrated less activity, except for aqueous and acetonic extracts, which were
inactive. Results are summarized in (Table 3.32 – 3.34) and depicted in (Figure 3.52 – 3.54) and
(Picture 3.36).
163
Table 3.32: Tabular depiction of anti-termite assay by Agave americana N
am
e o
f O
rg
an
ism
Co
ntr
ol
(mg
/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic Extract
Methanolic Extract
Aqueous Extract
Acetone Extract
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
Fo
rmosa
n
sub
terr
an
ean
term
ite
100
4
33
9
75
3
25
6
50
3
25
5
42
2
17
5
42
2
17
2
17
Figure 3.52: Graphical depiction of anti-termite assay by Agave americana
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
3325 25
17 17
75
5042 42
17
Per
cen
t In
hib
itio
n
Agave americana
Termite (12 hours)
Termite (24 hours)
164
Table 3.33: Tabular depiction of anti-termite assay by Mangifera indica N
am
e o
f O
rg
an
ism
Co
ntr
ol
(mg
/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous Extract
Acetone Extract
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
Fo
rmosa
n
sub
terr
an
ean
term
ite
100
5
42
8
67
3
25
8
67
1
8
3
25
0
0
2
17
0
0
2
17
Figure 3.53: Graphical depiction of anti-termite assay by Mangifera indica
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
42
25
80 0
67 67
2517 17
Per
cen
t In
hib
itio
n
Mangifera indica
Termite (12 hours)
Termite (24 hours)
165
Table 3.34: Tabular depiction of anti-termite assay by Mentha spicata N
am
e o
f O
rg
an
ism
Co
ntr
ol
(mg
/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous Extract
Acetone Extract
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
12
hours
24
hours
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
No
. o
f d
ead
% I
nh
ibit
ion
Fo
rmosa
n
sub
terr
an
ean
term
ite
100
3
25
6
50
1
8
4
33
0
0
3
25
0
0
0
0
0
0
0
0
Figure 3.54: Graphical depiction of anti-termite assay by Mentha spicata
0
20
40
60
80
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
25
80 0 0
50
3325
0 0
Per
cen
t In
hib
itio
n
Mentha spicata
Termite (12 hours)
Termite (24 hours)
166
Picture 3.36: Pictorial depiction of anti-termite activity against Formosan subterranean termite
167
3.4.10 ENZYME INHIBITION ASSAY
3.4.10.1 ACETYLCHOLINE ESTERASE INHIBITION
From acetylcholine esterase inhibitory appraisal, it was evinced that biosynthesized AgNPs and
crude extracts from A. americana possess moderate AChE inhibitory effects, 58% for AgNPs,
55% for aqueous extracts, 50% for acetone extracts and 49% for ethanolic and methanolic
extracts respectively. Moderate arresting aptness was also demonstrated by green AgNPs,
ethanolic, methanolic and acetone extracts of M. indica estimated as 46%, 41%, 39% and 38%
respectively. While aqueous extracts showed low inhibitory capacity against acetylcholine
esterase enzyme, 29%. Corresponding to A. americana and M. indica, bioinspired AgNPs and
crude methanolic, ethanolic and acetone extracts manifested moderate percent enzyme inhibition,
41% for methanolic extract, 36% for AgNPs, 34% for ethanolic extract and 32% for acetone
extract. Less inhibiting aptness was manifested by aqueous extracts, 26%. Results are
summarized in (Table 3.35 – 3.37) and depicted in (Figure 3.55 – 3.57).
Early studies on silver nanoparticles revealed that they act as robust reversible inhibitors for
acetylcholine esterase and butyrylcholinesterases which effect increases when increase in
concentration [219]. In some reported cases, these AgNPs acts as potential activators for
enzymes monoamino oxidases and choline esterases [220]. Herb M. spicata was reported to be
utilized by Palestinians to treat Alzehmeir’s disease as it has potency to inhibit acetylcholine
esterase upto 94.8% [221]. Similarly, stem and bark extracts of M. indica corroborated good
AChE and histamine inhibition potency upto 74.18% [222].
168
Table 3.35: Tabular depiction of acetylcholine esterase inhibition by Agave americana
Enzyme
Control
(mg/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
Electric
eel AChE 100 58 49 49 55 50
Figure 3.55: Graphical depiction of acetylcholine esterase inhibition by Agave americana
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
58
49 4955
50
Per
cen
t In
hib
itio
n
Agave americana
AChE
169
Table 3.36: Tabular depiction of acetylcholine esterase inhibition by Mangifera indica
Enzyme
Control
(mg/ml)
Percent inhibition by Mangifera indica (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
Electric
eel AChE 100 46 41 39 29 38
Figure 3.56: Graphical depiction of acetylcholine esterase inhibition by Mangifera indica
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
4641 39
29
38
Per
cen
t In
hib
itio
n
Mangifera indica
AChE
170
Table 3.37: Tabular depiction of acetylcholine esterase inhibition by Mentha spicata
Enzyme
Control
(mg/ml)
Percent inhibition by Mentha spicata (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
Electric
eel AChE 100 36 34 41 26 32
Figure 3.57: Graphical depiction of acetylcholine esterase inhibition by Mentha spicata
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
36 34
41
2632
Per
cen
t In
hib
itio
n
Mentha spicata
AChE
171
3.4.10.2 UREASE INHIBITION
From urease inhibitory appraisal, it was corroborated that AgNPs and aqueous extracts from A.
americana exhibited good enzyme impeding capability owing 73% and 75% percent inhibition.
While moderate restraining aptness was displayed by acetone extracts 52%, ethanolic extracts
44% and methanolic extract 42%. Good urease inhibition, 63% was also manifested by crude
ethanolic leaf extracts of M. indica while moderate percent inhibition was displayed by M. indica
AgNPs 56%, acetone extract 52%, methanolic extract 51% and aqueous extract 46%.
Ecofriendly nanoparticles and crude leaves extracts from M. spicata demonstrated moderated
urease inhibition prowness owing 59% for green AgNPs, 55% for aqueous extract, 45% for
ethanolic extract, 44% for methanolic extract and 41% for acetone extract respectively. Results
are summarized in (Table 3.38 – 3.40) and depicted in (Figure 3.58 – 3.60).
Investigations conducted earlier suggests that genus Agave possess urease activating and
inhibiting properties. A. cantala and A. sisalana performed as dynamic inhibitors while A.
americana performed as potential activator but these peculiarities had not been characterized to
date [223]. Strong urease inhibiton was also manifested by M. spicata upto 70% at minimum
concentration of 10 mg/mL, which can aid to treat H. pylori infection [224]. Likewise, green
AgNPs from S. xanthocarpum berry were also documented to successfully manage H. pylori
infections by significantly inhibiting urease activity [225]. Urease sensitivity towards silver ions
leached from AgNPs in soil negatively affects agriculture sectors due to manipulation of soil
microflora and other exo-enzymes [226].
172
Table 3.38: Tabular depiction of urease inhibition by Agave americana
Enzyme
Control
(mg/ml)
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
Jack bean
urease 100 73 44 42 75 52
Figure 3.58: Graphical depiction of urease inhibition by Agave americana
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
73
44 42
75
52
Per
cen
t In
hib
itio
n
Agave americana
Urease
173
Table 3.39: Tabular depiction of urease inhibition by Mangifera indica
Enzyme
Control
(mg/ml)
Percent inhibition by Mangifera indica (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
Jack bean
urease 100 56 63 51 46 52
Figure 3.59: Graphical depiction of urease inhibition by Mangifera indica
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
56
63
5146
52
Per
cen
t In
hib
itio
n
Mangifera indica
Urease
174
Table 3.40: Tabular depiction of urease inhibition by Mentha spicata
Enzyme
Control
(mg/ml)
Percent inhibition by Mentha spicata (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
Jack bean
urease 100 59 45 44 55 41
Figure 3.60: Graphical depiction of urease inhibition by Mentha spicata
0
10
20
30
40
50
60
70
80
90
100
Control AgNPs Ethanolic
Extract
Methanolic
Extract
Aqueous
Extract
Acetone
Extract
100
59
45 44
55
41
Per
cen
t In
hib
itio
n
Mentha spicata
Urease
175
3.4.11 HEMAGGLUTINATION ASSAY
Hemagglutination evaluation revealed that the crude ethanolic, methanolic, aqueous and acetone
extracts and synthesized AgNPs from the aerial parts of A. americana, M. indica, and M. spicata
had a negative outcome at different sample dilutions of 1:2, 1:4, 1:8, and 1:16. The inability to
agglutinate experimental samples and RBC suspension into smooth buttons demonstrated the
absence of any hemagglutinin. Results are summarized in (Table 3.41 – 3.43) and depicted in
(Picture 3.37).
176
Table 3.41: Tabular depiction of hemagglutination assay by Agave americana
Blo
od
Gro
up
s
Percent inhibition by Agave americana (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous Extract
Acetone Extract
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
A+ - - - - - - - - - - - - - - - - - - - -
A- - - - - - - - - - - - - - - - - - - - -
B+ - - - - - - - - - - - - - - - - - - - -
-
B- - - - - - - - - - - - - - - - - - - - -
AB+ - - - - - - - - - - - - - - - - - - - -
AB- - - - - - - - - - - - - - - - - - - - -
O+ - - - - - - - - - - - - - - - - - - - -
O- - - - - - - - - - - - - - - - - - - - -
Note: (-) sign manifest absence of bioactive phyto-lectins
177
Table 3.42: Tabular depiction of hemagglutination assay by Mangifera indica
Blo
od
Gro
up
s
Percent inhibition by Mangifera indica (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous Extract
Acetone Extract
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
A+ - - - - - - - - - - - - - - - - - - - -
A- - - - - - - - - - - - - - - - - - - - -
B+ - - - - - - - - - - - - - - - - - - - -
-
B- - - - - - - - - - - - - - - - - - - - -
AB+ - - - - - - - - - - - - - - - - - - - -
AB- - - - - - - - - - - - - - - - - - - - -
O+ - - - - - - - - - - - - - - - - - - - -
O- - - - - - - - - - - - - - - - - - - - -
Note: (-) sign manifest absence of bioactive phyto-lectins
178
Table 3.43: Tabular depiction of hemagglutination assay by Mangifera indica
Blo
od
Gro
up
s
Percent inhibition by Mentha spicata (Leaves)
AgNPs
Ethanolic
Extract
Methanolic
Extract
Aqueous Extract
Acetone Extract
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
1:2
1:4
1:8
1:1
6
A+ - - - - - - - - - - - - - - - - - - - -
A- - - - - - - - - - - - - - - - - - - - -
B+ - - - - - - - - - - - - - - - - - - - -
-
B- - - - - - - - - - - - - - - - - - - - -
AB+ - - - - - - - - - - - - - - - - - - - -
AB- - - - - - - - - - - - - - - - - - - - -
O+ - - - - - - - - - - - - - - - - - - - -
O- - - - - - - - - - - - - - - - - - - - -
Note: (-) sign manifest absence of bioactive phyto-lectins
179
Picture 3.37: Pictorial depiction of hemagglutination activity against ABO blood group
180
CONCLUSION
From the current study findings, it can be concluded that leaves of A. americana, M. spicata and
M. indica possess cardinal phyto-chemicals such as flavonoids, flavonones, phenolic compounds,
steroids, glycosidases and saponins which contribute to reducing and capping capabilities
resulting green AgNPs. These AgNPs were purified and characterized resulting in maximum
absorbance at 430 nm for A. americana and M. indica while 410 nm for M. spicata. The
biofabricated nanostructures were further characterized as polycrystalline, monodispersed,
thermally sensitive, mostly spherical in conformation and having diameter of 30–150 nm.
Optimum pH range was recorded as 6.0–9.0 while favorable temperature was recorded as 70oC –
90oC. These biosynthesized AgNPs were biologically evaluated in contrast to aqueous, ethanolic,
methanolic and acetone leaf extracts of selected plants. From the comparative analysis it was
reckoned that green AgNPs own excellent antibacterial potentials against bacterial species such
as E.coli and MRSA. While aqueous and methanolic extracts from A. americana, ethanolic and
acetone extracts from M. indica and acetone extract from M. spicata possess remarkable
antibacterial potentials against the test organisms. Estimated MIC for AgNPs and crude extracts
against test bacteria were in the range of 40–320 µL. These bacteriostatic and bactericidal
potentials can be exploited in the field of medicine to manufacture topical creams, gels and
ointments to manage chronic cutaneous infections and burns. Excellent antifungal capacity was
also manifested against Verticillium (82%), Fusarium oxysporum (89%) and Aspergillus niger
(80%) which can also be exploited by industries to tackle issues related to verticillum and
fusarium wilts in crops and aspergillosis infection in animals. At intense 1000 µL sample
concentration, eminent cytotoxic values were exposed by ethanolic, methanolic and aqueous
extracts of A. americana (100%) and methanolic extract of M. spicata (83%). Explicit moderate
181
to good anticancer potency was manifested by both leaves extracts and biofabricated AgNPs
against prostate cancer cell line. These anti-neoplastic potentials can assist to propose novel and
economic routes to favorably tackle various forms of malignant carcinomas. At elevated
concentration of 300 µL, boosted antioxidant potentials were recorded which can be exploited in
food industries to increase shelf-life of perishable items. Similarly at elevated concentration of
1000 µL, intense phytotoxic, insecticidal and anti-termite activity was recorded which can aid in
agriculture sectors to manufacture economic and environment amicable herbicides and
pesticides. Moderate acetylcholine esterase inhibition and good urease inhibition was manifested
by green AgNPs and crude extracts which can aid to alleviate neurodegenerative, gastric, hepatic
and renal maladies. Negative hemagglutination reactions manifest absence of phyto-glutinins.
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