SYNTHESIS AND CHARACTERIZATION OF ZINC OXIDE NANOPARTICLE

FROM SOLANAUM VIRGINIANUM TO EVALUATE INHIBITION OF

BIOFILM FORMATION.

Dissertation submitted to the SASTRA Deemed to be University

in partial fulfilment of the requirements

for the award of the degree of

M.Sc., MICROBIOLOGY

Submitted by

VARSHA.S

(Reg.No.221064022)

JULY 2021

DEPARTMENT OF CHEMISTRY AND BIOSCIENCES

SRINIVASA RAMANUJAN CENTRE

KUMBAKONAM - 612 001

I
 DEPARTMENT OF CHEMISTRY AND BIOSCIENCES

SRINIVASA RAMANUJAN CENTRE

KUMBAKONAM - 612 001

Bonafide Certificate
This is to certify that the dissertation titled “Synthesis and characterization of zinc
oxide nanoparticle from solanaum virginianum to evaluate inhibition of biofilm formation”
submitted in partial fulfilment of the requirements for the award of the degree of M.Sc.,
Microbiology to the SASTRA Deemed to be University, is a bonafide record of the work done
by Ms. VARSHA.S. (Reg. No. 221064022) during the final semester of the academic year
2020-2021, in the Department of Chemistry and Biosciences, under my supervision. This
dissertation has not formed the basis for the award of any degree, diploma, associate ship,
fellowship or other similar title to any candidate of any University.

Project Supervisor :

Name :

Date :

Project Viva voce held on :

Examiner 1 II Examiner 2
 DEPARTMENT OF CHEMISTRY AND BIOSCIENCES

SRINIVASA RAMANUJAN CENTRE

KUMBAKONAM - 612 001

Declaration
I declare that the dissertation titled “Synthesis and characterization of zinc oxide
nanoparticle from solanaum virginianum to evaluate inhibition of biofilm formation”
submitted by me is an original work done by me under the guidance of Dr. S.BALAKUMAR ,
during the final semester of the academic year 2019- 2020, in the Department of Chemistry
and Biosciences. The work is original and wherever I have used materials from other sources, I
have given due credit and cited them in the text of the dissertation. This dissertation has not
formed the basis for the award of any degree, diploma, associate ship, fellowship or other similar
title to any candidate of any University.

Signature of the candidate :

Name of the candidate : Varsha.S

Date :

III
 Acknowledgements

First and foremost, I take this opportunity to thank GOD, the almighty who blessed
me with mental and physical strength for the successful completion of this project work.

I wish to express my sincere thanks to our honorable Vice-Chancellor,

Dr. S. Vaidhyasubramaniam, SASTRA Deemed to be University,
Thirumalaisamudrum, Thanjavur for giving me an opportunity to undertake my
dissertation work in SASTRA Deemed to be University, SRC, Kumbakonam.

It gives me immense pleasure to express my hearty thanks our Dean,

Prof. V. Ramaswamy, SRC, SASTRA Deemed to be University, Kumbakonam for
providing me the necessary facilities and for his support throughout the course.

This momentous task would have not seen that light of the day without the dynamic
guidance and encouragement of my guide, Dr. S. Balakumar, Assistant professor, Department
of Chemistry and Biosciences for his active involvement, stimulating suggestion and
encouragement.

I am indebted to him for his helpful advice, constructive suggestions and valuable
discussion in every step of project work and preparation of this written dissertation. The
unflinching support and the immeasurable patience displayed by him during the project work are
exemplary.

I wish to thank members of faculty , Dr. R.Sivakumar, Dr. N.Mahesh,

Dr. M. Arunkumar, Dr. S. Bhuvaneshwari, Dr. C.S. Sumathi, Dr. N. Ravichandran,
Dr. P. Sampathkumar, Dr. S. Sheik Abdulla, Dr. C.D. Venkatakrishanan,
Dr. P. Thiruvasagam,Dr. M. R. Suchitra, Dr. V. Venkataramasubramanian,
Dr. R. Renuka, Department of Chemistry and Biosciences, SRC, SASTRA Deemed to be
University for their kind help, encouragement and support.

I extend my hearty thanks to Mr. R. Vijayaragavan, Technical Assistant,

Miss. R. Nanthini and Mr. P. Santhakumar, Lab Technician, Department of Chemistry and
Biosciences, SRC, SASTRA Deemed to be University for all their help, support, interest and
valuable hints.

IV
 My feelings go beyond the limit of my language in acknowledging the cooperation,
encouragement and affection showed on me by my father Mr. T. Soundharajan and my
mother Mrs. S. Rajeswari.

A faithful friend is the medicine of life” special thanks go to my friends for their
constant support. I would like to express my gratitude for all those who gave me the possibility
to complete this thesis.

“Done” when expressed “well done” is nothing but the fullest co-operation of one and
all. Hence I wish to express my profound thanks to parents, brother, friends and well-wishers for
their help and co-operation for completing this project work successfully.

Varsha.S
 Table of Contents

Title Page No

Bona-fide Certificate II

Declaration III

Acknowledgements iv

List of Figures viii

List of Tables ix

List of abbreviations x

Abstract xi

1. Introduction 1

1.1 Metallic nanoparticles synthesis 1

1.2 Zinc oxide nanoparticular 2

1.3 Biological approaches for nanoparticle synthesis 2

1.4 Characterization of Nanoparticles 3

1.5 Ultraviolet-visible spectroscopy 3

1.6 Scanning electron microscopy 4

1.7 Energy dispersive X-ray (EDX ) 5

1.8 Plant mediated synthesis of nanoparticles 5

1.9 Biofilm formation 6

1.10 Biofilm forming bacteria 6

1.11 The role of biofilm in pathogen 6

1.12 Medicinal uses 7

2. Aim and objectives 9

vi
 3. Review of literature 10

4. Materials and Methods 13

4.1 collection of sample 13

4.2 Preparation for sample 13

4.3 Phytochemical screening 13

4.4 Quantitative analysis of phytochemicals 15

4.5 Charaterization of zinc oxide nanoparticles from solanum virginianum 16

4.6 Assessment of minimum inhibition concentration (MIC) and turbidity 17

4.7 Biofilm formation inhibition 18

5. Results and Discussion 19

6. Conclusion 35

7. References 37

vii
 List of Figures

Figure No. Title Page No.

1 Solanum virginianum 8

2 Qualitative analysis of phytochemical in Solanum virginianum 21

extract

3 Histochemical analysis of Solanum virginianum power 23

4 Synthesis of zinc oxide nanoparticles (ZnONPs) using 24

Solanum virginianum extract

5 Uv- vis.analysis of ZnONPs using Solanum virginianum 25

aqueous extract

6 Fourier transform infra-red spectral analysis of ZnONPs 26

7 Scanning electron microscopy (SEM) analysis of 27

ZnONPs

8 Histogram showing particle size distribution of ZnONPs 28

9 Energy dispersive spectroscopy (EDS) analysis of ZnONPs 29

10
Plate.1: Confocal laser scanning microscopy of bacterial 33
biofilms grown in the presence of ZnCl2, plant extract extract
and ZnONPs.

viii
 List of Tables

Table No. Table name Page No.

1 Qualitative analysis of phytochemicals in Solanum 20

virginianum power

2 Quantitative photochemical in Solanum virginianum power 20

3 Histochemical analysis of Solanum virginianum power 23

4 Fourier transform infra-red spectral analysis of ZnONPs 26

5 Determination of MIC of ZnONPs against bacterial strains 30

(Turbidity in broth)

6 Inhibition of biofilm formation 31

ix
 List of Abbreviation
NPs - Nanoparticles

ZnONPs - Zinc oxide nanoparticls

EDS - Evaporative decomposition of solution

ZnCl2 - Zinc chloride

AFM - Atomic force microscopy

MIC - Minimum inhibitory concentration

UV - Ultra Violet

XRD - X – Ray Diffraction

FT-IR - Fourier Transform Infra red

SEM - Scanning Electron Microscope

DLS - Dynamic Light Scattering

SBF - Specific biofilm formation

µg - Microgram

mL - Milliliter

nm - Nanometer

x
 ABSTRACT
In this study biosynthesis the Zinc oxide nanoparticle (ZnONPs) from plant extract of Solanum
virginianum , it eco-friendly processes to synthesis nano sized materials is major feature of
nanotechnology. Zinc chloride (0.05M) solution was mixed with plant extract for the synthesis of
Zinc oxide nanoparticle. The synthesized ZnONPs was characterized by SEM, FTIR, UV-Vis
spectroscopy, XRD and DLS. The characterization results confirmed the presence of Zinc oxide
nanoparticle (ZnONPs). The qualitative phytochemical characterization result confirmed the
presence of several phytochemicals such as Tannin, Saponin, Flavonoid, Steroids , Terpenoids
and polyphenol. The anti-biofilm activity of green synthesized Zinc oxide nanoparticles was
observed against the Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis,
Klebsiella pneumoniae and Staphylococcus aureus . These result showed that the Solanum
virginianum based ZnONPs could be potent inhibitor of bacterial biofilm.
Keywords: green synthesis, Zinc oxide nanoparticle , phytochemiclas , Anti-biofilm.

x
 CHAPTER 1

INTRODUCTION

Nanotechnology is a multidisciplinary field, as it combines the information from various

controls: science, natural chemistry, physical science, and science among others (Schmid et al.,
2006; Schmid, 2010). The expression "Nanoparticles" is utilized to portray a molecule with size
in the scope of 1nm - 100nm, in any event in one of the three potential measurements. In this size
range, the physical, substance and organic properties of the nanoparticles change in principal
ways from the properties of both individual ions/atoms and of the relating mass materials.
Nanoparticles can be made of materials of assorted synthetic nature, the most well-known being
metals, metal oxides, silicates, non-oxide ceramics, polymers, organics, carbon and
biomolecules. Nanoparticles exist in a few unique morphologies like circles, chambers, platelets,
tubes and so forth (Shakeel Ahmed et al., 2016).

Generally the nanoparticles are designed with surface modifications tailored to meet the
needs of specific applications they are going to be used for. The enormous diversity of the
nanoparticles arising from their wide chemical nature, shape and morphologies, the medium in
which the particles are present, the state of dispersion of the particles and in particular, the
various conceivable surface changes the nanoparticles can be exposed to make this a significant
dynamic field of science now-a-days. The most successfully studied nanoparticles today are
those made from noble metals, in particular Cu (copper), Ag (Silver), Zn (Zinc), Ti (Titanium),
Pt (Platinum), Au (Gold) and Pd (Palladium). Nanoparticles of noble metals, such as copper,
gold, silver, and platinum, are widely applied in products that directly come in contact with the
human body, such as shampoos, soaps, detergent, shoes, cosmetic products, and toothpaste,
besides medical and pharmaceutical applications (Ankanna et al., 2010).
1.1 Metallic Nanoparticle synthesis
Nanoparticles can be extensively assembled into two, to be specific, natural nanoparticles which
incorporate carbon nanoparticles (fullerenes) and inorganic nanoparticles which incorporates
attractive nanoparticles, honorable metal nanoparticles (like copper, gold and silver) and semi-
channel nanoparticles (like titanium oxide and zinc oxide). There is a developing interest in
inorganic nanoparticles for example of respectable metal nanoparticles (Gold and silver) as they
give predominant material properties useful flexibility. Because of their size, highlights and
1
benefits over accessible compound imaging drug specialists and medications, inorganic particles
have been inspected as expected apparatuses for clinical imaging just as for treating infections.
Inorganic nonmaterial have been broadly utilized for cell conveyance because of their adaptable
highlights like wide accessibility, rich usefulness, great similarity, and capacity of focused
medication conveyance and controlled arrival of medications (Xu et al., 2006).
1.2 Zinc oxide nanoparticles
Zinc oxide (ZnO) is utilized as a heterogeneous impetus, have a high catalytic action, non-
harmful, insoluble, and furthermore a modest impetus (Xu et al., 2006) which is a significant n-
type (Kanade et al., 2006 and Wang et al., 2016) semiconductor. ZnO isn't just a material of
particular interest therefore its unique optical and electronic properties, yet in addition it has a
few qualities including: (I) wide-band hole semiconductor (3.3 eV at room temperature) (Yusan
et al., 2016 and Flores et al., 2016), (ii) enormous exaction binding energy of 60 MeV (Kanade
et al., 2006 and Nagaraju et al., 2010) and (iii) great possibility for various potential applications,
especially as dainty film, nanowires, nanorods, or nanoparticles (Goh et al., 2011) in solar cells
gas sensor (Kanade KG, et al., 2006), photo detectors, luminescent oxides (Zareie et al., 2013),
photovoltaic, lasers, optical gadgets, sensors, cathode (Nagaraju et al., 2010 and Matsushima et
al., 2007), drug, and in the creation of shades and beautifying agents (Darezereshki et al., 2011).
The employments of ZnO as a photocatalytic debasement material has been widely
contemplated (Goh et al., 2011). Planning of nano-size ZnO has been completed by various
strategies like hydrothermal method (Xu et al., 2004), aerosol, micro- emulsion, ultrasonic, sol–
gel technique, evaporation of solution and suspensions, evaporative decomposition of solution
(EDS), solid state response, regular, conventional ceramic fabrication, wet synthetic combination,
spray pyrolysis strategy (Kanade et al., 2006 and Kale, 2016). Among these manufactured
courses, precipitation approach contrasted and other conventional strategies gives a simple
method to minimal expense and large scale production, which doesn't require costly crude
materials and confounded gear.
1.3 Biological approaches for Nanoparticle synthesis
Physical and synthetic techniques have been utilizing high radiation and profoundly thought
reductants and balancing out specialists that are destructive to natural and to human wellbeing.
Consequently, organic amalgamation of nanoparticles is a solitary advance bioreduction
technique and less energy is utilized to combine eco-accommodating NPs (Sathishkumar et al.,

2
2009). As of late, the improvement of proficient green science techniques utilizing characteristic
diminishing, covering, and balancing out specialists to plan nanoparticles with wanted
morphology and size have become a significant focal point of analysts. Natural strategies can be
utilized to union nanoparticles without the utilization of any unforgiving, poisonous and costly
synthetic substances (Ankamwar et al., 2005). The bioreduction of metal particles by mixes of
biomolecules (e.g., proteins, amino acids, polysaccharides, and nutrients) found in the
concentrates of specific organic entities is earth considerate, yet synthetically unpredictable.
1.4 Characterization of Nanoparticles
Nanoparticles are generally characterized by their size, shape, surface area, and dispersity (Mittal
et al., 2013). The common techniques to evaluate nanoparticles characteristics can be classified
into two main groups namely, quantitative and qualitative. These methods include a range of
various sophisticated techniques like, dynamic light scattering (DLS), scanning electron
microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectroscopy
(EDS), UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction
(XRD), atomic force microscopy (AFM), surface enhanced raman spectroscopy (SERS), atomic
absorption spectroscopy (AAS), high angle annular dark field (HAADF), inductively coupled
plasma (ICP) and X-ray photoelectron spectroscopy (XPS) (Rajasekharreddy et al., 2010; Mittal
et al., 2013).
1.5 Ultraviolet-Visible Spectroscopy
Ultraviolet - visible spectroscopy or ultraviolet - visible spectrophotometer (UV- Vis)
involves the spectroscopy of photons in the UV Visible region (Ramana Reddy et al., 2015). It
uses light in the visible and adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this
region of the electromagnetic spectrum, molecules undergo electronic transitions. UV-Vis
Spectrophotometers are mainly used to measure transmission or absorption in liquids and
transparent or opaque solids. It does so by sending a beam of light through the sample and then
monitoring the remaining light in a detector. In the case of a UV-Vis spectrophotometer the light
is in the wavelength of 800 - 200nm, examine electronic transitions in the sample.
It is difficult to arrive at a lower frequency than 200nm as oxygen begins to absorb light beneath
that frequency. When the light passes through the sample some of the molecules in the sample
will absorb lights at various wavelengths of this spectrum, depending on their chemical bonds
and structure (Lue and Juh Tzeng, 2007). When in doubt, vigorously preferred electron

3
advancement will be from the most highest occupied molecular orbital (HOMO) to the lowest
unoccupied molecular orbital (LUMO), and the subsequent species is called an excited state. A
spectrophotometer records the frequencies at which absorption happens, along with the degree of
absorption at every frequency. The resulting spectrum is presented as a graph of absorbance
versus wavelength (Mittal et al., 2013).
1.6 Scanning Electron Microscopy (SEM)
Scanning electron microscopy (SEM) is quite possibly the most famous and generally utilized
procedures for the portrayal of nanomaterials and nanostructures (Nagarajan et al., 2014). SEM
can be adequately used to describe examples down to a goal of a few of nanometres, with picture
amplifications reachable in the scope of ~10 to more than 300,000. In additional information on
surface geography, SEM can likewise give valuable data on science, precious stone direction and
inward pressure circulation. SEM comprises extremely fine spot size of ~5 nm. Electrons are
sped up to energy esteems in the scope of a couple hundred eV to 50 KeV, the surface of the
specimen by deflection coils As the electrons strike and penetrate the surface, various
connections that outcome in the emission of electrons and photons from the sample happen, and
SEM pictures are created by collecting the radiated electrons on a cathode beam tube (Gondal et
al., 2013). Different SEM methods are separated based on what is consequently identified and
imaged
The various parts of crystallite data that can be researched utilizing SEM are recorded
underneath:

Geography: The surface highlights of an object or 'what it looks like'; recognizable highlights are
restricted to a couple manometres.

Morphology: The shape, size and disposition of the particles making up the item that are lying on
the surface of the sample or have been uncovered by granulating or synthetic scratching;
discernible highlights restricted to a couple manometres.

Structure: The components and mixtures the sample is made out of and their relative proportions,
in regions ~ 1 micrometer in measurement and profundity. Crystallographic data: The

4
arrangement of atoms in the sample and their degree of order; just helpful on single-gem
particles >20 micrometers
1.7 Energy dispersive X-ray (EDX)
Energy Dispersive X-beam Spectroscopy (EDS or EDX) is an analytical instrument
exceptionally utilized for compound characterization.. Its portrayal abilities are because of the
central property (Wang et al., 2002) that every component of the occasional table has a
remarkable electronic design and, consequently, an extraordinary reaction to electromagnetic
waves. It relies upon the examination of an sample through communications among light and
matter, analyzing X-rays in its specific case. An EDS indicator contains a crystal that assimilates
the energy of approaching X-beams by ionization, yielding free electrons in the crystal that
become conductive and produce an electrical charge predisposition. To invigorate the emission
of character X-beams from an sample, a high-energy light emission particles, for example,
electrons or protons or a light emission beams is engaged onto the example being contemplated
(Wua et al., 2010). Very still, a molecule inside the sample contains ground state (or unexcited)
electrons in discrete energy levels or electron shells bound to the core.
The incident beam may excite an electron in an inward shell, launching it from the shell
while making an electron opening where the electron was. An electron from an external, higher-
energy shell at that point fills the opening, and the distinction in energy between the higher-
energy shell and the lower energy shell might be delivered as a X-beam (Gondal et al., 2013). As
the energy of the X-beams are normal for the distinction in energy between the two shells, and of
the nuclear construction of the component from which they were transmitted. This permits the
natural organization of the sample to be estimated (Deng et al., 2012).
1.8 Plant mediated synthesis of nanoparticles

Recently, the plant mediated nanomaterial has drawn more attention due to its vast application in
various fields due to their physic-chemical properties. The different metallic nano-particles such
as gold, silver, platinum, zinc, copper, titanium oxide, magnetite and nickel were synthesized
from natural resources and have been studied exclusively. The different parts of plant such as
stem, root, fruit, seed, callus, peel, leaves and flower are used to syntheses of metallic
nanoparticles in various shapes and sizes by biological approaches (Dubey et al., 2010). In the
present study the synthesis of zinc oxide nanoparticles using Solanum virginianum.

5
1.9 Biofilm formation

Biofilm is a relationship of micro-organisms in which microbial cells stick to one another on a

living or non-living surfaces inside a self-created matrix of extracellular polymeric substance.
Bacterial biofilm is irresistible in nature and can causes about nosocomial diseases. Biofilm
development is a multi-step measure beginning with connection to a surface then arrangement of
micro-colony state that prompts the development of three dimensional design lastly finishing
with development followed by separation. During biofilm development numerous types of
microbes can speak with each other through this mechanism system called quorum sensing. It is
an arrangement of boost to co-ordinate differ gene expression. Bacterial biofilm is less available
to antibiotics and human immune system and thus poses a big threat to public health because of
its involvement in variety of infectious diseases (Muhsin et al., 2005).

1.10 Biofilm forming bacteria

Nearly all (99.9%) of micro-organisms beings can form biofilm on a wide scope of surfaces for
example organic and latent surfaces (Sekhar et al., 2009). At the point when micro-organisms
bind to a surface, they produce extracellular polymeric substance (EPS) and structure biofilm.
Biofilm representing an incredible issue for general wellbeing because of its safe nature to
antimicrobials and illness related with inhabiting clinical gadgets (Khan et al., 2014). Biofilm
forming ability has been accounted for in large number of bacterial species like P. aeruginosa, S.
epidermidis, E. coli, S. aureus, E. cloacae, K. pneumonia. (Ma et al., 2009; Fux et al., 2005).
1.11 The role of biofilm in pathogenesis
Biofilms can be found anyplace and may affect human wellbeing both decidedly and adversely.
One illustration of a beneficial outcome incorporates the biofilms of commensal microscopic
organisms, for example, Staphylococcus epidermidis, which can hinder the colonization of
conceivably pathogenic microbes through the incitement of host-cell safe protections and the
avoidance of attachment. In any case, biofilms are all the more regularly connected with
numerous pathogenic types of human sicknesses and plant contaminations. One regular model is
cystic fibrosis, the most every now and again passed hereditary problem in Western Europe.
Cystic fibrosis (CF) patients experience the ill effects of persistent P. aeruginosa contaminations.
While contaminating the CF lung, P. aeruginosa goes through a trademark progress from an

6
intense destructive microorganism to a CF adjusted microbe, permitting it to persevere in the
lung for quite a long time or even many years
Scientific classification of Solanum virginianum L.
Kingdom : Plantae
Order : Solanales
Family : Solanaceae
Genus : Solanum
Species : virginianum

1.12 Medicinal uses

Solanum Virginianum L. (Kantkari) is a very prickly perennial plant. In ayurvedic it is utilized
in various medicial application This plant is utilized in treatment of epilepsy ,migraine, pain
relieving, headache, hair fall, bronchial asthma, skin issues, cough and different sicknesses.
Ayurvedic medication Swasari Kwath contain Kantkari alongside different plant. It is likewise
utilized in readiness of chyavanaprasam. Dashmularishta which is ayurvedic tonic contain root of
this plant. Kantkari is awesome switch tonic. Its decoction is exceptionally useful in liver
swelling and disease. It is utilized in cough, epilepsy, liver swelling and contamination, vomiting
and sickness during pregnancy, Improves odds of imagining child, Migraine, Hair fall,
Toothache.

7
 Fig.1:Solanum virginianum

8
 CHAPTER 2

AIM AND OBJECTIVE

AIM
 To synthesis and characterization of Zinc oxide nanoparticle(ZnONPs) by using plant
exact of Solanum virginianum and to evaluate inhibition of biofilm formation.
OBJECTIVE
 To synthesis nanoparticles from plant extract
 Characterization of ZnONPs by UV,FTIR,EDS and SEM
 To evaluate of biofilm formation inhibition of ZnONPs against Pseudomonas
aeruginosa, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae and
Staphylococcus aureus .

9
 CHAPTER 3

REVIEW OF LITERATURE

Jayachandran et al.,(2021) have reported that the green synthesis of zinc oxide nanoparticles
from the plant extract of Cayratia pedata. Synthesis nanoparticle confirmed by UV visible
spectrometer (UV–Vis) absorption peak was observed at 320 nm. In XRD pattern average size of
the nanoparticles is 52.24nm. (FT-IR) spectroscopic analysis peak of Zn–O bonding between
400 and 600 cm−1.Biosynthesis nanoparticles are cost effective.

Chatterjee et al.,(2021)gold nanoparticle effectively inhibit biofilm of vibrio cholera by

disturbing production and structure of cholera toxin. different size and shape of gold
nanoparticleare targeted against Vc biofilm. Spherical gold nanoparticle kill the intestinal
colonization of Vibrio cholera in gastrointestinal tract. Mice ileal loop assay used to disruption
of Cholera toxin function by gold nanoparticle.

Nguyen et al.,(2021) alpha-mangostin loaded nanoparticles inhibition of biofilm formation

Staphylococcus aureus including the methicillin-resistant strain MRSA252.The results of the
study treatment with 24 μmol/L nanoAMG inhibited the biofilm biomass by 53–61%, compared
to 41–44% for free AMG (p < 0.05).

Mahamuni-Badiger et al.,(2020) review the role of zinc oxide-based nanoparticles inhibition

biofilm formation Bacterial biofilm formed on various medical devices which include central
venous catheters, contact lenses, cardiovascular implantable devices, prosthetic joints,
intrauterine contraceptive devices, dental implants, urinary catheters and breast implants cause
persistent infections. So required to use of some antimicrobial agent which inhibit biofilm
formation in medical instruments.The application of Zinc oxide-based material in the prevention
of biofilm on the medical devices.

Liang, Ziwei, et al., AgWPA-NPs effectively inhibition of Staphylococcus aureus growth in

biofilms AgWPA-NPs destroy the structure of Staphylococcus aureus cell membrane. real-time
PCR analysis result that the expression of the biofilm-related genes, icaA, sarA and cidA, was

10
down regulated. This study result AgWPA-NPs have the higly potential to be an antibacterial S.
aureus biofilm agent.

Guisbiers et al.,(2017) in this study pure selenium nanoparticle synthesis by pulsed laser
ablation in liquids .The nanoparticle have inhibit biofilm formation by candida albicans
electron microscopy images demonstrate the selenium nanoparticles is adhere and penetrate into
the pathogen, and damage the cell structure by substituting with sulfur.
Obeizi, et al.,(2020) study the antimicrobical and antibiofilm activity by green synthesis zinc
oxide nanoparticle from essential oil of Eucalyptus globules .By using XRD, DLS, FT-IR, SEM,
EDX, TEM and UV-vis synthesis nanoparticle characterized. The size of synthesis nanoparticle
40nm is analyzed by DLS. The result show for ZnONPs is maximum zone of inhibition of
19.35 ± 0.45 mm for K. pneumoniae at a concentration of 100 μg / ml. The anti-biofilm activity
of zinc oxide nanoparticle in S. aureus ATCC 25923 and P. aeruginosa ATCC 27853 found
85% and 97%.So ZnO NPs high potential to used as alternative for antibiotics
Umamaheswari et al.,(2021) evaluation of their anticancer property in A549 cell lines and Green
synthesis of zinc oxide nanoparticles using leaf extracts of Raphanus sativus var. Longipinnatus.
Investigate synthesis nanoparticle by UV–vis, FTIR, particle size analysis, SEM, XRD and its
anticancer activity using A549 cell lines. Synthesized ZnO NPs using Raphanus sativus var.
Longipinnatus leaves extract proved to have cytotoxicity against A549 cell lines.
Jobie et al.,(2021)Biosynthesis Zinc oxide nanoparticle using Amygdalus scoparia. Spach stem
bark extract and applications as an alternative antimicrobial, anticancer, and anti-diabetic agent.
The ZnONPs showed surprising inhibitory action against P. oryzae, F. thapsinum, and F.
semitectum, E. coli, E. aerigenes, S. aureus compared to antibiotic standards.
Raja et al.,(2014) Solanum virginianum L. study the invivo antitussive activity of a pectic
arabinogalactan. Aqueous extraction protocol followed and using the biological, spectroscopic,
chromatographic and chemical method are analyzed an antitussive pectic arabinogalactan
confined from its leaves.

Patel et al.,(2019) Biosynthesis silver nanoparticle by using stem part of Solanum virginianum L
to evaluate the antibacterial (Bacillus Subtalis, Actinomysis, Proteous vulgaris, Pseudomonas
aeruginosa,) and antifungal (Aspergillus niger, Candida crusi). Phytochemical analysis of

11
Solanum virginianum L in Methanolic extract alkaloids, flavonoids, glycosides, saponins, sterols
etc. identified. total phenolic content is 127.27±0.231 and total flavonoid 88.33±0.577.

Sharifi-Rad et al., (2020) study was to evaluate the ethanolic, methanolic and water
extracts of the leaves, roots and flowers of Nepeta juncea Benth and screen the amount of
secondary metabolites. The study demonstrated that the content of total flavonoid was 41.37 ±
0.17 (mg quercetin equivalents (QE)/g dry weight), total phenol was 69.54 ± 0.31 (mg gallic acid
equivalents (GAE)/g dry weight), tannin was 47.36 ± 0.33 (mg catechin\g dry weight) and
anthocyanin was 6.52 ± 0.21 (mg cyanidin/100 g dry weight) concentrations were recorded in
methanolic extract of N. juncea leaves.
Kamalika mazumder et al., (2018) described the pharmacognostic and phytochemical
analysis by the standard monograph and to explore it’s in vivo analgesic and in vitro antioxidant
activity in ethyl acetate extract. Aerial roots of F. benghalensis have been found the source of
steroidal glycosides, cardiac flavonoids, tri-terpenoids glycosides, and phenols. The xylem,
phellem, phellogen and phloem has been found after microscopic investigation. The absence of
heavy metals were observed. The potent antioxidant activity at 100 µg/ml concentration in ethyl
acetate extract was observed and greater analgesic activity at the concentration of 400 mg/kg
than 200 mg/kg.
Fe3O4 NPs has slight antibacterial properties, and their effective concentrations reach 10–
20 mg/ml (Raghunath and Perumal, 2017).Fe 3O4 NPs against biofilms showed mostly
insignificant effects. To obtain significant antibiofilm effects, Fe 3O4 NPs have to be used in high
concentrations. It has been shown that iron-oxide NPs had the option to decrease biofilm
development by S. aureus, E. coli, P. aeruginosa, S. epidermidis (Thukkaram et al., 2014; Taylor
and Webster, 2009).

CHAPTER 4

MATERIAL AND METHODS

12
4.1 Collection of sample
The Solanum virginianum were collected in February from Kumbakonam, Thanjavur
district, Tamil Nadu, India.
4.2 Preparation for extract
1 gram of the powder of Solanum virginianum were transferred in to different conical
flask (250ml). The conical flask containing 50ml of different solution (water and methanol). The
conical flask were shaken well for 30 minutes by free hand. After 24 hrs, the extracts were
filtered using Whatman filter paper No.1 and filtrate is used for further analysis.
4.3 Phytochemical screening
Chemical tests were carried out on the plant extract (Solanum virginianum) using
standard procedures to identify the constituents as described by Sofowara (1993), Trease and
Evans (1989) and Harborne (1973 and 1984).
Test for tannins
1ml of extract was dissolved in 5ml of distilled water and heated for few minutes and
cooled. The mixture was treated with 1-2 drops of 0.1% ferric chloride (FeCl 3) solution and
observed for brownish green or a blue black coloration that indicates the presence of tannins.
Test for saponins
1ml of sample and 2ml distilled water was shaken vigorously with 8 drops of olive oil to
obtain stable persistent foam. The formation of emulsion indicates the presence of saponins.
Test for flavonoids
2.5ml of ammonia solution was added to 1 ml of extract in a test tube and few drops of
concentrated sulfuric acid was carefully added along the wall of the test tube. A yellow
coloration indicates the presence of flavonoids.
Test for steroids
0.5ml sample was added with 1ml of acetic anhydride and 2ml of concentrated sulfuric
acid. The formation of violet to blue or green indicates the presence of steroids.

Test for terpenoids

0.5ml of sample was added with 2ml of chloroform and few drops of concentrated
sulfuric acid was carefully added along the wall of the test tube to form two layers. An interface
with a reddish brown colors layer confirms the presence of terpenoids.

13
Test for triterpenoids
0.5ml of sample was mixed with 1ml of chloroform in a test tube. Then 1ml of acetic
anhydride and 2ml Concentrated sulfuric acid was carefully added along the wall of the test tube
to form a reddish violet color. It confirms the presence of triterpenoids.
Test for Alkaloids (Mayer’s test)
To 1ml of plant extract 5 drops of Mayer’s reagent was added. Creamy white precipitate
obtained which shows that the alkaloid groups are present.
Test for Anthraquinone
1ml leaf extract was mixed with 2ml of concentrated sulfuric acid and 1ml 10% ammonia
solution was added. The presence of a rose pink color indicates the anthraquinone.
Test for polyphenol
1ml of sample was added with 4ml of ethanol. The test tubes were kept in boiling water
bath for 15 minutes and cool. Then 3 drops of ferric cyanide was added. The appearance of blue
green colour indicates the presence of polyphenol.
Test for glycosides
To 1ml of plant extract, 1ml of acetic acid, few drops of 5% ferric chloride, and 1ml of
concentrated sulfuric acid was added .The formation of brown ring indicates the presence of
cardiac glycosides.
Test for coumarins
2ml of sample was added with 3ml of 10% NaOH. The appearance of yellow color show
the presence of coumarins.

4.4 Quantitative analysis of phytochemicals

Determination of total phenols by spectrophotometric method
Total phenols evaluated by the method of Edeoga et al., (2005)
Reagent

14
 1. Ether
2. Ammonium hydroxide
3. Amyl alcohol
Procedure
Plant powder (250 mg) was boiled with 10 ml of ether for the extraction of the phenolic
component for 15 min. 2.5 ml of the extract was pipetted into a 50 ml flask, then 5 ml of distilled
water was added. 1 ml of ammonium hydroxide solution and 2.5 ml of concentrated amyl
alcohol were also added. The samples were made up to mark and left to react for 30 min for
colour development. The optical density was measured at 505 nm.
Determination of Flavonoid
Flavonoid determine by the method of Bohm and Kocipai-Abyazan (1994)
Reagents
1. 80% aqueous methanol
Procedure
250 mg of the plant sample was extracted repeatedly with 10 ml of 80% aqueous
methanol at room temperature. The whole solution was filtered through Whatman filter paper No
42 (125 mm). The filtrate was later transferred into a crucible and evaporated into dryness over a
water bath and weighed to a constant weight.
Estimation of total terpenoid content
Total terpenoid content in the leaf extracts were assessed by standard method (Ferguson,
1956).
Reagents
1. Methanol
2. Petroleum ether
Procedure
250 mg of plant powder was taken separately and soaked in alcohol (10ml) for 24 hrs.
After filtration, the filtrate was extracted with petroleum ether (1: 3 ratio) for 2 hours. The dried
ether extract was evaporated by complete elimination of petroleum ether under reduced pressure.
The dried ether extract was treated as total terpenoid.

Histochemical tests (John Peter Paul, 2014; Gersbach et al., 2001) .

15
 A small quantity of dried and finely powdered sample was placed on a grease free
microscopic slide and treated with specific chemicals and reagents and waited for 1-2 minutes. A
positive test for histochemical was indicated by the appearance of the appropriate colour change
after application of the reagent. Using a light microscope to observe and record any colour
changes.
The powder of Solanum virginianum was treated with specific chemicals and reagents.
The treated plant powder further analyzed in light microscope. The Solanum virginianum
powder treated with diluted ammonia and H 2SO4 gave yellow colour indicates flavonoids. Plant
powder treated with Toludine blue to give Blue green/Red colour indicates the presence of
polyphenol. Plant powder treated with Dinitrophenol hydrazine (few drops) to give Orange
colour indicates the presence of Terpenoids.
Synthesis of Zinc oxide nanoparticles using plant extract (Thema et al., 2015)
20 ml of the aqueous extract of Solanum virginianum was added to 80ml of 0.05 M
ZnCl2. The solution was then transferred to a 200 ml conical flask and boiled at 70°C for 20 min
with a magnetic stirrer and a heater until a brown colored precipitate, which marked the
completion of the reaction, was observed. The precipitate was collected while the product was
dried in the oven at 80 °C for 6 h given a powder.
4.5 Characterization of Zinc oxide nanoparticles from Solanum virginianum
UV-Visible Spectroscopic and FTIR analysis
The Zinc oxide nanoparticles were examined under UV and visible spectrophotometer
analysis. The Zinc oxide nanoparticles were scanned in the wavelength ranging from 380-800
nm using Perkin Elmer Spectrophotometer and the characteristic peaks were detected. FTIR
analysis was performed using Spectrophotometer system, which was used to detect the
characteristic peaks in ranging from 400-4000 cm -1 and their functional groups. The peak values
of the UV and FTIR were recorded. Each and every analysis was repeated twice for the spectrum
confirmation.
Scanning Electron Microscope (SEM)
In this research work, Jeol JSM-6480 LV SEM machine was used to characterize mean
particle size and morphology of nanoparticles. The freeze dried sample of ZnONPs solution was
sonicated with distilled water and small drop of this sample was placed on glass slide and
allowed to dry. A thin layer of platinum was coated to make the samples conductive. Jeol JSM-

16
6480 LV SEM machine was operated at a vacuum of the order of 10-5torr. The accelerating
voltage of the microscope was kept in the range 10-20kV. Compositional analysis on the sample
was carried out by the energy dispersive X-ray spectroscopy (EDS) attached with the SEM.
Electron microscopy and EDX analysis of Zinc oxide nanoparticles
The particle size and morphology of nanoparticles were analyzed by ZEEISS-SEM
machine. The dried form of Zinc oxide nanoparticles were sonicated with distilled water, small
droplet of Zinc oxide nanoparticles were placed on glass slide and permitted to dry. The
ZEEISS-SEM machine was worked at a vacuum of the order of 10-5 torr. The accelerating
voltage is 10 kV. The TEM study was done by CM30–Philips at functioning voltage of 80 kV.
Compositional analysis on the sample was carried out by the energy dispersive X-ray
spectroscopy (EDS) attached with the SEM. The EDX analysis of ZnONPs sample was done by
the SEM machine.
4.6 Assessment of Minimum Inhibitory Concentration (MIC) and turbidity
Minimum Inhibitory Concentration was determine by the method of Pradeep, et al.,
(2015)
Preparation of Nutrient Media
2.8 grams of Nutrient Media was weighed.100 ml of distilled water was taken in a
conical flask and to the distilled water , the weighed nutrient media was powder was added. The
conical flask was stirred thoroughly.
Preparations of sample solutions:
Sample (ZnONPs): 2μg/ml, 4μg/ml 8μg/ml, 12/ml 16μg/ml and 20μg/ml
Inoculation of bacterial culture:
5mL of the prepared and sterilized nutrient media was poured into test tubes. To this, 1
mL of bacterial culture were inoculated (inoculums suspension contained 1 x 10 6 cells/mL). A
blank solution or control was prepared containing only the nutrient media i.e 5mL.
Procedure
To the nutrient media containing the inoculums, 1mL of 10μg/ml concentration of sample
was added at 0 minutes. Similarly, 1mL from 2μg/ml, 4μg/ml 8μg/ml, 12/ml 16μg/ml and
20μg/m were added to the respective test tubes. The procedure was repeated at intervals of 30
minutes i.e. 30 minutes and 60 minutes. This was done for all the concentrations of Sample. The
contents of each tube were mixed on a shaker at 250 rpm for 2 minutes. After carrying out the

17
above procedure, the test tubes containing the inoculums and the sample were placed in the
incubator overnight at 35°C. After 24 hrs incubation, the test tubes were taken out of the
incubator. The lowest concentration of MIC tubes with no visible bacterial growth (turbidity).
They were then subjected to UV Spectrophotometry at 540 nm.
4.7 Biofilm Formation Inhibition
The effect of extracts on biofilm formation was evaluated in 96-well polystyrene
flatbottom plates followed by the method of Antunes, et al., (2010) with minor modification.
Briefly, 300 𝜇L of inoculated fresh NB medium strains of Staphylococcus aureus (MTCC 3160),
Pseudomonas aeruginosa (MTCC 358), Escherichia coli (MTCC 119), Enterococcus faecalis
(MTCC 439) and Klebsiella pneumoniae (MTCC 4031) final concentration (106 CFU/mL) was
aliquoted into each well of microplate and cultured in presence of sublethal concentrations
(50.00µg/ml of ZnONPs, plant extract and ZnCl₂) previously determined. Wells containing
medium and those without extracts and only with water were used as controls. Plates were
incubated at 37° C for 48 h. After incubation, supernatant was removed and each well was
washed thoroughly with sterile distilled water to remove free-floating cells; thereafter plates
were air-dried for 30 min and the biofilm formed was stained during 15 min at room temperature
with 0.1% aqueous solution of crystal violet. Following incubation, the excess of stain was
removed washing the plate three times with sterile distilled water. Finally, the dye bound to the
cells was solubilized by adding 250 𝜇L of 95% ethanol to each well and after 15 min of
incubation , absorbance was measured using wavelength at 570 nm.
Biofilm determination was made using the formula SBF = (AB - CW)/G.
SBF is the specific biofilm formation,
AB is the OD570 nm of the attached and stained bacteria,
CW is the OD570 nm of the stained control wells containing only bacteria-free medium,
and
G is the OD630 nm of cell growth in broth (Niu and Gilbert, 2004).
The SBF classification categories were mentioned by Mittal et al. (2010) who mention
that strong biofilm producers (SBF index > 2.00), intermediate biofilm producers (SBF index
between 1 and 2), and weak biofilm producers (SBF index < 1.00).

18
 CHAPER 5

RESULTS AND DISCUSSION

The use of nanotechnology has stretched across various streams of basic Sciences like Physics,
Biology, Chemistry, Material Science to almost all branches of Engineering starting from
Mechanical, Electrical, Chemical, and Electronics to IT, Medicine and Robotics.
Nanotechnology has transformed science and engineering over the past two decades. The
capacity to know nanosized material has opened up a universe of potential in a verity of
industries and scientific endeavors. It has explored a wide range of applications in the field of
energy, data storage, medicine, food, agriculture and cosmetic industries (Altan et al., 2004).
Nanoparticles are mainly synthesized by chemical methods that usually involve toxic
reactants as reducing agents that further produce toxic by-products, which in turn are hazardous
to the environment. During the last decade, the development of eco-friendly alternative chemical
methods based on microorganisms like bacteria or fungi, or more recently biological molecules
extracted from plants (Di Sia, 2015). On the wide range of applications, present study to
synthesis and characterization of Zinc oxide nanoparticles.
Phytochemicals screening of Solanum virginianum
Plants supply a diversity of resources that contribute to the essential needs of both human
being and animals like eating, clothing and shelter. Among plants of economic importance are
medicinal plants. Medicinal and aromatic plants have played vital role in relieving human
tribulation (Soni et al., 2010). In the present study qualitative, quantitative and Histochemical
screening of Solanum virginianum. Show the table 1 Qualitative analysis of Solanum
virginianum aqueous and Alcoholic extract presence of Tannin, Saponin, Flavonoids, Steroids,
Terpenoids, Triterpenoids, Antroquinone, Polyphenol, Glycoside and Coumarins while Alkaloids
was absent in aqueous extract.

19
 Table.1: Qualitative analysis of Phytochemicals in Solanum virginianum extract

Extracts
S. No Phytochemicals Methanol Aqueous
1 Tannin + +
2 Saponin ++ ++
3 Flavonoids ++ ++
4 Steroids ++ +
5 Terpenoids ++ ++
6 Triterpenoids ++ +
7 Alkaloids ++ -
8 Antroquinone ++ +
9 Polyphenol ++ ++
10 Glycoside ++ +
11 Coumarins ++ ++
(-) Indicates Absence; (+) Indicates Presence; (++) Moderately present

Quantitative analysis
Quantitative analysis revealed that the Solanum virginianum has flavonoids, terpenoids
and phenol. Significant amount of flavonoids (120.00 mg/gm), terpenoids (20.00 mg/gm), and
phenol (295.00 mg/gm) were presented (Table 2). The above phytoconstituents were tested
according to the standard strategies.
Table.2: Quantitative phytochemical analysis of Solanum virginianum powder
S. No Phytochemicals Results (mg/gm)
1 Flavonoids 120.00
2 Terpenoids 20.00
3 Phenol 295.00

20
 Aqueous extract

Methanolic extract

(1.Tannin , 2. Saponin , 3. Flavonoids , 4. Steroids , 5. Terpenoids ,

6.Triterpenoids , 7.Alkaloids , 8. Anthroquinone , 9.Polyphenol , 10.Glycoside and 11.
Coumarins)

Fig.2: Qualitative analysis of Phytochemicals in Solanum virginianum extract

21
 Phytochemicals are basically parted into two gatherings that are primary and secondary
metabolites. primary metabolites incorporate general sugar, amino acids, proteins and
chlorophyll while secondary metabolites comprise of alkaloids, flavonoids, tannins etc.,
(Dhongade and Chandewar, 2013). Secondary metabolites are normal for unrivaled plants. The
fundamental quality of the predominant plants is that they have blossom and, consequently,
seeds. Natural items have organic properties, and they are described by their various uses and
applications as prescriptions, insect sprays, herbicides, fragrances or colors, among others. The
biosynthesis of secondary metabolites is generally limited to explicit phases of plant
advancement and times of stress. Some plant cells produce significant secondary metabolites of
the collaborations of the plant with the climate (insurance against hunters, microorganisms or
ecological pressure).

Histochemical analysis of Solanum virginianum powder

.Histochemistry is the part of histology managing the recognizable proof of synthetic
segments of cells and tissues , it is a useful asset for localization of trace quantities of substances
present in natural tissues. Histochemical strategies have been utilized to describe design and
development , and to contemplate time course of statement and dissemination of major
phytocompounds (Krishnan and Dayanandan 2003.)
In the current investigation, Solanum virginianum powder were treated with specific
synthetic substances and reagents (Table 3 and figure 3). This outcomes further affirmed the
presence of phytochemicals. (Paul and John Peter 2014) endeavor was taken for histochemical
and fluorescence examination of Turbinaria ornata (Turner). Histochemical examinations of the
plant were completed utilizing light microscopy and fluorescence study was dissected by UV
light. Consequences of histochemical tests showed positive response to phenol compounds,
polyphenol and tannin in the thallus.

22
 Table.3: Histochemical analysis of Solanum virginianum powder
S. No Phytochemicals Results
1 Tannin ++
2 Flavonoids ++
3 Terpenoids ++
4 Alkaloids ++
5 Glycoside ++
6 Polyphenol ++
Note: (+) Presence; (++) present with high intensity of the colour

Fig.3: Histochemical analysis of Solanum virginianum powder

Synthesis of Zinc oxide nanoparticles

The aqueous Zinc oxide when exposed to Solanum virginianum extract was reduced in solution,
there by leading to the formation of Zinc oxide hydrosol. During the visual observation, Zinc
oxide and Solanum virginianum extract stirred magnetically showed the yellow mixture after
1hr. The appearance of yellowish-brown (Fig. 4) color is clear indication for the development of
water soluble monodispersed Zinc oxide nanoparticles.

23
 Fig.4: Synthesis of Zinc oxide nanoparticle (ZnONPs) Using Solanum virginianum extract
The time duration of changes in colour varies from chemical to chemical. It is well
known that Zinc oxide nanoparticles in aqueous solution due to excitation of surface plasmon
vibrations in Zinc oxide nanoparticles (Awwad et al.,2020)
Characterization of ZnONPs
Characterization of nanoparticles is important to understand and control nanoparticles
synthesis and applications. Characterization is performed using a variety of different techniques
such as scanning electron microscopy (SEM), EDX, Fourier transform infrared spectroscopy
(FTIR) and UV–Vis spectroscopy (Vijayakumar et al..2018),( Elumalai, and Velmurugan, 2015)
Ultra violet and visible spectrometric analysis
Ultraviolet/Visible/Infrared (UV/Vis/IR) spectroscopy is a technique used to quantify the
light that is absorbed and scattered by a sample (a quantity known as the extinction, which is
defined as the sum of absorbed and scattered light). Nanoparticles have optical properties that are
delicate to sensitive to size , shape, concentration, agglomeration state, and refractive list close
the nanoparticle surface, which makes UV/Vis/IR spectroscopy an important apparatus for
distinguishing, describing, and considering these materials. Nanoparticles made from certain
metals strongly interact with specific wavelengths of light and the unique optical properties of
these materials is the foundation for the field of plasmonics.
Metal nanoparticles have free electrons, which yield a surface plasmon resonance (SPR)
assimilation band, because of the common vibration of electrons of metal nanoparticles in
reverberation with light wave. The appearances of the peaks show the characteristics of surface

24
plasmon resonance of Zinc oxide nanoparticles. In the UV–Vis spectra of the reaction mixture of
ZnCl2 solution with Solanum virginianum extract the peak was observed at 370nm (figure 5)
indicating the presence of ZnO nanoparticles which is synthesized by Solanum virginianum
extract.

Fig.5: UV-Vis. analysis of ZnONPs using Solanum virginianum aqueous extract

The peak was raised because of the impact of surface plasmon resonance of electrons in the
response combination and the expanding of peak showed that the particles are polydispersed.
Appearance of this peak indicated that the surface plasmon is well-documented for numerous
metal nanoparticles with size starting from 2nm to a 100nm (Rajabairavi et al.,2017).
Frequencies somewhere in the range of 300 and 800 nm are generally utilized for portraying
metallic nanoparticles going in size from 2 nm up to around 100 nm.
Fourier Transform Infra-Red spectral analysis of ZnONPs
FTIR spectrum of ZnO nanoparticles was scanned to identify the probable biomolecules
responsible for efficient stabilization and capping of the Zinc oxide nanoparticles synthesized by
Solanum virginianum extract. The peaks observed (Figure 6) for phytochemicals capped ZnO
nanoparticles formed through reduction by ZnONPs. The results of FTIR analysis evidenced the
presence of phenol, alcohol, alkenes, carboxylic acid, aromatics and aliphatic amines compounds
(Table 4).

25
 Fig.6:
Fourier

Transform Infra-Red spectral analysis of ZnONPs

Table.4: Fourier Transform Infra-Red spectral analysis of ZnONPs
Frequency (cm-1) Functional group
3258.34 Alcohols, Phenols
2103.18 Alkynes
1641.25 Alkenes
1260.39 Aromatic amines
708.67 Aromatics
598.97 Alkyl halides

The analysis of IR spectrum also provided an idea about biomolecules bearing different
functionalities which are present in the underlying system. The peaks observed for ZnO
nanoparticles formed through reduction by Solanum virginianum suggest the presence of
flavonoids and phenols adsorbed on the surface of ZnO nanoparticles. FTIR analysis of ZnONPs
showed the presence of alcohols, phenols, alkenes, alcohols, carboxylic acids, aliphatic amines
and aromatics compounds. This suggests the attachment of some polyphenolic components on to
ZnO nanoparticles.

26
Scanning Electron Microscopy (SEM) and Particle size analysis of ZnONPs

SEM analysis was carried out to understand the topology and the size of the ZnONPs,
which showed the synthesis of higher density polydispersed spherical ZnONPs of various
sizes that ranged from 52 to 70nm respectively as well cubic and crystalline nature of the
nanoparticles. Most of the nanoparticles gathered and only a little of them were dispersed,
when observed under SEM (Figure 7). To find out the particle size of the nanoparticles the
dynamic light scattering measurement was performed. Laser diffraction had shown that particle
size was found in the range of 34 to 96 nm (Figure 8).

Fig.7: Scanning Electron Microscopical (SEM) analysis of ZnONPs

27
 Fig.8: Histogram showing particle size distribution of ZnONPs
The histograms plotted on the obtained data to study the particle size distribution reveals
that the size of the nanoparticles ranged from 34 to 96 nm and the average particle size was
found to be 63.88 ± 17.72 nm. Overall Particle size of ZnONPs were highly distributed between
40nm to 80nm range which is the evidence that the NPs synthesized less than 100nm (NPs <
100nm).
Energy Dispersive Spectroscopy (EDS) analysis of ZnONPs

EDX were used to determined elemental composition in the reaction mixture. EDS of

ZnONPs revealed the presence of pure zinc (Zn 93.70%) and was the major constituent element

compared to oxide (O 6.30%). The EDX reading proved that the required phase of zinc (Zn) was

present in higher percentage in the ZnONPs (figure 9).

28
 Fig.9: Energy Dispersive Spectroscopy (EDS) analysis of ZnONPs

The energy dispersive spectroscopy (EDX) data show very strong Zinc and weak signals of
Oxide peak, which indicate that the reduction of zinc ions to elemental zinc possibly originated
from the molecules attached to the surface of the ZnONPs. The dense peak of Zinc strongly
confirmed the reduction of Zinc chloride to ZnO nanoparticles. Zinc peak is thicker than other
peak. This confirms the complete reduction of Zn compounds to ZnONPs as shown in the
spectrum. The EDX reading proved that the required phase of zinc (Zn) is present in the sample.

Determination of MIC of ZnONPs against bacterial strains.

Minimum inhibitory concentration (MIC) can be determined by culturing

microorganisms in liquid media or on plates of solid growth medium. A lower MIC value
demonstrates that less drug is required for inhibiting growth of the organism; consequently,
drugs with lower MIC scores are more effective antimicrobial agents. The identifying
appropriate drugs and their effective concentrations, MIC scores aid in improving outcomes for

29
patients and preventing evolution of drug-resistant microbial strains. In the present study was
determination of MIC of ZnONPs against Pseudomonas aeruginosa, Escherichia coli,
Enterococcus faecalis, Klebsiella pneumoniae and Staphylococcus aureus for bacterial strains.
Present study MIC can be determined lower MIC was Enterococcus faecalis (< 2 µg/ml)
compare with Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and
Staphylococcus aureus in ZnONPs (Table 5).
Table.5: Determination of MIC of ZnONPs against bacterial strains (Turbidity in
broth)
Concentration Bacterial strains
(µg/ml) Pseudomonas Staphylococcus Escherichia Enterococcus Klebsiella
aeruginosa aureus coli faecalis pneumoniae
2 (µg/ml) + + + - +
4 (µg/ml) + - - - -
8 (µg/ml) - - - - -
12 (µg/ml) - - - - -
16 (µg/ml) - - - - -
20 (µg/ml) - - - - -
MIC (µg/ml) 4 or < 8 2 or < 4 2 or < 4 <2 2 or < 4

+ = Growth; - = no growth

Present study concludes determined lower MIC of Enterococcus faecalis (< 2 µg/ml),
Pseudomonas aeruginosa (4 or < 8 µg/ml), Escherichia coli (2 or < 4 µg/ml), Klebsiella
pneumoniae (2 or < 4 µg/ml) and Staphylococcus aureus (2 or < 4 µg/ml) in ZnONPs. Similarly
concentrations of antibiotic that showed no bacterial growth or turbidity after the first 24 h
incubation but showed growth (on agar plate) or turbidity (in tube) after the addition of equal
volumes of sterile nutrient broth and further 24 h incubation, were said to have minimum
inhibitory concentration, while tubes with least antibiotic concentration that showed no growth
or turbidity by Chikezie Ihebuzoaju Owuama (2017).

30
Evaluation of biofilm formation inhibitions of ZnONPs against bacterial strains.

In nature, microorganisms exist fundamentally by connecting to and developing after

living and lifeless surfaces. These surfaces may take numerous structures, remembering those
found for soil and aquatic systems, those on the range of inhabiting clinical gadgets, and those of
living tissues, for example, tooth lacquer, heart valves, or the lung, and center ear. The basic
component of this connected development state is that the cells create a biofilm. Biofilm
arrangement is an interaction whereby microorganisms irreversibly connect to and develop on a
surface and produce extracellular polymers that work with connection and matrix development
(Donlan, Rodney 2001).
The aim of present study evaluation of biofilm formation inhibitions activity of ZnONPs
against Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Klebsiella
pneumoniae and Staphylococcus aureus for bacterial strains. Biofilm formation inhibition
results by addition of ZnCl2, plant extract and ZnONPs against Pseudomonas aeruginosa,
Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae and Staphylococcus aureus
indicated that the obtained effect. The best biofilm formation inhibitions high observed in
ZnONPs compare with ZnCl2 and plant extract (Table 6).

Table.6: Inhibition of biofilm formation

50 (µl)
Bacterial strains ZnCl2 Plant extract ZnONPs
Pseudomonas 0.623 0.139 0.037
aeruginosa (SBF)
Staphylococcus 0.607 0.489 0.209
aureus (SBF)
Escherichia coli 0.333 0.166 0.064
(SBF)
Enterococcus 0.177 0.129 0.123
faecalis (SBF)
Klebsiella 0.073 0.048 0.016
pneumoniae (SBF)

31
Note: The Specific biofilm formation (SBF) classification categories were mentioned by Mittal
et al. (2010) who mention that strong biofilm producers (SBF index > 2.00), intermediate biofilm
producers (SBF index between 1 and 2), and weak biofilm producers (SBF index < 1.00).
The best biofilm formation inhibitions high observed in ZnONPs compare with ZnCl2
and plant extract. Similar results were reported by Issac Abraham et al. (2011) who reported that
methanolic caper extract significantly inhibited biofilm formation and EPS production in E. coli,
Serratia marcescens, Pseudomonas aeruginosa, and Proteus mirabilis. The SBF classification
categories were mentioned by Mittal et al. (2010) who mention that strong biofilm producers
(SBF index > 2.00), intermediate biofilm producers (SBF index between 1 and 2), and weak
biofilm producers (SBF index < 1.00).

32
 Klebsiella pneumoniae

Plate.1: Confocal laser scanning microscopy of bacterial biofilms grown in the presence
of ZnCl2, plant extract extract and ZnONPs.

The development and persistence of surface-attached microbial communities, known as

biofilms, are responsible for 75% of human microbial infections (National Institutes of Health).
Biofilm lifestyle confers several advantages to the pathogens, notably during the colonization

33
process of medical devices and/or patients’ organs. In addition, sessile bacteria have a high
tolerance to exogenous stress including anti-infectious agents. Biofilms are highly competitive
communities and some microorganisms exhibit antibiofilm capacities such as bacterial growth
inhibition (Miquel et al., 2016). Present study good biofilm formation inhibitions against
Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae and
Staphylococcus aureus in ZnCl2, plant extract and ZnONPs.

34
 CHAPTER 6

SUMMARY AND CONCLUSION

Nanotechnology manages the production and utilization of materials with size of up to

100 nm. They are broadly utilized in various cycles that incorporate material science,
agribusiness, food industry, restorative, clinical, and symptomatic applications. Zinc is a
fundamental mineral of "extraordinary biologic and general wellbeing significance. Zinc oxide
nanoparticle is one such inorganic metal oxide which satisfies all the above prerequisites, and
thus, it can securely be utilized as medication, additive in bundling, and an antimicrobial agent. .
In the present study to synthesis and characterization of ZnONPs from Solanum virginianum to
evaluation of biofilm formation inhibitions.
The conclusion obtained from the study Qualitative analysis of Solanum virginianum aqueous
and Alcoholic extract presence of Tannin, Saponin, Flavonoids, Steroids, Terpenoids,
Triterpenoids, Antroquinone, Polyphenol, Glycoside and Coumarins while significant amount of
flavonoids, terpenoids and total phenol were quantified and histochemical analysis of terpenoids,
flavonoids, polyphenol, alkaloids, glycoside and tannin was further confirmed the presence of
phytochemicals. The appearance of brown color in Solanum virginianum extract treated with
zinc chloride indicates the formation of Zinc oxide nanoparticles. UV Visible spectrum analysis
of synthesized nanoparticles absorbance occurs at 370 nm indicates the nanoparticles FTIR
analysis of synthesized nanoparticles confirmed the presence of functional groups regarding
phenols, aromatic and alkyl halides, etc. SEM analysis was carried out to understand the
topology and the size of the ZnONPs, which showed the synthesis of higher density
polydispersed spherical ZnONPs of various sizes that ranged from 52 to 70 nm and
average size 63.88 ± 17.72 nm. EDX were used to determined elemental composition in the
reaction mixture. EDS of ZnONPs revealed the presence of pure zinc (Zn 93.70%) and was the
major constituent element compared to oxide (O 6.30%). The EDX reading proved that the
required phase of zinc (Zn) was present in higher percentage in the ZnONPs. MIC was
determined and showed lower MIC was found in Enterococcus faecalis compared with other
bacterial strains. The potential of biofilm formation inhibitions of zinc oxide nanoparticles

35
confirmed against Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Klebsiella
pneumoniae and Staphylococcus aureus.
From the above results it was concluded that Solanum virginianum extract as a source of
green synthesis for the zinc oxide nanoparticles and possess potential biofilm formation
inhibitions.

36
 CHAPTER 7

REFERENCES

Agarwala, M., Choudhury, B., & Yadav, R. N. S. (2014). Comparative study of antibiofilm
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