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INTRODUCTION Nanotechnology is a fascinating word in the field of current science and research

April 2, 2019 0 Comment

INTRODUCTION

Nanotechnology is a fascinating word in the field of current science and
research. Nanotechnology deals with dimensions and tolerances of less than 100
nanometers (nm), especially the manipulation of individual atoms and molecules.
Nanotechnology became an intense area of scientific research due to its potent
applications in biomedical, optical and electronic fields, among others. Business
Communications Company (BCC) proposed that in the year 2013 the nanotechnology
industry was approximately 7.6 billion USD market. With the growing business impact
of nanotechnology, it was further expected to have a potential rise up to 1 trillion USD
by 2020 (Sabourin, 2015).

The U.S. National Nanotechnology Initiative (NNI) defines nanotechnology as:
“The understanding and control of matter at dimensions between approximately 1 and
100 nanometers, where unique phenomena enable novel nanotechnology applications.
Encompassing nanoscale science, engineering, and technology, nanotechnology
involves imaging, measuring, modeling, and manipulating matter at this length scale. A
nanometer is one-billionth of a meter. Nanoscale dimension is approximately between
1 and 100 nanometers. Unusual physical, chemical, and biological properties may rise
in nanomaterials. These properties of nanoparticles differ in specific ways from the
single atoms or molecules and properties of bulk materials”.

Nanotech products that are available in today’s market today includes
gradually improved products (using evolutionary nanotechnology) where some form of
nano-enabled materials (such as carbon nanotubes, nanocomposite structures or
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nanoparticles of a particular substance) or nanotech process (e.g. quantum dots or
nanopatterning for medical imaging) is used in the manufacturing process.
Nanoparticles are of great scientific interest since they are effective bridge between
bulk materials and atomic or molecule at structures. Nanoparticles are known to
possess unexpected visible properties due to their small size that confine their electrons
and produce quantum effects. The nanoparticles possess a unique physical, chemical
and biological properties due to their relatively high surface area to volume ratio,
increased reactivity or stability in chemical process and enhanced mechanical strength
(Anu Mary Ealia, 2017). The nanoparticles are of different shape, size and structure.
The surface of the nanoparticles includes surface variations which may be uniform or
irregular. Some nanoparticles are crystalline or amorphous with single or multicrystal
solids either loose or agglomerated (Machado, 2015).

Metal and metal oxide nanoparticle have unique properties compared to the
bulk material which has recently become the focus of current scientific research. Metal
nanoparticles are synthesised either by destructive or constructive methods. The metals
used for nanoparticle synthesis include Gold (Au), Silver (Ag), Copper (Cu), Iron (Fe),
Zinc (Zn), Cadmium (Cd), Cobalt (Co), Lead (Pb) and Aluminium (Al). Metal oxide
nanoparticles possess increased reactivity and efficiency when compared to their metal counterparts. This includes Cerium oxide(CeO2), Silicon dioxide (SiO2), Iron oxide (Fe2O3), Zinc oxide (ZnO), Magnetite (Fe3O4), Titanium di oxide (TiO2) and Aluminium oxide (Al2O3).

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Titanium di oxide (TiO2) persuaded the research front, due to its excellent photocatalytic activity. Researchers further initiated fabrication of nanoparticles with
various morphologies including nanowires, nanorods, nanotubes and many other
nanostructures. Extensive methods are being developed to synthesis nanoparticles with
enhanced physical, chemical, optical and mechanical properties at low cost. These
include UV irradiation, chemical vapour deposition, pyrolysis, laser ablation and
photochemical reduction which are expensive and involves use of toxic chemicals.
These toxic chemicals might pass down to nanoparticles that pose threat to the
environment and living organims. Therefore, there is a compelling demand in the
development of eco friendly and sustainable synthesis. Recently, synthesising
nanoparticles using microorganisms and plants has been recognised as greener and
efficient way of synthesis.

The biosynthesis of nanoparticles is regarded as a rapid, ecofriendly and easily
scaled-up technology. Metal nanoparticles synthesised using microorganisms and plant
extracts are found to be stable and monodispersed using controlled synthetic parameters,
such as pH, temperature, incubation period, and mixing ratio. Recently, biosynthesised
nanoparticles were suggested to be therapeutically more active than chemically or
physically synthesized nanoparticles. Of all the biosynthesised nanoparticles, those that
are produced by extracts of medicinal plants have been found to be pharmacologically
active. This pharmacological activity might be due to the attachment of several
bioactive compounds from the plant extracts (Singh, 2016).

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Phytonanotechnology was considered advantageous because it is eco-friendly, safe,
rapid, fast, simple and inexpensive. Synthesizing nanoparticles using the aqueous
solvent of the plant extract, as a reducing medium is advantageous since they are
biocompatible (Noruzi, 2015). There is a need to decode the exact mechanism and the
components involved in biosynthesis of nanoparticles using plant extracts. It has been
suggested that amino acids, proteins, vitamins, organic acid, as well as secondary
metabolites such as terpenoids, flavonoids, polyphenols, alkaloids, heterocyclic
compounds and polysaccharides might have involved in reduction of metal salts and
furthermore believed to act as stabilizing and capping agents for synthesized
nanoparticles (Duan, 2015).

Marine organisms possess unique bioactive components due to their complex
marine environment. In an aqueous environment, solubility in water plays an important
role in the communication between marine organisms. As a consequence, the chemical
compounds used in the communication and perpetual competition can have complex
structures and produce an enormous range of biological activity. Around 1,14,000
extracts from an estimated 35,000 plant samples have been screened against a wide
range of cancers by The National Cancer Institute (NCI) of the United States of
America (USA)(Cragg, 1996). Of the 92 anti-cancer drugs commercially available
approximately 62% can be related to natural origin (Cragg, 1997). Though the marine
environment represents 95% of the biosphere, marine compounds are less explored in
the current pharmacopoeia. It is further expected that the marine environment will
become a hub of remarkable novel compounds in future. In current pharmacopoeia,
marine compounds are less exploited, it is anticipated that the marine environment will
become an invaluable source of novel compounds in the future, as it represents 95% of
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the biosphere (Jimeno, 2004). Marine floras include microflora (actinobacteria, bacteria,
fungi and cyanobacteria), microalgae, macroalgae (seaweeds), and flowering plants
(mangroves and other halophytes).

Seaweeds also known as marine macroalgae, are a group of photoautotrophic,
multi-cellular algae occurring in marine environments. Marine algae are used for many
purposes, such as in the food industry, animal feeding, medicine, the cosmetic industry,
and for soil enrichment. Apart from these colloids, there is a remarkable array of
bioactives in current use and development. These include lectins, polyphenols, sulfated
polysaccharides, terpenes, fatty acids, proteins, and several other bioactives. These
seaweed derivatives have emerged in recent years as a rich and important source of
natural bioactive compounds, and, for this reason, the production and applications of
these bioproducts as therapeutic agents in the pharmacological industry have been the
subject of intense research (Fenical, 1983). Thus the current research interest is in this
area of drug development. All of the macroscopic seaweeds red, brown, green, and
blue-green algae possess structurally unusual, biologically active metabolizers.
Compounds that show impressive antibiotic activities, and a number of unique
metabolizers that show impressive cytotoxicities, have been isolated from algae. Being
a potent sources of vitamins, protein, minerals and iodine, they have bioactive
compounds that possess anticancer activity (Mans, 2000)

Many researchers have suggested synthesis of nanoparticles using the
marine seaweeds to be safe, rapid, low cost and eco friendly due to their unique
bioactive compounds. Though there are many research in metal nanoparticle synthesis,
yet they are confined to silver and gold nanoparticles. Similarly only few reports have
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been made in metal oxide nanoparticle synthesis using marine algae. Compared to other metal oxide nanoparticles TiO2 has attracted much attention due to its photocatalytic activity. TiO2 (one of the most important compounds in nanotechnology) have also been obtained by greener methods. Titanium oxide nanoparticles (100–150 nm) have
been prepared from titanium isopropoxide solution using nyctanthes leaf extract (Sundrarajan, 2011). Alternatively, TiO2 nanoparticles (25–100 nm) have been prepared by using 0.3% aqueous extract prepared from latex of Jatropha curcas
(Hudlikar, 2012).

The advent of nanoparticles has led to wide range of applications including
biomedical, optical, electronics, therapeutic and bioremediation. Most highly used
nanoparticles, around 37% of the total Nanotechnology Consumer Products in the
world market constitute of inorganic nanoparticles that includes both metal
nanoparticles (MNPs), and metal oxide nanoparticles (Vance, 2015). Among these, the
most extensively produced nanoparticle is titanium dioxide due to it’s wide application
in various consumer products like sunscreens, toothpaste and cosmetics and industrial
catalysis and degradation of pollutant in wastewater (Shi, 2013)

Despite the numerous advantages there are concerns about their
bioaccumulation and toxicity. Nanoparticles if commercialized, will be liberated into
the environment, this might pose possible risks. In accordance, until 2013, 1814
nanoparticles products are commercially available in the market (Vance, 2015). The
regulatory aspects of nanoparticle should also be considered in nanotoxicity. It is
recommended to highlight the increased likelihood of nanoparticle exposure and their
harmfull effects on consumers and environment (Maynard, 2011). Thus better
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understanding of the nanoparticle toxicity becomes inevitable, before the use of
nanoparticles as therapeutic drug. Assessment of nanoparticle toxicity using in vivo
mammalian model is particularly of much importance.
Zebrafish model is one of the best models to assess the nanoparticle toxicity
due to its unique set of characteristics like easy handling, low maintenance, very high
reproducibility, rapid development, embryo transparency and genetic similarity to
humans. The disruption of endocrine system, gills and skin by nanoparticles is another
parameter to understand nanoparticle induced toxicity. Compared with other model
organisms, such as the fruit fly Drosophila melanogaster and the worm Caenorhabditis
elegans, there is strong conservation between zebrafish and humans, which makes
zebrafish an excellent model organism for studying complex biological processes, such
as generation of the nervous system, kidney, heart, hematopoietic system, and
notochord as well as assessing angiogenesis, apoptosis, and toxicity response (Chen,
1996; Granato, 1996).

Thus, the present study focused on the synthesis, characterisation and invivo toxicity assessment of TiO2 nanoparticles, using the seaweed, Caulerpa racemosa. The seaweed, Caulerpa racemosa was collected from the south east coast of India with an intent to synthesis TiO2 nanoparticles capable of exhibiting antioxidant and anticancer activity. TiO2 nanoparticles was efficiently synthesised and stabilised using the seaweed Caulerpa racemosa. TEM and SEM images of TiO2 nanoparticles revealed the shape, size and structure as bimorphic: tetragonal and rod shaped, with an average
particle size of 21.4nm and anatase crystal structure. The surface of the biosynthesised TiO2 nanoparticles revealed various bioactive compounds of seaweed origin. Further the biosynthesised TiO2 nanoparticles exhibited potential antioxidant and anticancer
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activity. In this regard, the nanotoxicity of the TiO2 nanoparticles was assessed invivo using zebrafish model. The histopathological study of the gill, liver and brain tissues
revealed no remarkable morphological changes with repect to nanoparticle exposure. The present study was designed to synthesis and characterise TiO2 nanoparticles using the seaweed, Caulerpa racemosa, Phytochemical profiling and identification of
bioactive compounds using UV-Vis, FTIR and GC-MS in the seaweed (Caulerpa racemosa), study on antioxidant and anticancer activity of TiO2 nanoparticles and in vivo assessment of nanotoxicity using zebrafish.

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2. REVIEW OF LITERATURE
2.1 NANOTECHNOLOGY

Nanotechnology converge knowledge from material science, physics,
chemistry, biology, health sciences, engineering and technology conducted at the
nanoscale of about 1 to 100nm. This deals with the synthesis, modulation and
application of nanoparticles that can be used across all other science fields such as
chemistry, biology, physics, material science and engineering (Rao et al., 2015).
Nanotechnology is sometimes referred to as a general-purpose technology. This is due
to its advanced form that have significant impact in almost all industries and in all
walks of life. This technology will offer better built, longer lasting, cleaner, safer and
smarter products for the home, for communications, for medicine, for transportation,
for agriculture and for any industry in general. The crucial element of nanotechnology
is synthesis and modification of nanoparticles that are made up of metals, metal oxides
or carbon (Hasan et al., 2015).

There are two main reasons for The properties of materials can be different in
nanoscale: First, nanomaterials have a relatively larger surface area when compared to
the same mass of material produced in a larger form. Materials can be made more
chemically reactive by this (in some cases materials that are inert in their larger form
are reactive when produced in their nanoscale form), and affect their or electrical
strength properties.

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Second, quantum effects can begin to dominate the behavior of matter at the
nanoscale – especially at the lower end, affecting the optical, electrical and magnetic
behavior of materials. Materials can be produced are nanoscale in one dimension (for
example, very thin surface coatings), in two dimensions (for example, nanowires and
nanotubes) or in all three dimensions (for example, nanoparticles) (Anu Mary Ealia et
al., 2017).

2.2 HISTORY OF NANOTECHNOLOGY

The nanoparticles history dates back to 9th century in Mesapotamia. Artisans
used these to produce a glittering effect on the surface of pots. This glitter over pottery
is because of a metallic film that was applied to the transparent surface of a glazing.
Artisans added silver oxides and gold and vinegar on the glazed pottery. They would
then keep these in a kiln at a temperature of 600°C.With the heat, the glaze would
soften and migrate into the outer layers with the ions to glaze.

In 1959, Richard P Feyman, an American Physicist in his famous lecture
“There’s Plenty of Room at the Bottom” described the ability to manipulate individual
atoms and molecules. Later in a 1974 conference, a Japanese scientist, Norio Taniguchi
of Tokyo University of Science first used the term nanotechnology and defined that it
mainly consists of the processing of, separation, consolidation, and deformation of
materials by one atom or one molecule (Nori et al., 1974).

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The golden era of nanotechnology began in 1980s when Eric Drexler (1981)
encountered Feynman’s provocative 1959 talk “There’s Plenty of Room at the Bottom”
while preparing his initial scientific paper on the subject, published in the Proceedings
of the National Academy of Sciences “Molecular Engineering: An approach to the
development of general capabilities for molecular manipulation,”. His vision of
nanotechnology is often considered as molecular manufacturing or Molecular
nanotechnology .

Fullerenes were discovered in 1985 by Harry Kroto, Richard Smalley,
and Robert Curl, who together won the 1996 Nobel Prize in Chemistry (Kroto et al.,
1985, Adams et al., Wade et al., 2005). The discovery of carbon nanotubes is largely
attributed to Sumio Iijima of NEC in 1991, although carbon nanotubes have been
produced and observed under a different conditions prior to 1991 (Monthioux et al.,
2006). Iijima’s discovery of multi-walled carbon nanotubes in the insoluble material of
arc-burned graphite rods in 1991 (Iijima et al., 1991) and Mintmire, Dunlap, and
White’s independent prediction that if single-walled carbon nanotubes could be made,
then they would exhibit remarkable conducting properties (Mintmire et al., 1992)
helped create the initial spark that is now associated with carbon nanotubes.

In the early 1990s Kraetschmer and Huffman, of the University of Arizona,
discovered the process of synthesize and purification of large quantities of fullerenes.
This unfolded their characterization and functionalization by hundreds of researchers in
government and industrial laboratories. In the Materials Research Society meeting held
in 1992, Dr. T. Ebbesen (NEC) described his discovery and characterization of carbon
nanotubes that enthralled the audience. Using the same or similar tools as those used by
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Huffman and Kratschmer, hundreds of researchers further developed the field of
nanotube-based nanotechnology (Bethune et al., 1993).

The beginning of 21st century saw an increased interest in the emerging fields
of nanotechnology. The National Nanotechnology Initiative is a United States federal
nanotechnology research and development program that serves as the central point of
communication, cooperation, and collaboration for all Federal agencies engaged in
nanotechnology research, bringing together the expertise needed to advance this broad
and complex field.

The project on Emerging nanotechnologies (2008) states that the early 2000s
saw the beginnings of the use of nanotechnology in commercial products that
include titanium dioxide and zinc oxide nanoparticles in cosmetics, sunscreen and some
food products. Similarly silver nanoparticles are utilised in food packaging, clothing,
household appliances and disinfectants, while cerium oxide as a fuel catalyst and
carbon nanotubes in production of stain-resistant textiles.

2.3 NANOPARTICLES

Nanoparticles are anthropogenic in nature with a dimension range of 1-100nm.
Nanoparticles exhibit unique properties compared to their bulk, due to the high surface
area to volume ratio that increases its reactivity, stability and mechanical strength (Cho
et al., 2013). The nanoparticles have different size, shape, and dimensions that can
either be zero (nanodots), or one (graphene), or two (nanotubes) or three (nanorods)
dimensional. It can be spherical, tubular, cylindrical, conical, flat, spiral or irregular in
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shape with uniform or irregular surface variations. Some nanoparticles may be
crystalline or amorphous, as monodispersed or aggregated single or multi crystals
(Machado et al., 2015). Nanoparticles are being used in various industry that includes
medicine, cosmetics, catalysis, biomedical, paint, paper electronics, optics, food,
construction, clean energy and many more. Nanoparticles are synthesised and modified
by various methods to enhance the optical, physical and chemical properties and also to
reduce the production cost (Dubchak et al., 2010). It has been reported that more than
1,800 consumer products worldwide are ENM-related up till 2015 (Vance et al., 2015) .
The global market value of ENMs was $3.4 billion in 2014 and is predicted to reach
$ 11.8 billion by 2025 (Lai et al., 2018).

2.4 CLASSIFICATION OF NANOPARTICLES

Nanoparticles are classified as organic, inorganic and carbon based.
Organic nanoparticles – They are present in nature, non-toxic and biodegradable.
Liposomes, dendrimers, ferritin and micelles are some of the known organic
nanoparticles. Liposomes and micelles have a hollow core which makes it a suitable
choice for drug delivery (Tiwari et al., 2008).

Figure 1. Organic nanoparticles (Mary Ealias and Saravanakumar 2017)
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2.4.1 INORGANIC NANOPARTICLES

Metal based – Nanoparticles that are derived from metals either by constructive
or destructive methods. Many metals are used to synthesise nanoparticles which
includes Gold (Au), Silver (Ag), Copper (Cu), Iron (Fe), Zinc (Zn), Cadmium (Cd),
Cobalt (Co), Lead (Pb) and Aluminium (Al) (Salavati-niasari et al., 2008)
Metal oxides based – Metal oxide nanoparticles are modified metal nanoparticles for enhanced efficiency and reactivity. This includes Cerium oxide(CeO2), Silicon dioxide (SiO2), Iron oxide (Fe2O3), Zinc oxide (ZnO), Magnetite (Fe3O4), Titanium di oxide (TiO2) and Aluminium oxide (Al2O3) (Tai et al., 2007).

2.4.2 CARBON BASED NANOPARTICLES

Carbon nanoparticles are completely made of carbon, that forms fullerenes,
graphenes, carabon nanotubes and carbon nanofibers.

Figure 2. Carbon based nanoparticles (Mary Ealias and Saravanakumar 2017)

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2.5 SYNTHESIS OF NANOPARTICLES

Nanoparticles are synthesised by various methods that are classified into Top
down and bottom up methods.
Top down method – This is also known as destructive method which involves
breaking up of bulk particles into nanosized particles. Mechanical milling, laser
ablation, nanolithography, sputtering and thermal decomposition are some of the
common methods (Hulteen et al., 1999; Amendola et al., 2009; Shah et al., 2006;
Yadav et al., 2012; Pimpin et al., 2012).
Bottom up method – This is known as constructive method in which build up
of material from atoms to nanosized particles. Sol-gel, chemical vapour deposition,
spinning, pyrolysis and biosynthesis are some of the common methods (Mann et al.,
1997; Mohammadi et al., 2014; Motoaki et al.,2003; Amato et al., 2013; Kuppusamy et
al, 2014).
Figure 2. Synthesis of nanoparticles (Mary Ealias and Saravanakumar 2017)
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Table 1. Nanoparticles synthesised by different methods (Mary Ealias and
Saravanakumar 2017)
Category Method Nanoparticles Bottom Up Sol-gel Carbon, metal and metal oxide based Spinning Organic polymers Chemical Vapour Deposition Carbon and metal based Pyrolysis Carbon and metal oxide based Biosynthesis Organic polymers, metal and metal oxide based Top Down Mechanical milling Metal oxide and polymer based Nanolithography Metal based Laser ablation Carbon based and metal oxide based Sputtering Metal based Thermal decomposition Carbon and metal oxide based

Nanoparticle synthesis can be physical, chemical or biological based on the
process involved. Chemical methods have disadvantage of adverse effects due to the
chemicals adhered to the nanoparticles being toxic. Physical methods involve high end
machines which are costly (Shankar et al., 2004). Best alternative for physical and
chemical method is the biosynthesis or ecofriendly synthesis of nanoparticles using
microorganisms (Klaus et al., 1999; Konishi et al., 2007), fungi (Vigneshwaran et al.,
2007), plant extracts (Ahmad et al., 2011) and enzymes (Willner et al., 2006).

2.6 CHARACTERISATION

The unique characteristics determines the potential and application of a
nanoparticle. The nanoparticle characterisation is carried out by various measurement
techniques that is summarised in Table 2.
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Table 2. Characterisation methods for nanoparticles in solid, liquid and gas phase
(Mary Ealias and Saravanakumar 2017)
Characteristics Solid Liquid Gas Size Electron microscope and laser diffraction for bulk samples Photon correlation spectroscopy and centrifugation SMPS and optical particle counter Surface area BET isotherm Simple titration and NMR experiments SMPS, DMA Composition XPS and chemical digestion followed by wet chemical analysis for bulk samples Chemical digestion for mass spectrometry, atomic emission spectroscopy and ion chromatography Particles are collected for analysis by spectrometric or wet chemical techniques Surface Morphology Image analysis of electron micrographs Deposition onto a surface for electron microscopy Capture particles electrostatically or by filtration for imaging using electron microscopy Surface charge Zeta potential Zeta potential DMA Crystallography Powder X-ray or neutron diffraction Concentration CPC

BET – Brunauer–Emmett–Teller model, CPC – Condensation Particle Counter, DMA –
Differential Mobility analyser, NMR – Nuclear Magnetic Resonance Spectroscopy,
SMPS – Scanning Mobility Particle Sizer, XPS – X-ray Photoelectron Spectroscopy

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2.7 BIOSYNTHESIS OF NANOPARTICLES

Biosynthesis of nanoparticles are non-toxic, safe, eco-friendly and low cost
(Duan et al., 2015). Hence, many researchers started exploring natural sources for
nanoparticle synthesis that includes plant and plant extracts (Iravani et al., 2011),
marine source (Fawcett et al., 2017), bacteria (Santos et al., 2017) and fungi
(Uddandarao et al., 2017). In order to reduce the inevitable expenses in downstream
processing of the synthesized nanomaterials and to increase the application of
nanoparticles, the scientific community targeted the bioresources (biological organisms
such as Algae, Microbes, Plants etc.) (Ravinder Singh et al., 2015). Biosynthesis is a
green and eco friendly approach for the synthesis of nontoxic and biodegradable
nanoparticles (Kuppusamy et al., 2014). Hasan S, (2015) reported that the biosynthesis
of nanoparticles utilises bacteria, plant extracts, fungi, yeast, etc. instead of
conventional chemicals for bioreduction. Further, he proposed that the biosynthesised
nanoparticles has enhanced and unique properties that might be useful in the field of
biomedical applications. Additionally, intracellular and extra cellular inorganic
nanoparticles can be synthesised by the unicellular and multicultural organisms.
Various biological sources of nanoparticles synthesis has been listed in Table 3 (Ingale
and Chaudhari 2013).

2.7.1 BIOSYNTHESIS USING PLANTS

Phytonanotechnology, is the synthesis of nanoparticles using plants or plant
extracts, which is safe, non toxic, rapid, simple, eco friendly and inexpensive. It is
advantageous due to its single step biosynthetic process and readily available natural
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capping agents. This is advantageous over other conventional synthesis due to its
biocompatability, medical applicability and scalability of the nanoparticles synthesised
using aqueous extract of the plants or its extracts (Singh P et al., 2016). Biosynthesis of
gold and silver nanoparticles using plant extracts was the most studied nanoparticle
synthesis. Gold and silver nanoparticles synthesis using Aloe vera plant extracts
(Chandran et al., 2006), Geranium extract (Shankar et al., 2004), sundried
Cinnamomum camphora (Huang J et al., 2007) and Azadirachta indica leaf extract
(Shankar et al., 2004) has been reported. Medicinal plant, Panax ginseng is also used in
the biosynthesis of silver and gold nanoparticles, suggested the use of medicinal plants
as sources (Singh P et al., 2015).

Patil et al., 2012 synthesised silver nanoparticles using Plumeria rubra plant
latex. Szyygium aromaticum bud extract (Singh et al., 2010), Azadirachta indica
(Poopathi et al, 2015), Pistacia atlantica (Sadeghi et al, 2015) Nyctanthes arbortristis
(Gogoi et al., 2015), Anogeissus latifolia gum (Kora et al, 2012) and Murraya koenigii
leaf extract (Christensen et al, 2011) were also used in the synthesis of silver
nanoparticles. Vankar and Bajpai, 2010 prepared gold nanoparticles using Mirabilis
jalapa flowers. Gold nanoparticles were synthesised using pear fruit extract (Ghodake
et al., 2010), and Cymbopogon citratus (Murugan et al., 2015). Gurunathan et al., 2014
synthesised biocompatible gold nanoparticles using Ganoderma sp. Palladium
nanoparticles was synthesised using Catharanthus roseus (Kalaiselvi et al., 2015). The
exact mechanism and the biomolecules involved in nanoparticles biosynthesis using
plants were yet to be deciphered. Yet, many researchers have proposed that bioactive
compounds viz proteins, vitamins, amino acids, organic acids, secondary metabolites
viz flavanoid, alkaloids, polyphenols, terpenoids, heterocyclic compounds and
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polysacharides might have a convincing part in stabilizing and capping the
nanoparticles, in addition to metal bioreduction (Duan et al., 2015). El-Kassas et al.,
also showed that the carbonyl group of protein and hydroxy group of polyphenols that
is present in Corallina officinalis assisted in synthesis of gold and silver nanoparticle.

Table 3. Biosynthesis of nanoparticles using various Plant species
Plant species Nanoparticles References Azadirachta indica Ag, Au Shankar et al.,2004. Aloe vera Au Chandran et al.,2006. Cinnamomum camphora Ag Huang et al.,2007. Szygium aromaticum Ag, Au Kalpana devi et al.,1994. Murraya koenigii Ag Christensen et al.,2011. Plumeria rubra Ag Patil et al.,2012. Citrus aurantium Ag Pala et al.,2010. Geranium leaf plant extract Ag Shankar et al.,2004. Jatropha curcas Ag Pala et al.,2010. Tridax procumbens Ag Pala et al.,2010. Hibiscus rosa sinensis Ag Daizy,2010. Euphorbia prostrata Au, TiO2 Zahiret al., 2015. Sargassum algae Palladium Momeni, S. et al.,2015. Ginkgo biloba Cu Nasrollahzadeh et al.,2015. Panax ginseng Au, Ag Singh, P. et al.,2015. Red ginseng Ag Singh, P. et al.,2015. Cymbopogon citratus Au Murugan, K. et al.,2015. Azadirachta indica Ag Poopathi, S. et al.,2015. Nigella sativa Ag Amooaghaie, R. et al.,2015. Cocos nucifera Lead Elango, G. et al.,2015. Catharanthus roseus Palladium Kalaiselvi, A. et al.,2015. Pistacia atlantica Ag Sadeghi, B. et al.,2015. Banana Cadmium sulfide Zhou, G.J. et al.,2014. Nyctanthes arbortristis Ag Gogoi et al.,2015. Anogeissus latifolia Ag Kora et al.,2012. Abutilon indicum Ag Ashokkumar et al.,2015. Pinus densiflora Ag Velmurugan et al.,2015. Artocarpus gomezianus Zinc Suresh et al.,2015. Citrus medica Copper Shende et al.,2015. Orange and pineapple Silver Hyllested et al.,2015. Lawsonia inermis Fe Naseem et al.,2015. Gardenia jasminoides Fe Naseem et al.,2015.

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2.7.2 BIOSYNTHESIS USING BACTERIA

Microorganisms are nanofactories which can accumulate and detoxify heavy
metals using various reductase enzyme that can reduce metal salts to metal
nanoparticles with narrow size and less polydispersity. Microorganisms can synthesis
nanoparticles both intra- and extracellularly, of which extracellular is preferred because
it eliminates the downstream process in the nanoparticles recovery (Singh et al., 2016).
Synthesis of silver and gold nanoparticles using Weissella oryzae (Singh P et al., 2015),
Pseudomonas deceptionensis (Jo et al., 2015), Bacillus methylotrophicus (Wang C et
al., 2015), Bhargavaea indica (Singh P et al., 2015) and Brevibacterium firgoritolerans
have been reported. Bacillus sp have been reported to synthesis silver nanoparticles
extracellularly (Sunkar and Nachiyar, 2012). Pseudomonas stutzer, isolated from silver
mines, was reported to accumulate silver nanoparticles in its periplasmic space
(Slawson et al., 1994). Sharma et al. (2012) synthesised gold nanoparticles using
Marinobacter pelagius that are stable and monodispersed. Titanium nanoparticles have
been synthesised using Lactobacillus strains by Prasad et al., 2007.

2.7.3 BIOSYNTHESIS USING FUNGI

Mycosynthesis is gaining importance considered advantageous than bacterial
synthesis due to their higher bioaccumulation, economic, easier synthesis method with
rapid downstream processing and biomass handling. Biological production of
nanoparticles using fungi The underlying mechanism in mycosynthesis of metal
nanoparticles includes electron shuttle quinones, nitrate reductase action or both
(Alghuthaymi et al., 2015). Biosynthesis of silver, gold and bimetallic nanoparticles
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using filamentous fungus Neurospora crassa was reported by Castro-Longoria et al.,
(2011). Silver nanocrystals are synthesised extracellularly by Aspergillus niger (Gade
et al., 2008), Aspergillus oryzae (Binupriya et al., 2010), Fusarium solani (Ingle et al.,
2009) and Pleurotus sajor caju (Nithya and Ragunathan, 2009). Trichoderma viride
was used to synthesis spherical nanoparticles (Thakkar et al., 2010). Fusarium
oxysporum leads to synthesis of stable silver hydrosols (Ahmad et al., 2003).

Table 4. Biosynthesis of nanoparticles using various Bacterial species
Bacterial species Nanoparticles References Bacillus cereus Ag Ganesh Babu et al. and Gunasekaran et al.,2009. Bacillus thuringiensis Ag Jain et al.,2010. Escherichia coli Ag CdS Gurunathan et al.,2009. Sweeney et al.,2004. Lactobacillus strains Ag, Au Sintubin et al.,2009. Pseudomonas stutzeri Ag Klaus et al.,1999. Corynebacterium Ag Zhang et al.,2005. Staphylococcus aureus Ag Nanda and Saravanan et al.,2009. Ureibacillus thermosphaericus Ag Juibari et al.,2011. Pseudomonas deceptionensis Ag Jo, J.H. et al.,2015. Weissella oryzae Ag Singh, P. et al.,2015. Bacillus methylotrophicus Ag Wang, C. et al.,2015. Brevibacterium frigoritolerans Ag Singh, P. et al.,2015. Bhargavaea indica Ag, Au Singh, P. et al.,2015. Bacillus amyloliquefaciens Cadmium sulphide Singh, B.R. et al.,2011. Bacillus pumilus Ag Elbeshehy, E.K. et al.,2015. Bacillus persicus Ag Elbeshehy, E.K. et al.,2015. Bacillus licheniformis Ag Elbeshehy, E.K. et al.,2015. Listeria monocytogenes Ag Soni, N. et al.,2015. Bacillus subtilis Ag Soni, N. et al.,2015. Streptomyces anulatus Ag Soni, N. et al.,2015.

23

Phoma glomerata was reported to synthesis silver nanoparticles that exhibits
antibacterial activity (Birla et al., 2009). The genus Penicillium was found to synthesis
silver nanoparticles via extracellular mechanism (Sadowski et al., 2008). Silver
nanoparticles synthesized using Verticillium, Fusarium oxysporum, or Aspergillus
?avus were found to be in the form of a ?lm or seperated in solution or accumulated on
the surface of the fungal cell (Jain et al., 2011; Vigneshwaran et al., 2007; Bhainsa et
al., 2006; Senapati et al., 2004)

2.7.4 BIOSYNTHESIS USING YEAST

Biosynthesis of gold nanoparticles by the tropical marine yeast Yarrowia
lipolytica was reported by Agnihotri et al, 2009. Zheng et al., 2010 synthesised
silver-gold alloy nanoparticles using yeast cells. Mesoporous magnetic Fe3O4
materials were synthesized using yeast cells by coprecipitation method (Zhou et al..
2009). Intracellular CdS nanoparticles were synthesised using Candida glabrata and
Schizosaccharomyce pombe with cadmium salt solution (Dameron et al., 1989). Yeasts
were also used as biotemplates in zinc phosphate nanopowders synthesis (Pandian et al.,
2009). Zinc phosphate nanopowders with narrow size distribution were obtained by
inducing yeasts in an effective way, thereby inhibiting excess agglomeration of
Zn3(PO4)2 crystals (Yan et al., 2009). Kowshik et al. (2003) isolated silver tolerant
yeast strain that are capable of synthesising (2-5 nm) silver nanoparticles. An
extremophilic yeast strain from mine drainage was reported in silver and gold
nanoparticles biosynthesis (Mourato et al., 2011). Stable lead sulfide nanoparticles
were synthesised inside the marine yeast Rhodosporidium diobovatum (Seshadri et al.,
2011).
24

Table 5. Biosynthesis of nanoparticles using various Fungal species
Fungal Species Nanoparticles References Phoma sp. Ag Saba Hasan et al.,2015. Fusarium oxysporum Au Saba Hasan et al.,2015. Verticillium sp. Ag Saba Hasan et al.,2015. Aspergillus fumigates Ag Saba Hasan et al.,2015. Trichoderma asperellum Ag Saba Hasan et al.,2015. Phaenerochaete chrysosporium Ag Saba Hasan et al.,2015. Aspergillus niger Ag Gade et al.,2008. Aspergillus oryzae Ag Binupriya et al.,2010. Fusarium oxysporum Ag Duran et al.,2007. Fusarium solani Ag Ingle et al.,2009. Pleurotus sajor-caju Ag Nithya and Ragunathan.,2009. Trichoderma viride Ag Thakkar et al.,2010. Klebsiella pneumoniae Se Fesharaki et al.,2010. Neurospora crassa Ag, Au, bimetallic silver and gold Castro-Longoria, E. et al. 2011.

Table 6. Biosynthesis of nanoparticles using various Yeast species
Yeast species Nanoparticles References Silver-tolerant strain Ag Kowshik et al.,2003 Candida glabrata CdS Dameron et al.,1989. Schizosaccharomyce pombe CdS Dameron et al.,1989. Extremophillic yeast Ag Mourato et al.,2011. Rhodospiridium dibovatum PbS Seshadri et al.,2011. Yarrowia lipolytica Ag Apte, M. et al.,2013. Rhodosporidium diobovatum Lead Seshadri, S. et al.,2011. Extremophilic yeast Ag, Au Mourato, A. et al.,2011. Candida utilis Ag Waghmare, S.R. et al.,2015.

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2.7.5 BIOSYNTHESIS USING ALGAE

Algae may reduce any metal salt into nanoparticles by either extracellular or
intracellular pathways with the help of their enzymes or biochemicals (Siddiqi et al.,
2016). Biosynthesis of gold, silver, gold-silver alloy, selenium, tellurium, platinum,
palladium, silica, titania, zirconia, quantum dots, magnetite and uraninite NP are being
reported (Shankar et al., 2016). Algae are a rich source of biomolecules and frequently
used for the extracellular synthesis of nanoparticles (Singaravelu et al., 2007; Ghosha
et al., 2008; Davis et al., 2003; Vijayaraghavan et al., 2011). Phyconanotechnology has
also become one of the prominent fields of research in nanoparticle synthesis
(Narayanan and Sakthivel, 2011). Hydroxyl, carboxyl and amino functional groups that
are present in algal phytochemicals serves both as capping agentsand as effective metal
reducing agents that provides a vigorous coating on the metal nanoparticles in a single
step (Mahdavi et al., 2013)

Synthesis of metal and metal oxide nanoparticles of well-defined shape and
size depends on the concentration of algal extract/biomass, metal salt, pH of the
reaction mixture, temperature and incubation time. Biosynthesis of metal and metal
oxide nanoparticles using various algal species are presented in Table 1 (Siddiqi et al.,
2016). Silver nanoparticles were synthesised using the aqueous extract of Caulerpa
Racemosa and proved excellent catalytic activity towards degradation of methylene
blue (Edison et al., 2016) and antibacterial activity against human pathogens
(Kathiraven et al., 2015).

26

Metal oxide nanoparticles can be composed of a variety of diverse materials,
including titanium, zinc, cerium, aluminium and iron oxides. The size of such particles
is integral to their exploitation, but size is also responsible for prompting concern
surrounding their potential toxicity (Johnston et al., 2009). Metal oxide nanoparticles
can be synthesized by a variety of biosynthetic methods under ambient conditions
(Deravi et al., 2010). There are several types of metal oxide nanoparticles such as ZnO, TiO2, CuO, MgO, NiO, ZrO2 nanoparticles.

The metal oxide nanoparticles have high density and limited size on the
surface sites due to which they exhibit unique chemical and physical properties. In
order to display mechanical stability, nanoparticles must have a low surface free energy.
As a consequence of this requirement, phases that have a low stability in bulk materials
can become very stable in nanostructures (Kung et al., 1989; Noguera et al., 1996). The
size-induced structural alterations related with changes in cell parameters have been observed, for example, in nanoparticles of CuO, ZnO, TiO2, SnO2, Al2O3, MgO, AgO, CeO2, ZrO2 etc. As the nanoparticles size decreases, the increasing number of surface and interface atoms generates strain or stress and concomitant structural perturbations
(Gleiter et al., 1995; Valden et al., 1998; Rodriguez et al., 2002; Trudeau et al., 1996;
Schoiswohl et al., 2004; Song et al., 2003).

Other metal oxides like ZnO, MgO and TiO2 nanoparticles were synthesised using fungus (Raliya and Tarafdar, 2014). In these syntheses, enzymes replaces the
chemicals which removes the toxic materials and is more eco-friendly. Of this marine
algae have always fascinated the researchers, due to its unique and rare bioactive
compounds.
27

Table 7. Biosynthesis of nanoparticles using various Algal species
Algal species Nanoparticles References Sargassum wightii Au Singaravelu et al.,2007. Sargassum muticum Au, ZnO, FeO Namvar et al.,2015. Azizi et al.,2014. Mahdavi et al.,2013. Spirulina platensis Au, Ag Govindaraju et al.,2008. Uma Suganya et al.,2015. Stoechospermum marginatum Au Rajathi et al.,2012. Navicula atomus Au Schröfel et al.,2011. Cladosiphon okamuranus Au Lirdprapamongkol et al.,2011. Tetraselmis kochinensis Au Senapati et al.,2012. Ecklonia cava Au Venkatesan et al.,2014. Chlorella vulgaris Au Annamalai and Nallamuthu .,2015. Padina gymnospora Au Singh et al.,2013. Fucus vesiculosus Au Mata et al.,2009. Turbinaria conoides Au Vijayan et al.,2014. Oscillatoria willei Ag Mubarak Ali et al.,2011. Caulerpa racemosa Ag, Au Kathiraven et al.,2015. Cystophora moniliformis Au Prasad et al.,2013. Chlamydomonas reinhardtii Au Barwal et al.,2012. Turbinaria conoides Au Vijayan et al.,2014. Pithophora oedogonia Au Sinha et al.,2015. Bifurcaria bifurcata CuO Abboud et al.,2014. Streptomyces sp. LK3 Ag Karthik, L. et al.,2014

2.8 MARINE ALGAE

More than 70% of earth is surrounded by ocean and in that around 30,000
species of marine algae are found. Algae occur all over the world where there is light
and moisture and are found in abundance in sea. They are the major source of oxygen
supply to the biosphere and good source of food for fishes, cattle and human. Algae are
diverse group of photosynthetic organism that range from unicellular microalgae to
multicellular macroalgae. Based on the pigmentation of algae are classified into three
28

groups, namely Red algae (Rhodophyceae), brown algae (Phaeophyceae) and green
algae (Chlorophyceae). Most of the brown and red algae are found in salt water,
whereas green algae is found in shallow waters near the tidal zone. Marine algae are
used as food, medicine and fertiliser and act as sustainable resource (Kamath et al.,
2018). Marine algae are well-known for its potential medicinal uses against cancer,
oxidative stress, lipidemia, allergy, thrombosis, inflammation, hypertensive and other
degenerative diseases because of their richness in certain vitamins, lipids, minerals and
other bioactive substances like proteins, polysaccharides and polyphones (A.W.
Zuercher et al., 2006; Perez et al., 1998; Nishino et al., 1999; Miyashita et al., 2009;
Wada et al., 2011; Mohamed et al., 2012; Namvar et al., 2012). Extensive research has
been done on the secondary metabolites of marine algae (Faulkner et al., 1999).
Reports are available for substances derived from marine algae that includes amino
acids (Fattorusso et al., 1983), guanidine derivatives (Chevolot et al., 1981), phenolic
substances (Higa et al., 1981), bioluminescence (Goto et al., 1980), carotenoids
(Liaaen-Jensen et al., 1978), diterpenoids (Fenical et al., 1978), indoles, bioactive
polymers (Schimizu et al., 1983) and halogenated compounds (Faulkner et al., 1980).

Many bioactive substances of marine organisums are due to ecological
pressures on seaweeds. These include competition for space, grazing pressures by
herbivores, tolerance to dangerous levels of sunlight or UV-B radiation, desiccation
during exposure at low tide or highly saline waters and conditions resulting from
thallus breakage and wound formation. The algae overcome these problems by
chemical means that can be potentially useful to humans and may result in new
technologies such as natural antifoulants and novel UV-sunscreens. The chemistry of
marine natural products has grown enormously in the last fifty years (Smit et al., 2004).
29

2.9 SEAWEEDS

Marine macroalgae, also known as seaweeds, are consumed as food and used
in traditional medicine since ancient times. These macroalgae are a rich source of the
colloids that are used in foods. The main commercial “phycocolloids” of edible
seaweeds are agar, alginate, and carrageenan, which are used as thickening agents in
foods. Apart from this marine macroalgae possess remarkable biological activity. An
anthelmintic drug was the first example of a pharmacological product to be extracted
from marine algae. A remarkable percentage of algal polysaccharides possess antiviral
activity. The extracts of red seaweeds from the family Dumontiaceae, particularly the
extract of Constantine simplex, contain a specific and potent antiviral substance against
herpes simplex virus (Fenical et al., 1983). Some sulphated polysaccharides from red
algae Aghardhiella tenera and Nothogenia fastigiata show antiviral activities towards
human immunodeficiency virus (HIV), Herpes simplex virus (HSV) types 1 and 2 and
respiratory syncytial virus (RSV) (Witvrouw et al., 1994; Damonte et al., 1994 and
Kolender et al., 1995).
Chemicals responsible for antibiotic activities are widespread in macroalgae.
Interesting substances in particular are the halogenated compounds such as haloforms,
halogenated alkanes and alkenes, alcohols, aldehydes, hydroquinones and ketones
(Lincoln et al., 1991). The dipeptides kahalalide A and F from Bryopsis sp. were noted
for their in vitro activity against Mycobacterium tuberculosis (Sayed et al., 2000). A
halogenated furanone, or fimbrolide, from Delisea pulchra is a promising antibacterial
agent (Kjelleberg and Steinberg, 2001) and also used to treat chronic Pseudomonas
aeruginosa infection. Seaweed derived bioactive compounds were known to possess
fertilisating effect and inhibitory effect in the larval or embryonic development in both
30

invertebrates and vertebrates. Fucoidan prevents the initial binding of sperm and
subsequent recognition events that are essential to penetrate human zona pellucida layer
(Oehninger et al., 1991; Patankar et al., 1993).

Kainoids, isolated from the macroalgae Digenea simplex, Chondria armata,
Chondria baileyana, Alsidium corallium, Amansia glomerata, Vidalia obtusiloba,
Laurencia papillosa and Centroceras clavulatum (Ceramiales) (Takemoto and Daigo,
1958; Impellizzeri et al., 1975; Laycock et al., 1989; Smith and Kitts, 1994; Sato et al.,
1996) are unusual amino acids structurally related to, and having similar functions as,
glutamic and aspartic acids, both well-known neuronal excitants (agonists) or
neurotransmitters (Laycock et al., 1989).

Mitogenic activities, have been demonstrated in mouse lymphocytes using
lectins from the seaweed Eucheuma serra (Kawakubo et al., 1997). Kainoids are
important tools in research (Higa and Kuniyoshi, 2000) into neurophysiological
disorders such as Alzheimer’s and Parkinson’s disease and epilepsy (Ben-Ari and
Cossart, 2000; Hopkins et al., 2000; Carcache et al., 2003). Macroalgae, especially red
algae, are rich in 20-carbon atom polyunsaturated fatty acids (PUFAs), chiefly
eicosapentaenoic and docosahexanoic acids (Stefanov et al., 1988; Gerwick and
Bernart, 1993). Renn et al., (1994a and 1994b) have reported lowering of systolic blood
pressure (antihypertensive responses) and lower levels of total cholesterol, free
cholesterol, triglyceride and phospholipid in the liver was reported by Nishide and
Uchida, 2003.

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2.10 CAULERPA RACEMOSA

Caulerpa racemosa (Forsskål) J.Agardh, is a species of edible green alga,
a seaweed in the family Caulerpaceae. It can be seen at depths from near the surface to
100 m that is from shallow muddy shore to clear water reef environments. Thus this
seaweed can be seen in most of the shallow seas around the world. It is commonly
known as sea grapes (along with the related Caulerpa lentillifera). There are about 75
identified species of Caulerpa so far. Many of them shows polymorphism, which
makes them difficult to identify due to different growth forms in different habitats .
Caulerpa racemosa, C. peltata and C. laetevirens form a species complex. A number
of forms and varieties for C. racemosa have been identified but further investigation is
expected to elucidate their exact phylogenetic relationships (Marc et al., 2003).

Taxonomy
Kingdom: Protoctista
Phylum: Chlorophyta
Genus: Caulerpa
Species: Caulerpa racemosa

C. racemosa plant have many branches that are linked to stolons which are
anchored to the soil substrate by rhizoids. These branches are few centimetres apart in
the stolon and can grow to a height of 12inch (30 cm). The side shoots branch off these
stolons which are spherical or oval in shape, thus the seaweed s named as sea grapes.
Each C. racemosa plant consists of a large number of nuclei inside a single enormous
cell that resembles other members of the order Bryopsidales. The chloroplasts with
32

chlorophyll are free to move within the organism from one part to another that is
facilitated by a network of fibrous proteins that supports the organelle movement
(Robert et al., 1996). Many countries including Japan, Philippines, Fiji and Thailand
consume it as salads, especially local fishermen in Indonesia and Malaysia. Further this
seaweed was known to be low in fat and rich in minerals (calcium and magnesium),
proteins, fiber, folic acid (Vitamin B9), retinoic acid (Vitamin A), ascorbic acid
(vitamin C) and thiamine (vitamin B1) (Diini et al., 2015). C. racemosa was first
observed in Sousse Harbor, Tunisia, by Hamel (1926). It was considered a Lessepsian,
though not an invasive, migrant at that time (Por et al., 1978; Verlaque et al., 1994;
Verlaque et al., 2004). But after 1990, an unknown form of C. racemosa was observed
in Mediterranean and this form has been invading about 11 Mediterranean countries
(Albania, Croatia, Cyprus, France, Greece, Italy, Libya, Malta, Spain, Tunisia and
Turkey) and some big islands (Balearic Islands, Corsica, Crete, Cyprus, and Sicily)
(Verlaque et al., 2003a,b) since then.

2.11. BIOLOGICAL ACTIVITY OF MARINE ALGAE

Bioactive metabolites are also isolated from marine algae which possess a
remarkable array of biological activites. Chemically the bioactive metabolites of marine
algae include brominated phenols, oxygen heterocyclics, nitrogen heterocyclics,
sulphur nitrogen heterocyclics, sterols, terpenoids, polysaccharides, peptides and
proteins (Heilbron et al., 1935; Santos et al., 1971; Upham et al., 1968; Butler et al.,
2004). Mayer et al., 2017) compiled the biological activity of the marine compounds in
a two year time period. He reported that the bioactive compounds isolated from marine
33

organisms possess antioxidant, anticancer, anti-inflammatory, anti-microbial,
antidiabetic, anti-Alzheimer, anti-aging, antiprotozoal and antituberculosis.
Several sulphated macroalgal polysaccharides have cytotoxic properties. Fucoidans
are known to have antitumour, anticancer, antimetastatic and fibrinolytic properties in
mice (Coombe et al., 1987; Maruyama et al., 1987), and they also reduce cell
proliferation (Religa et al., 2000). Sulphated fucoidan has several advantages over
heparin. It shows concentration-dependent inhibition of thrombin generation from
platelets; it exhibits concentrationdependent inhibition of thrombin-induced platelet
aggregation; it lacks the hypotensive effect found in thrombin; it reduces the sticking of
polymorphonucleated leukocytes to rabbit aorta; and it shows a dosedependent
inhibition of thrombin-induced thrombosis (Trento et al., 2001).

Caulerpa racemosa extract possess antiproliferative and apoptotic effects on
neuroblastoma cell lines SHSY5Y and Kelly (Levent et al., 2006). The C.
racemosa polysaccharide (CRP) was known to be composed of sulfated polysaccharide
with 3.9–7.9% uronic acid and protein. Bioassay indicated that CRP had strong
antitumor activity in both in vitro and in vivo, and its inhibition rate of K562 cells in
vitro was 59.5–83.8% (48 h) (Hongwu et al., 2008). Methanolic extract of Caulerpa
racemosa was found to possess antioxidant activity (Chewa et al.,2008). Dekhil et al.,
(2011) suggested that dried Caulerpa racemosa could adsorb lead and cadmium ions
from aqueous solution.

Zhongrui (2012) found that the phenolic compounds of Caulerpa racemosa
was linked to its antioxidative activity. Two new prenylated para-xylenes, named
caulerprenylols A and B, isolated from the green alga Caulerpa racemosa were proved
34

to possess broad spectrum of antifungal activity (Liu et al., 2013). The bioactive
compounds 2-(-3-bromo-1-adamantyl) acetic acid methyl ester and Chola-5,
22-dien-3-ol isolated from the methanolic extracts of Caulerpa racemosa showed
antibacterial and larvicidal activity (Sowmya et al., 2014). Ribeiro et al., (2014) proved
that the isolated sulphated polysaccharides from Caulerpa racemosa possess
antinociceptive and anti inflammatory activity. Hui Yang et al., (2014) isolated a novel
minor bisindole alkaloid, racemosin C, which was found to possess significant PTP1B
(Protein tyrosine phosphatase -1B) inhibitory activity. Polysaccharide fractions
(uncharacterised) obtained from Caulerpa sp., Hypnea charoides, Corallina sp.,
Padina arborescens and Sargassum patens was reported to have low levels of
cytotoxicity but also possess high antiviral activity against HSV (type 1 and 2) (Zhu et
al., 2003).

Terpenoids are reported to possess exceptional cytotoxic and antitumour
activities. This includes Caulerpenyne from Caulerpa taxifolia which is cytotoxic
towards several human cell lines and as such has anticancer, antitumour and
antiproliferative properties (Fischel et al., 1995; Parent-Massin et al., 1996; Barbier et
al., 2001). Seaweeds can metabaolize various polyunsaturated fatty acids (C20-PUFAs)
via oxidative pathways (Gerwick et al., 1993). Hypocholesterolemic and hypolipidemic
responses have been reported in macroalgal polysaccharides and fibres such as alginate,
fucoidan, laminaran, carrageenan, funoran, porphyran and ulvan which is due to
reduced cholesterol absorption in the gut (Kiriyama et al., 1968; Lamela et al., 1989;
Panlasigui et al., 2003). Hypolipidemic activities have been identified in ethanolic
extracts of Solieria robusta, Iyengaria stellata, Colpomenia sinuosa, Spatoglossum
35

asperum and Caulerpa racemosa, as shown by decreases in the serum total cholesterol,
triglyceride and low density lipoprotein cholesterol levels in rats (Ara et al., 2002).

Different solvent extracts of Caulerpa racemosa was reported to possess
antibacterial activity whereas the methanolic extract of Caulerpa racemosa was proved
to exhibit maximum activity against various pathogenic organisms (Jebasingh et al.,
2011). The methanolic extract of Caulerpa racemosa showed larvicidal activity against
Culex tritaeniorhynchus larva by disintegrating the mid gut epithelium cells and
columnar cells (Nagaraj and Osborne, 2014).

Caulerpa racemosa is one type of green algae that has the ability to defend
against radiation of ultraviolet (Nurlina et al., 2017). Caulerpa racemosa posses
fertilizer activity to enhance the germination and seedling growth in different legumes
and cereal crops due to presence of growth promoting hormones (Bharat et al., 2017).

2.12 BIOLOGICAL ACTIVITY OF NANOPARTICLES

Several literature reports on the mechanism of toxicity of nanomaterials
through oxidative stress, lipid peroxidation and reaction of DNA with reactive oxygen
species that causes DNA collapse, disintegration of cell membrane that leads to cell
death (Oberdorster et al., 2004; Hsin et al., 2008; Li et al., 2008; Sharma et al., 2009).
Ashraf et al., 2013 reported that silver nanoparticles exhibit significant cytotoxicity in
cell culture media. In contrast, silver nanoparticles synthesised using chrysanthemum
flower extract exhibited no or minimal cytotoxicity towards 3T3 cell lines (Arokiyaraj
et al., 2014). Maddinedi et al., (2015) synthesised gold nanoparticles using the enzyme
36

diastase and suggested that the increased biocompatibility towards A549, HCT116 cell
lines might be due to the presence of biomolecules on the surface of nanoparticle.
Again Maddinedi et al., in 2017, synthesised silver nanoparticles using the enzyme
diastase and observed dose and size dependent cytotoxicity in mice fibroblast 3T3 cell
line.

TiO 2 was found binding to plasma proteins and migrate into the bloodstream, through the lymphatic system after phagocytosis by macrophages, or to the bone
marrow via monocytes (Urban et al., 2000; Olmedo et al., 2002). Few researchers proved synergistic effect of UV radiation and TiO2 nanoparticles on several cells such as CHO (Uchino et al., 2002), glioma (Yamaguchi et al., 2010) and HeLa (Matsui et al., 2010). Lopez et al., 2013 reported that the TiO2 nanoparticles accumulates inside the cell vacuoles, endosomes and lysosomes, might also be localised in the cytoplasm due
to lysosome rupture (Saimon et al., 2013). This was confirmed by the work of Rene et al., 2016 who reported that the TiO2 nanoparticles were found in the vacuoles where they became aggregated again. The only nanoparticle that can cross the blood brain barrier is TiO2, when compared to other nanoparticle (Chakraborty et al., 2009). In the field of nanomedicine, biocompatibility is one of the most important criteria for any
nanomaterial, so extensive research in biosynthesis using microorganisms, fungi and
plants is a current trend (Lang et al., 2007). Rajiv et al., (2013) synthesised ZnO
nanoparticles from leaf extracts of Parthenium hysterophorus and proved its antifungal
activity against plant pathogens.

37

2.13 ZEBRAFISH

Zebrafish is a freshwater fish belonging to the minnow family of the order
Cypriniformes (Froese et al., 2007). It is a popular aquarium fish, native to Himalyan
region, sold with a trade name of zebra danio. The zebrafish is the widely used
vertebrate model organism in scientific research, due to its regenerative abilities
(Goldshmit et al., 2012) and many modified transgenic strains had been developed by
researchers (White et al., 2008). The zebrafish belongs to the family of Cyprinidae and
the genus Danio. It has a sister-group relationship with Danio aesculapii (McCluskey et
al., 2015). Zebrafish are also closely related to the genus Devario, as demonstrated by
a phylogenetic tree of close species (Parichy et al., 2006).

The zebrafish is named for the five uniform, pigmented, horizontal, blue
stripes on the side of the body, including an incomplete lateral line, two pairs of barbels,
which resembles zebra’s stripes that extends till the end of the caudal fin. They have
fusiform, laterally compressed bodies with its mouth directed upwards, that reach an
average length of 25 mm.

Scientific name: Danio rerio
Kingdom: Animalia
Order: Cypriniformes
Family: Cyprinidae
Class: Actinopterygii
38

The largest recorded zebrafish reached 64 mm in constraint. They have thin
elongate mandibles with a protrusive lower jaw that causes the mouth to point upwards
and centrally located eyes. The male is torpedo-shaped, with gold stripes between the
blue stripes; the female has a whitish belly, larger and silver stripes instead of gold.
Like other cyprinids, zebrafish are toothless and stomachless. As a result, they depend
on gill rakers to break up food. In addition, they are obligate suction feeders. They are
omnivores. They get most of their food from the water column, mainly aquatic insects
and eating zooplankton. Zebrafish also surface feed, eating arachnids and
terrestrial insects. Zebrafish especially eat mosquito larvae. The degree of sexual
dimorphism in zebrafish is minimal, as males tend to have larger anal fins than females
and tend to have more yellow coloration. Adult females distributes a small genital
papilla in front of the anal fin origin. Its lifespan in constraint is around two to three
years, although in ideal conditions, this may be extended to over five years (Spence et
al., 2008; Gerhard et al., 2002).

2.13.1 ZEBRAFISH IN RESEARCH
The development of Danio rerio as a model organism for modern biological
investigation began with the pioneering work of George Streisinger and colleagues at
the University of Oregon (Streisinger et al., 1981; Briggs et al., 2002), who recognized
39

many of the virtues of Danio rerio for research. Streisinger developed methods to
produce homozygous strains by using genetically inactivated sperm, performed the first
mutagenesis studies, and established that complementation methods (in which
heterozygous mutant fish are paired) could be used to assign mutations to genetic
complementation groups. Subsequently, the use and importance of Danio rerio in
biological research has exploded and diversified to the point that these fish are
extremely important vertebrate models in an extraordinary array of research fields
(Runkwitz et al., 2011; Vascotto et al., 1997).

2.13.2 ZEBRAFISH FOR NANOTOXICITY ASSESSMENT

In recent years, zebrafish (Danio rerio) as an established animal model system for
nanoparticle toxicity assay is epidemic. The world wide acceptance of zebrafish both
adult as well as embryonic stage, as an experimental animal model in the fields of
toxicology and biomedical research is becoming popular. Being small in size zebrafish
is handled without any difficulty. Rapid hatching and quick feeding of larvae (120h
post fertilization) makes it unique, since experiments can be initiated on zebrafish
larvae from that point (Easter et al., 1996). Additionally all cells are detectable due to
the transparency of the embryos counting from early larval stages. Apart from this,
organs and tissues can be examined instantly due to its in vivo visiblity (Fishman et al.,
1996; Parichy et al., 2009).

Moreover, zebrafish is known for its high fertility rate that spawn large
number of embryos, around 300 eggs per week under optimum conditions. Withal, it
can be hatched in the laboratory aquarium just by adding flora and gravel into the tank
40

(Hsu et al., 2007; Spence et al., 2007). Since organogenesis is quick, the major organs
are developed within 5-6 days post-fertilization (dpf) in larvae. At the average of 350
dpf, females can attain size of 38 mm while males can attain a maximum mean size of
35 mm with a weight of 0.9 and 0.6 g, respectively (Ribas et al., 2014). Similarity of
cardiovascular, nervous and digestive systems to mammals, makes zebrafish a
convenient choice (Hsu et al., 2007).

A high level of genomic homology is found between zebrafish and human in
conserved signaling pathways (Beliaeva et al., 2010). Henceworth, genetic analysis
assessment of a particular gene function by transgenic development and knockdown
experiments is convenient through zebrafish (Varshney et al., 2013). National Institutes
of Health (NIH), USA, has started to encourage the zebrafish model organism for the
analysis of different diseases with a genetic program (Rasooly et al., 2003).

Ali-Boucetta et al., 2011 studied CNT (carbon nanotubules) toxicity by MTT
and LDH assays and observed that there is synergy between CNT and cell cultures that
lead to cytotoxicity. On the contrary, multi-walled CNTs (MWCNTs) (agglomerated
with a standard diameter of 500nm) were surprisingly found to be non-genotoxic to
zebrafish model with reversible inflammation (FilhoJde et al., 2014).

Silver ions generated by silver nanoparticles inhibits Na+/K+-ATPase action
and the enzymes related to Na+ and Cl– uptake. Silver ions affects the osmoregulation
in the gills and are thus toxic to the gills of zebrafish (Bury et al., 1999; Wood et al.,
1999). Invivo quantitative study of silver nanoparticles revealed that the nanoparticle
toxicity and transport is size dependent in zebrafish (Lee et al., 2012). Inversly,
41

Bar-Ilan et al., (2009) proved that the toxicity of silver nanoparticles are size
independent. The size, shape, crystallinity and chemical parameters of gold
nanoparticles are attributed to the mortality and developmental disorders in zebrafish
(Harper et al., 2011). Dedeh et al., (2015) suggested modifications in metabolism of
mitochondria, oxidative stress and neurotransmission in zebrafish when exposed to
different concentrations of gold nanoparticles.

Griffitt et al., (2007) proposed that insoluble forms of copper nanoparticles
might damage gills lamellae and are toxic to zebrafish. Researchers found deposition of
nanoparticles in zebrafish skin upon exposure. The nanoparticles enter through
diffusion or endocytosis into the embryo and accumulates in the larval epidermis layer.
This provoke cell death which precedes abnormalities in the skin (Asharani et al.,
2008). Jin and Zheng (2011) suggested that zebrafish can also be used to evaluate
immunotoxicity. Carcinogens have long term toxic effects that leads to genotoxicity
which can also be studied using zebrafish model. Genotoxic effects of cadmium
(Cambier et al., 2010) and gold nanoparticle (Dedeh et al., 2014) was studied through
RAPD and RT-PCR based methodology using zebrafish model. Till date only limited
research have been reported in nanoparticle genotoxicity using zebrafish and
considerable studies are expected in this field of research. Nanoparticle neurotoxicity of
dendrofullerene nanoparticle (DF-1), a C60 fullerene derivative, was studied using
zebrafish embryo in dose-limiting toxicity level (Daroczi et al., 2006).

Toxicity of metal oxide nanoparticles was investigated by Jeng and Swanson
(2006) using ZnO nanoparticles, which killed the cells by apoptosis. ZnO nanoparticles
delayed hatching of zebrafish embryos and skin lesions in addition to mortality in
42

zebrafish (Zhu et al., 2008). Shape dependent toxic effects of zinc oxide nanoparticles
was noticed by Hua et al., (2014). ZnO nanosticks are found to be more toxic than
other nanoparticles that resulted in reasonable mortality and reduced hatching in zebrafish. Wang et al., (2011) reported that TiO2 nanoparticles affects male and female reproductivity and fetal development in zebrafish. After 13 weeks of chronic exposure to TiO2 nanoparticle, there is a reasonable decrease in the number of zebrafish eggs. Similarly increased mortality of zebrafish embryos exposed to silica nanoparticles was
reported by Duan et al., (2013). Chen et al., (2011) observed that the toxicity level of TiO2 nanoparticle is dependent on both concentration and time, using zebrafish model. He further discussed about the distribution and accumulation of TiO2 nanoparticle in different body parts of zebrafish.

Presently, in the field of toxicological testing, zebrafish have become a smart
vertebrate model. As a result, Germany have introduced the zebrafish embryo test as a
standardized ISO program in chemical evaluation. This assay can be used to evaluate
the level of environmental contaminants in water testing (Kanungo et al., 2014).
Recently, zebrafish is proposed as one of the most successful invivo model since it is
faster, cheaper and more efficient animal model for more than a decade (Spitsbergen
JM et al, 2003) and notable advancement has been made in nanotoxicology studies
using zebrafish (Jang et al., 2014). The zebrafish might be able to become a significant
alternative of other mammalian models for toxicological testing of nanomaterial in
forthcoming years.

43

MATERIALS AND METHODS
3.1 SAMPLE COLLECTION

Fresh samples of the macro algae, Caulerpa racemosa were collected from the
Southeast coast of India mainly in the intertidal region of Mandapam, Ramanathapuram
District, Tamil Nadu (9° 22′ N, 78° 52 ‘ E) during the month of June. The collected
seaweed samples were washed thoroughly with tap water followed by distilled water
until the debris and associated epiphytes were removed. After subsequent washing, the
seaweed was shade dried for 3-weeks. Then the dried seaweed was powdered in a
blender and stored for further use.

3.2 SYNTHESIS OF TIO2 NANOPARTICLES

30 grams of dried seaweed (Caulerpa racemosa) was boiled with 150 ml of
deionised water. The boiled suspension was filtered through whattman no 1 filter paper. The final extract solution was collected and stored at 4?C for the synthesis of TiO2 nanoparticles. The erlenmayer flask containing 100mM of Titanium tetra isopropoxide
in 100 ml of seaweed extract solution was reacted under stirring at 50?C. The above
solution was heated on a hot plate at 80?C for two hours. The obtained product was
pounded into the powder form and calcinated in muffle furnace at 500?C for 5 hrs.

44

3.3 CHARACTERISATION OF TIO2 NANOPARTICLES
3.3.1UV-VISIBLE SPECTROSCOPY

Ultraviolet-visible spectroscopy (UV-Vis) refers to absorption spectroscopy or
reflectance spectroscoy in the ultraviolet-visible spectral region. UV-Vis absorption
spectroscopy measures the percentage of radiation that is absorbed at each wavelength.
Typically this is done by scanning the wavelength range and recording the absorbance.
It is also used for the measurement of electronic band gap of semiconductor films. UV-visible spectra of TiO2 nanoparticles was recorded using UV –Vis spectrophotometer (Perkin Elmer Lambda 35). The synthesis of TiO2 nanoparticles was monitored continuously using wavescan mode from 200nm – 1100nm wavelength and the band gap energy (Ebg) was calculated using

Ebg = h*c/?
where,
h= planck’s constant (6.626 x 10-34Js) c= speed of light (3.0 x 108 m/s)
?= cutoff wavelength
The band gap of the samples can also be determined by the equation, (Regan and
Gratzel, 1991)
Eg = 1239.8/?
where,
Eg is the band gap (eV)
? (nm) is the wavelength of the absorption

45

3.3.2 SCANNING ELECTRON MICROSCOPE (SEM) ANALYSIS

The surface study and the porosity of the nanoparticle was analysed in field
emission scanning electron microscope (FESEM) attached with EDAX to determine
the elemental characterisation at a specific position. For FE-SEM analysis, a drop of the
sample was placed on a stub using adhesive carbon tape with gold sputtering and
observed using Carl ZEISS SIGMA operating at a voltage of 30kV. SEM images have
greater depth of field yielding a characteristic 3D appearance useful for understanding
the morphology material. Magnification is of order 10,000 X and resolution 10 nm.

3.3.3TRANSMISSION ELECTRON MICROSCOPE (TEM) ANALYSIS

Transmission Electron Microscopy (TEM) is a useful technique in determining
crystal morphology and particle size of materials. TEM is used to directly image
nanoparticles at scales approaching a single atom. The morphology and size
distribution of the nanoparticle was analysed using transmission electron microscope
(TEM). For TEM analysis, the sample was air dried on a 200 mesh carbon coated
copper grid. TEM measurements were performed on Jeol/JEM 2100 instrument
operated at an accelerating voltage of 200kV. Selected area electron diffraction was
also carried out from the same transmission electron microscope (TEM).

3.3.4 X-RAY CRYSTALLOGRAPHY ANALYSIS

X-Ray crystallography determines the atomic and molecular structure of a
crystal whose crystalline nature diffracts the incident x-rays. The angles and intensities
46

of these diffracted beams are measured by a x-ray diffractometer. The powder XRD
analysis was done on AL- 2700B X-ray diffraction instrument. The X -ray diffractometer operated at a voltage 40kV and a current of 30mA using CuK? radiation of wavelength (?) 1.5405? over a wide range of Bragg angles (2?) (100 ? 2? ? 800). In XRD, a beam of X-rays with a wavelength ranging from 0.5 to 2 ?, incident on a
specimen is diffracted by the crystalline phases in a specimen according to Bragg’s
law:
n? = 2d sin ?
where, n is the order of reflection, ? the wavelength of X-Rays, ? Bragg angle and d the
interplanar spacing. In addition to characterization of various crystalline forms, XRD is
also used to find the average particle size of the nanocrystallites using Debye-Scherrer
formula,
D = K?/?cos?
Where,
D = mean crystallite diameter of the nanoparticle
K = shape constant (0.9)
? = wavelength of X-ray radiation
? = angular full width at half maximum of the XRD peak at the diffraction
angle

3.3.5 FOURIER TRANSFORM INFRA-RED (FT-IR) SPECTROSCOPY

FTIR spectroscopy is used to measure the absorption, emission, and
photo-conductivity of matter by shining a narrow beam of infrared light at the matter in
various wavelengths and detecting how the matter responds to each wavelength. The
47

data obtained is further converted into digital information using a mathematical
algorithm known as the “Fourier transform”. The functional groups present on the
surface of the TiO2 nanoparticles synthesized were analyzed using FT-IR (Perkin
Elmer Spectrum RX I) spectrophotometer. The TiO2 nanoparticles were pelleted by
centrifugation at 8000 rpm for 20 min. The pellet obtained was washed with Milli-Q
water. The centrifugation and re-dispersion was repeated thrice to remove any unbound components. The samples were then dried in hot air oven at 50oC and pelletized with
potassium bromide in the ratio of 1:100. All measurements were carried out in the
range of 400–4,000 cm-1 at a resolution of 4 cm-1.

3.4 PHYTOCHEMICAL ANALYSIS
3.4.1 THIN LAYER CHROMATOGRAPHY

TLC analysis of the seaweed was done with different solvent extracts of the
seaweed Caulerpa racemosa (Methanol, Dichloromethane, Acetone, Chloroform) to
check the presence of bioactive compounds. Four types of solvent system was used as
mobile phase: a) Chloroform in dichloromethane, b) Chloroform in methanol, c)
Chloroform in Dichloromethane and d) Chloroform in ethyl acetate. The seaweed
extract was spotted on silica coated TLC sheet and was separated using various
solvents from polar to non polar.

3.4.1.1VISUALIZATION OF THE TLC PLATE

The separated TLC Plates were visualized under UV light of both short and
long wavelength and then plates were analyzed and documented
48

3.4.2 PREPARATION OF SEAWEED SOLVENT EXTRACTS

About 10g of powdered seaweed material was soaked in dichloromethane and
chloroform, respectively for 3days at room temperature with mild shaking. Then the
solvent was filtered with Whatman filter paper (125mm). This was repeated 3-4 times
until the extract turned colorless. The extracts were collected and stored in refrigerator at 4oC for further analysis.

3.4.3 QUALITATIVE ANALYSIS OF PHYTOCHEMICAL CONSTITUENTS

Phytochemical screening was performed using standard procedures (Kokate,
1986, Harborne, 1998)

3.4.3.1 Test for terpenoids (Salkowski test)

5 ml of extracts were mixed with 2 ml of chloroform, and concentrated H2SO4 (3 ml) was carefully added to form a layer. Formation of a reddish brown color at the
interface shows the positive result for the presence of terpenoids.

3.4.3.2 Test for flavonoids

Alkaline Reagent Test – Extracts were treated with few drops of sodium
hydroxide solution. Formation of intense yellow colour, which becomes colourless on
addition of dilute acid, indicates the presence of flavonoids.
49

Lead acetate Test – Extracts were treated with few drops of lead acetate
solution. Formation of yellow colour precipitate indicates the presence of flavonoids.

3.4.3.3 Test for saponins (Foam Test)

Foam test – To 5 ml of extracts, added 5 ml of distilled water in a test tube. The
solution was shaken vigorously and observed for a stable persistent froth. The frothing
was mixed with 3 drops of olive oil and shaken vigorously after which it was observed
for the formation of an emulsion.

Froth Test – Extracts were diluted with distilled water to 20ml and this was
shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates
the presence of saponins.

3.4.3.4 Test for tannins

About 0.5 g of dried seaweed sample was boiled in 20 ml of water in a test
tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for
brownish green or a blue-black coloration.

3.4.3.5 Test for alkaloids

Mayer’s test – 1.2 ml of the extracts were taken in a test tube, 0.2 ml of dilute
hydrochloric acid and 0.1 ml of Mayer’s reagent (1.36 g of mercuric chloride was
dissolved in 60 ml of distilled water and 5 g of potassium iodide in 10 ml of water. The
50

two solutions were mixed and diluted to 100 ml with distilled water. Formation of
yellowish buff coloured precipitate confirms the presence of alkaloids.
Hager’s Test – Filtrates were treated with Hager’s reagent (saturated picric acid
solution). Formation of yellow coloured precipitate confirms the presence of alkaloids.

3.4.3.6 Test for Steroids (Salkowski Test)

To 2 ml of extracts, 2 ml of chloroform and few drops of concentrated sulphuric
acid were added. Reddish brown ring confirms the presence of steroids.

3.4.3.7 Test for Glycosides (Liebermann’s Test)

To 2 ml of extracts, 2 ml of chloroform and 2 ml of acetic acid were added.
Violet to blue to green color is regarded as positive for the presence of glycosides.

3.4.3.8 Test for Phlobatannins (Precipitate Test)

To 2 ml of extracts, 2 ml of 1% hydrochloric acid was added and boiled. Red
precipitate is regarded as positive for the presence of Phlobatannins.

3.4.3.9 Test for Proteins

Xanthoproteic Test – To 1 ml of extracts, 1 ml of concentrated sulphuric acid was
added and boiled. White precipitate was regarded as positive for the presence of proteins.
51

Ninhydrin Test – To the extract, 0.25% w/v ninhydrin reagent was added and
boiled for few minutes. Formation of blue colour indicates the presence of amino acid.

3.4.3.10 Test for Coumarins

To 2 ml of extract, 3 ml of 10 % sodium hydroxide was added. Yellow colour
was regarded as positive for the presence of coumarins.

3.4.3.11 Test for Cardiac Glycosides (Keller-Killani Test)

5 ml of extracts was treated with 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was under layered with 1 ml of H2SO4. A brown ring at the interface indicates the deoxy sugar characteristic of cardenolides. A violet ring may
appear below the brown ring, while in the acetic acid layer, a greenish ring may form
just gradually throughout the layer

3.4.3.12 Test for carbohydrates

Fehling’s test – The filtrates were treated with 1 ml of Fehling’s A and B and heated
in a boiling water bath for 5-10min. Appearance of reddish orange precipitate shows the
presence of carbohydrates.
Benedict’s Test – Filtrates were treated with Benedict’s reagent and heated gently.
Orange red precipitate indicates the presence of reducing sugars.

52

3.4.4 DETERMINATION OF TOTAL PHENOLIC CONTENT

The total phenolic content of the seaweed extract was determined
spectrophotometrically by Folin-Ciocalteu method (Singleton et al., 1999). Briefly, 0.5
mL of chloroform extract solution (50µg/ml, 100µg/ml, 150µg/ml, 200µg/ml and
250µg/ml) and 2.5 mL of 1:10 Folin-Ciocalteau reagent were mixed and then 2 mL of
sodium carbonate (75 g/L) were added. After 15 min of incubation at 45°C, the
absorbance at 765 nm was measured. The total phenolic concentration was calculated
from catechol calibration curve. Data were expressed as catechol equivalents (CE)/g of
extract averaged from 3 measurements. Total Phenolic content was calculated as
follows:
TPC= C*V/M
Where, TPC = total phenolic content, milligram per gram of sample extract, in
CE (catechol equivalent); C = the concentration of catechol established from the
calibration curve, (mg/mL); V = the volume of extract, (ml); M = the weight of sample
extract (g).

3.4.5 QUANTIFICATION OF TOTAL TERPENOIDS

About 2g of dried seaweed powder was weighed and soaked in 50 ml of 95%
ethanol for 24h. The extract was filtered and the filtrate was extracted with petroleum
ether (60 to 80 deg. Celsius) and concentrated to dryness. The dried ether extract was
treated as total terpenoids (Ferguson, 1956).

53

3.4.6 UV-VISIBLE SPECTROSCOPY

UV-Vis analysis of the chloroform extract of the seaweed Caulerpa racemosa
was made by scanning in the range of 200-800nm using UV –Vis spectrophotometer
(Perkin Elmer Lambda 35).

3.4.7 GC-MS ANALYSIS

A high resolution mass spectrum equipped with a data system in combination
with Gas Chromatography was used for the chemical analysis of the seaweed, Caulerpa
racemosa. Gas chromatograph is used to seperate the individual chemical components
and the mass spectrometer ionizes and identifies them by their structure and molecular
weight. GC-MS was performed using Perkin elmer clarus SQ8C mass spectrometer
coupled with a Shimadzu 17A gas chromatograph fitted with a split-splitless injector
and a DB-5 capillary standard non-polar coloumn (cross linked 5% methyl silicone of
30m, 0.25mm ID and phase thickness of 0.25µm). The total run time was 35 min. The
helium flow rate was maintained at 1.2ml min. The sample was injected in the splitless
mode and the splitter was opened after a 4min delay. The sample injection volume was
1µl. Mass spectra were obtained at 0.5 sec intervals. Electron ionisation (EI) was used
at 70eV, the mass range scanned was 50-800m/z and the base peak of each compound
was selected for quantifying under full scan acquisition mode.

54

3.4.7.1 IDENTIFICATION OF BIOACTIVE COMPOUNDS

Interpretation of the mass spectrum obtained using GC-MS analysis was
performed by comparing with the database of National Institute of Standard and
Technology(NIST) having more than 62000 patterns. The name, structure and
molecular weight of the identified components were ascertained.

3.4.8 FTIR-SPECTROSCOPY

FTIR spectroscopy is used to measure the absorption, emission, and
photo-conductivity of matter by shining a narrow beam of infrared light at the matter in
various wavelengths and detecting how the matter responds to each wavelength. The
data obtained is further converted into digital information using a mathematical
algorithm known as the “Fourier transform”. The FTIR analysis of the seaweed extracts
were performed using FT-IR (Perkin Elmer Spectrum RX I) spectrophotometer. The
dried seaweed extract was mixed with potassium bromide (KBr) in the ratio of 1: 100
and then compressed to prepare pellets. These pellets were recorded in the mid IR region 400 – 4000cm-1 at a resolution of 4cm-1. For each spectrum, 100 scans were CO added at a spectral resolution of 4cm-1. The frequencies for all sharp bands were accurate to 0.01cm-1.

55

3.5ANTIOXIDANT ACTIVITY
3.5.1 DETERMINATION OF ANTIOXIDANT ACTIVITY

The antioxidant activity of both the seaweed extract and TiO2 nanoparticle was characterized by five complementary biochemical methods to determine and compare
their antioxidant potential. The mechanism responsible for antioxidant is different for
each method used. Therefore, different antioxidant assays were performed with different concentrations of the extract and TiO2 (0.2, 0.4, 0.6, 0.8 and 1mg/ml). All antioxidant assays were performed in triplicates, and the absorbance was read with a
UV-vis microplate spectrophotometer (Epoch Biotek, USA). Results are expressed as
antioxidant percentage and calculated from the standard curve. Ascorbic acid is an
antioxidant, which is used as a standard in all the antioxidant assays.

3.5.2 DPPH. RADICAL SCAVENGING ASSAY

The scavenging ability of the seaweed extract and TiO2 nanoparticle was determined in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH. (1, 1-Diphenyl-2-picryl-hydrazyl) according to the method of Braca et al,
(2001). DPPH radical scavenging activity of the extract and TiO2 nanoparticle was
determine according to the method reported by Blois (1958). An aliquot of 0.5 ml of
sample solution in methanol was mixed with 2.5 ml of 0.5 mM methanolic solution of
DPPH. The mixture was shaken vigorously and incubated for 30 min in the dark at
room temperature. The tubes were allowed to stand in dark for 30mins at room
56

temperature. All tests were performed in triplicate. The absorbance of the sample was
measured at 517nm against the blank using UV spectrophotometer..

3.5.3 FERRIC ION REDUCING POWER (FRAP) ASSAY

Ferric reducing power of seaweed chloroform extract and TiO2 were
determined using FRAP assay Dudonne et al., 2009, Luqman et al., 2012. FRAP
assay is based on the ability of the antioxidants to reduce Fe3+ to Fe2+ in the presence
of 2,4,6- tri(2-pyridyl)-s-triazine (TPTZ), forming an intense blue Fe2+ -TPTZ
complex with an absorption maximum at 593nm (Dudonné et al., 2009). Different
concentrations of the sample extract and TiO2 nanoparticle with the standard (Ascorbic
acid) was added to 2.7mL of FRAP reagent (10 parts of 300 mM acetate buffer
(pH-3.6), 1 part of 10mM TPTZ solution and 1 part of 20 mM ferric chloride hexahydrate (FeCl3.6H2O) solution) and the reaction mixture is incubated at 370C in dark for 30 min. The absorbance of the samples and control was measured at 593 nm.
The antioxidant capacity of the samples based on the ability to reduce ferric ions is
calculated from the linear calibration curve and expressed as mM. All tests were
performed in triplicate.

3.5.4 NITRIC OXIDE RADICAL SCAVENGING ACTIVITY

Nitric oxide generated form sodium nitroprusside in aqueous solution at
physiological pH interacts with oxygen to produce nitrite ions, which were measured
by the Griess reaction (Green et al., 1982). The reaction mixture containing 10 mM
sodium nitroprusside, phosphate buffered saline with different concentrations of the
57

seaweed extract and TiO2 nanoparticles in a final volume of 3ml, were incubated at
25°C for 150 min. A 0.5ml aliquot of the incubated sample was removed at 30min
intervals and 0.5 ml Griess reagent(1% sulfanilamide, 0.1% naphthylethylene diamine
dihyrochloride in 2% H3PO4) was added. The absorbance of the chromophore formed
was measured at 546nm. All tests were performed in triplicate. Percent inhibition of the
nitric oxide generated was measured by comparing the absorbance values of control
and sample preparations. Ascorbic acid is used as a positive control.

% NO Scavenging activity = (A0 ? A1) / A0 × 100

where A0 was the absorbance of the control (without sample) and A1 was the absorbance of the sample or standard.

3.5.5 HYDROXYL RADICAL SCAVENGING ACTIVITY

Hydroxyl radical scavenging activity is commonly used to evaluate the free
radical scavenging effectiveness of various antioxidant substances. Hydroxyl radical
scavenging activity was measured by studying the competition between deoxyribose and the test compound by Fe3+–Ascorbate–EDTA–H2O2 system (Fenton reaction) according to this method. The generation of OH is detected by its ability to degrade
deoxyribose to form products, which on heating with TBA forms a pink colored
chromogen. Reaction mixture contained 60 ?l of 1.0 mM FeCl2, 90 ?l of 1mM
1,10-phenanthroline, 2.4 ml of 0.2 M phosphate buffer, 150 ?l of 0.17 M H2O2, and
1.5 ml of sample at various concentrations. The reaction was initiated by the addition of H2O2. After incubation at room temperature for 5 min, the absorbance of the mixture at
58

560 nm was measured with a spectrophotometer. The hydroxyl radical scavenging
activity was calculated.

% Hydroxyl scavenging activity = (A0 ? A1) / A0 × 100 where A0 was the absorbance of the control (without sample) and A1 was the absorbance of the sample or standard.

3.5.6 TOTAL REDUCING POWER ASSAY

The total reducing power was determined by the method of (Oyaizu M 1986).
Different concentrations of test material in 1 ml 200 µM potassium phosphate buffer,
pH 6.6, and 2.5 ml 1% potassium ferricyanide K3Fe (CN)6. The mixture was
incubated at 50°C for 20 min. A 2.5ml aliquot of 10% trichloroacetic acid was added to
the mixture, which was then centrifuged at 3000 g for 10 min. The upper layer of the
solution (2.5 ml) was mixed with 2.5 ml distilled water and 0.5 ml 0.1% FeCl3and
absorbance was measured at 700 nm. Butylated hydroxytoluene (BHT) 5, 10, 20, 40,
50 µg was used as a reference material. Higher value absorbance of the reaction
mixture indicated greater reducing power. All tests were performed in triplicate and the
graph was plotted with the average of the three determinations.

3.6 ANTICANCER ACTIVITY
3.6.1 CELLS AND CELL CULTURE

Colon cancer cell line (HCT15) was purchased from National Centre for Cell Sciences (NCCS), Pune. Cell lines were grown in 96 well plates at 37°C, 5% CO2 and
59

90% humidity in DMEM medium, containing 10% fetal bovine serum and 1X
Antibiotic Antimycotic Solution. The cells were grown confluence, which could be
observed under an inverted microscope and sub-cultured at three to four days interval.

3.6.2 MTT ASSAY (CELL VIABILITY TEST)

Cell survival test was determined by using 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenylteterazolium bromide (MTT) reduction assay (Mossman, 1983). The HCT15 cells were plated separately in 96 well plates at a concentration of 1 × 104 cells/well.
After 24 h, cells were washed twice with 200 µl of 1X PBS, then the cells were treated with TiO2 nanoparticles and the chloroform seaweed extract at different concentrations (25, 50, 100, 250 and 500?g/ml) in triplicates and incubated at 370C for 24 h in a CO2 incubator. At the end of the treatment period the medium was aspirated and 5µl of
MTT (0.5mg/mL) was added to each well and the plates were further incubated for 4 h at 37ºC in CO2 incubator. The MTT containing medium was then discarded and the cells were washed with PBS (200 µl). The formazan crystals that formed were then
dissolved by adding 100 µl of DMSO and this was mixed properly by pipetting up and
down. Spectrophotometrical absorbance of the purple blue formazan dye was measured
in a microplate reader at 570 nm (Biorad 680). The cytotoxicity was obtained by
comparing the absorbance between the samples and control. The values were then used to calculate the concentration of the sample required to cause a 50% reduction (IC50) in cell growth. Each treatment was measured in triplicate; arithmetic mean and standard
deviation were calculated automatically and cytotoxicity was determined using Graph
pad prism5 software.

60

3.6.3 FLUORESCENT ANALYSIS FOR APOPTOTIC DETECTION USING
DUAL STAINING

Acridine orange is taken up by both viable and nonviable cells and if
intercalated into double stranded nucleic acid it emits green fluorescence or if bound to
single stranded nucleic acid it emits red fluorescence. Ethidium bromide is taken up
only by nonviable cells and emits red fluorescence by intercalation into DNA. 0.2 X 106cells/ well were seeded in a 96 well plate and allowed to adhere overnight. Cancer
cells were treated with respective IC50 values of both chloroform seaweed extract and synthesised TiO2, incubated upto 24 hrs, harvested and washed three times with phosphate buffered saline (PBS). One µl of dye mixture (100 ?L/mg AO and 100
?L/mg EB in distilled water) was mixed with 9 µl of cell suspension on a clean
microscope slide. The suspension was immediately (fast uptake) examined by
fluorescence microscopy (blue filter) at 400X magnification.

3.7 INVIVO ASSESSMENT OF NANOTOXICITY
3.7.1 ZEBRAFISH BIOASSAY

Wild type zebrafish were obtained from the Fisheries Department of
Puducherry and kept under a constant photoperiod of 14:10 (L:D) and fed twice a day
with commercial fish food. Dechlorinated water with a dissolved oxygen concentration
of 6.0 ± 0.2 mg/L was used. The temperature and pH was maintained at 26 ± 2 °C and
7 ± 0.5 respectively. Adult zebrafish of similar lengths (38 ± 2 mm), age (5 months)
and body weight (1 ± 0.2 g) were selected and acclimated for three weeks in a glass
tank before experiments. Fish were then graded into 5 experimental glass aquaria
61

(volume, 5 L), (3 fish/tank), and allowed to rest for 24 h prior to the commencement of
the experiment.

3.7.2 NANOPARTICLE EXPOSURE

Zebrafish were exposed to a concentration of 10mg/L TiO2 nanoparticles for
24, 48, 72 and 96 hours. The concentrations of seaweed synthesised TiO2 nanoparticles
were selected, to match the highest concentration of toxic nanometals used (Griffitt et
al., 2009).

3.7.3 PREPARATION FOR HISTOPATHOLOGICAL EXAMINATIONS

Briefly, fishes (n = 3) were sacrificed at 24, 48, 72 and 96 hours after TiO2
nanoparticles exposure. Gills, lungs and brains were excised. Histological examinations
were performed according to the standard laboratory procedures. All tissues were fixed
in 4% paraformaldehyde (PFA) prepared in 10mM phosphate buffered saline (PBS) for 24h at 40C. Fixed tissues were then gradually dehydrated in increasing concentration of
ethanol and embedded in paraffin wax . Sections of 5 ? thicknesses were collected on
super Frost+ glass slides (Menzel-Gläser, Braunschweig, Germany). At least three
slides at each tissue were collected. The sections were stained with hematoxylin–eosin
(HE) and observed under optical microscope (Nikon U-III Multi-point Sensor System,
USA) to identify and analyze the tissue slides.

62

3.8 STATISTICAL ANALYSIS

For statistical analysis, each of the experimental values was compared to its
corresponding control. Results were expressed as mean ± standard deviation (S.D.).
Statistical significance for all tests was set at P < 0.05.

63

4. RESULTS
4.1 SAMPLE COLLECTION

After a expeditious literature survey and based on the available marine algal
data, the seaweed Caulerpa racemosa was decided to collect in the Bay of Bengal at
mandapam region, Ramanathapuram district, located in the east coast of Tamilnadu.
The seaweed was collected with the help of the local fisherman, around 100mts from
the shore line.
A B

C
Figure1: A. Fresh seaweed, Caulerpa racemosa. B. Dried seaweed C. Satellite view
of the seaweed sampling point
64

4.2 SAMPLE PREPARATION

The collected samples were washed thoroughly in the seawater to remove
attached epiphytes, sand and debris. After transporting to the laboratory, the samples
were washed again with normal water followed by distilled water. Following
subsequent washing, the samples were shade dried for around 3 weeks. The dried
sample was powdered in a blender (figure 2) and stored for further use.

Figure 2: Dried seaweed powder

4.3 NANOPARTICLE SYNTHESIS

Biosynthesis of TiO2 nanoparticles using 10% crude aqueous extract of Caulerpa racemosa is safe and ecofriendly when compared to chemical synthesis.
After the addition of 100mM of Titanium tetra isopropoxide to the crude seaweed
extract, the reaction mixture turned light yellow from dark brown colour in 2hrs,
indicated the formation of TiO2 nanoparticles.

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4.4 CHARACTERISATION OF TIO2 NANOPARTICLES
4.4.1 UV VISIBLE SPECTROSCOPY

The UV-Vis spectrum profile of the biosynthesised nanoparticles was
confirmed using the wavescan mode that range from 200 to 1100nm. The optical
properties of the nanoparticles were studied using Perkin Elmer Lambda 35
spectrophotometer. Nanoparticle light absorption is produced by the coherent
oscillation of their conduction band electrons, induced by their interaction with the
electromagnetic field of the incident light. The resultant oscillation modes are called
surface plasmons. Thus an absorption band results when the frequency of the incident
photon resonates with the conduction band electron oscillation, and this is known as
surface plasma resonance (SPR). Figure 3 shows the UV-Vis absorption spectra that
exhibits a well defined plasma resonance peak at 324nm with absorbance of 0.943. The band gap energy value of TiO2 was calculated using the formula and was found to be 3.8eV.

Figure 3. UV-Vis spectra of synthesised TiO2 nanoparticles

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4.4.2 SCANNING ELECTRON MICROSCOPE (SEM) ANALYSIS

Scanning Electron Microscopy (SEM) is a well established technique used to
study the topography, texture and surface features of powders. The SEM produces a
three dimensional view of specimen and this is very useful in examining the shape and
structure of a specimen. In SEM analysis an electron gun emits a beam of electrons
which then interacts with the surface leading to emission of electrons from the surface
of the specimen during the scanning with the electron beam. The electron from the
beam interacts with the sample resulting in deflection of secondary particles to a
detector which subsequently converts the signal to voltage and amplifies it.

The surface morphology, size and shape of the seaweed synthesized TiO2 nanoparticles were investigated using FESEM with the magnification of 10,000x and
resolution 10nm (Fig 4a,b,c,d). The EDAX spectrum recorded in the binding energy
region of 0-20 keV shows the clear elemental composition of the synthesised TiO 2 nanoparticle (Figure 4f). The FESEM images clearly shows agglomerated TiO2 nanoparticles having irregular and porous surface. The intense signal at 4.5 and 0.25
keV strongly suggests that Ti and O were the major elemental compounds which is due
to its surface plasmon resonance. The atomic percentage of Ti and O was found to be
17.94 and 65.33.

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A B

C D

E F
Figure 4. FESEM images of synthesised TiO2 nanoparticles (A) at 100?m (B) at 10?m (C) at 2?m (D) at 2?m (E) EDAX image showing the surface of the synthesised TiO2
nanoparticles (F) EDAX showing the chemical composition of TiO2 nanoparticles
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4.4.3 TRANSMISSION ELECTRON MICROSCOPE (TEM) ANALYSIS

In TEM a beam of electrons is propagated through a solid sample in a vacuum.
Electrons transmitted through the sample are then detected to produce three
dimensional images which represent the relative extent of penetration of electrons in a
specific sample. The morphology and the size of the nanoparticles are studied using TEM. The TEM micrograph of the synthesized TiO2 nanoparticles at 50 nm and 10 nm scales is shown in Fig 5 (a, b). It was found that the nanoparticles are polydispersed
with an average particle size of 21.4nm.

Rod and tetragonal shaped nanoparticles can be visualized in figure 5 (b, c, d, e). Figure 5 (e) depicts the selected area electron diffraction (SAED) pattern of TiO2 nanoparticles. TEM image revealed the presence of two major different morphologies
of TiO2: tetragonal and rod. Selective area electron diffraction (SAED) patterns exhibit
concentric Scherrer rings having all possible orientations with intermediate dots, indicating that the TiO2 nanoparticles are highly concentric and crystalline in nature Fig 5 (e). Single concentric rings further confirms the presence of anatase crystals in the
nanoparticles.

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A B
C D

E F Figure 5 TEM image of synthesised TiO2 nanoparticles (A) at 100nm (B) at 50nm (C) (D) at 20nm (e) at 5nm (f) SAED image of synthesised TiO2 nanoparticles
70

4.4.4 XRD ANALYSIS

Figure 6 shows the XRD pattern that confirms the crystalline nature of the TiO2 nanoparticles. The crystallite size of the TiO2 nanoparticle was calculated using Scherrer’s equation and found to be 21nm. It was found that most of the the sharp
peaks observed in XRD pattern, are in consistent with anatase phase. The three main
types of information in a diffraction pattern include the position, the intensity and the
shape of diffraction peaks. Comprehensive libraries of available characteristic d-
spacings and intensities (JCPDS-files) of previously studied solids are available for
comparative purposes.

The XRD pattern revealed peaks at 25.320, 28.410, 37.870, 40.580, 48.010, 55.060 and 62.510 that matches with the miller indices value at (101), (110), (004), (111), (200), (105) and (204) crystallographic plane respectively. The peaks at 25.320, 37.870, 48.01, 55.060 and 62.510corresponds to the anatase phase of TiO2 nanoparticles which
is in agreement with the standard spectrum of Joint committee on powder diffraction standards (JCPDS) card no. 21-1272 (anatase TiO2). Similarly peaks at 28.41 0 and 40.580 corresponds to the rutile phase of TiO2 nanoparticles which coincides with the standard spectrum of JCPDS card no. 21-1276 (Rutile TiO2). The major peaks corresponds to the anatase crystals of TiO2. The intensity of XRD peaks of the
synthesized nanoparticles are crystalline and broad diffraction peaks indicates small
crystallite. There is no any other fallacious diffraction peak found in the prepared
sample.

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Table. 1 Comparison of d-spacing values of seaweed synthesized rutile and anatase nanoparticles from our XRD with the reported d-spacing values given in the JCPDS cards (21-1272, 21-1276) and the corresponding 2? values Crystal phase hkl 110 101 200 111 210 211 220 Rutile d-spacing/? JCPDS 3.22 2.47 2.28 2.17 2.04 1.67 1.61 XRD 3.13 2.22 2? 28.41 40.58 hkl 101 103 004 200 105 211 204 Anatase d-spacing/? JCPDS 3.51 2.42 2.37 1.89 1.69 1.66 1.47 XRD 3.51 2.37 1.89 1.66 1.48 2? 25.32 37.87 48.01 55.06 62.51

4.4.5 FOURIER TRANSFORM INFRA RED SPECTROSCOPIC ANALYSIS

Infrared spectroscopy has been extensively used for identifying the various
functional groups on the catalyst itself, as well as for identifying the adsorbed species
and reaction intermediates on the catalyst surface. It is one of the few techniques
capable of exploring a catalyst both in its bulk and its surface, and under actual reaction
conditions. It is widely used for characterizing the acid sites of the catalyst, which are
responsible for their catalytic properties. A typical FTIR spectra of seaweed
synthesised TiO2 nanoparticles is given in Figure 7.
Fourier transform infrared spectroscopy was performed to identify the
associated functional groups that are present in the TiO2 nanoparticles. The results of
FTIR analysis of the TiO2 nanoparticle and their corresponding functional groups are illustrated in the Table 1. The FTIR spectra of TiO2 nanoparticle revealed 9 prominent bands at 3408, 2926, 1630,1402, 1193, 1109, 991, 660 and 620 cm-1 that represents O-H, dimer O-H, CH=CHR, C-O, C-O, Si-OR, CH=CH2, C-Br and C-H respectively.
72

Figure 6 X-ray diffraction patterns of synthesised TiO2 nanoparticles
CL-Tio2-SeaweedName Description
4000 400 3500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
620.16cm-1
660.02cm-1
3408.79cm-1 1109.65cm-1
1630.83cm-12926.98cm-1 1193.44cm-1
1402.24cm-1
991.74cm-1

Figure 7. FTIR band of seaweed synthesized TiO2 nanoparticles
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Table: 2 Functional group analysis of the FTIR spectra for the TiO2 nanoparticles
Sl.No Peak values (cm-1) Functional groups Component
1. 3408.79 Phenols O-H stretch
2. 2926.98 Carboxylic acids Dimer O-H
3. 1630.83 Alkenes CH=CH-
4. 1402.24 Carboxylic acids C-O stretch
5. 1193.44 Esters C-O stretch
6. 1109.65 Miscellaneous Si-OR broad
7. 991.74 Alkenes CH=CH2
8. 660.02 Alkyl halides C-Br
9. 620.16 Alkynes C-H

4.5 PHYTOCHEMICAL PROFILING
4.5.1 QUALITATIVE AND QUANTITATIVE ANALYSIS

The phytochemicals present in chloroform and dichloromethane extracts of
Caulerpa racemosa is tabulated in Table.3. The qualitative analysis of the two different
extracts (chloroform and dichloromethane) of Caulerpa racemosa showed the presence
of phytochemical constituents such as terpenoids, saponins, glycosides, cardiac
glycosides and phenols. These phytochemicals were present in both the solvent extracts
of Caulerpa racemosa but higher in the chloroform extract. From the preliminary
results, the chloroform extract was further analyzed for total phenolic and terpenoid
contents. The FCR method depends on the reduction of Folin-Ciocalteu reagent by the
phenols in the sample to a mixture of blue oxides which have a maximal absorption in
the region of 765 nm. Total phenolic content of the seaweed extract was calculated to
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be 7.84 mg/g of catechol equivalent. Total terpenoid content of the chloroform extract
of Caulerpa racemosa was found to be 11.5 mg/g.

Table3. Qualitative phytochemical analysis of Caulerpa racemosa solvent extracts
(+: present, ++: highly present; -: absent)
S.No Phytoconstituents Test performed
Presence/ Absence CHCl3 DCM 1 Terpenoids Salkowski test ++ +
2 Flavonoids
Alkaline Reagent Test – –
Lead acetate Test – –
3 Saponins
Foam Test + +
Froth Test + +
4 Tannins – –
5 Alkaloids
Mayer’s test – –
Hager’s Test – –
6 Steroids Salkowski Test – –
7 Glycosides Liebermann’s Test ++ +
8 Phlobatannins Precipitate Test – –
9 Proteins
Xanthoproteic Test – –
Ninhydrin Test – –
10 Coumarins – –
11 Cardiac Glycosides Keller-Killani Test ++ +
12 Carbohydrates
Fehling’s test – –
Benedict’s Test – –
13 Phenols ++ +
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4.5.2 UV VISIBLE SPECTROSCOPY

The UV-Vis spectra of the chloroform seaweed extract was recorded in the
wavescan mode of Perkin Elmer Lambda 35 spectrophotometer that range from 200 to
1100nm. Figure 8 illustrates the UV-Vis spectra that exhibits a sharp peak at 262nm
with absorbance of 0.571.

Figure 8. UV-Vis spectra of the chloroform extract of Caulerpa racemosa

4.5.3 FOURIER TRANSFORM INFRARED SPECTROSCOPY (FT-IR)

FT-IR spectra of the chloroform seaweed extract showed the presence of
phenol as the major functional group. The FTIR spectra with significant spectral bands at 3457cm-1, 2970cm-1, 1637cm-1, 1109cm-1, and 657 cm-1 represents O-H, C-H, N=C,
C-O, and C-H respectively. (Figure 9) (Table 4)

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CL-Seaw-ExtName Description
4000 400 3500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
3457.24cm-1
1637.54cm-1
657.67cm-1
2970.67cm-1
2069.99cm-1
1109.98cm-1

Figure 9. FTIR band of the chloroform extract of Caulerpa racemosa

Table: 4 Functional group analysis of FTIR spectra for Caulerpa racemosa
Sl.No Peak values (cm-1) Functional groups Component
1. 3457.24 Phenols O-H stretch
2. 2970.67 Alkanes C-H stretch
3. 2069.99 Alkenes C=C stretch
4. 1637.54 Amide C=O stretch
5. 1109.98 Esters C-O stretch
6. 657.67 Alkynes C-H bend

4.5.4 BIOACTIVE COMPOUND IDENTIFICATION BY GC-MS ANALYSIS

The chromatogram of the chloroform extract of the seaweed Caulerpa
racemosa is shown in figure 10. GC-MS chromatogram revealed 30 chemical
compounds that was detected using NIST library. Out of which 20compounds were
selected based on the % area of the compound. A broad range of compounds such as
phenol, alkanes, amide, esters and alkyl halides could be deciphered from the
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chromatogram. The active principles with their retention time (RT), molecular weight
(MW) and molecular formula are presented in Table 3.

Figure 10 GC-MS chromatogram of chloroform extract of Caulerpa racemosa

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79

80

Figure 11. GC-MS spectrum and structure of the 20 bioactive compounds in the
chloroform extract of Caulerpa racemosa

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Table 3: Bioactive compounds in chloroform extract of Caulerpa racemosa
S.No Name of the Compound Retention Time
Molecular Weight
Formula
1 2,4-di tert-butylphenol 11.897 206.329 C14H22O
2 1-nonadecene 13.728 266.513 C19H38
3 2,4-Diphenyl-4-methyl-1-pentene 17.714 236.350 C18H20
4 3-Octadecene 17.944 252.486 C18H36
5 Phthalic acid, butyl hept-4-yl ester 21.091 320.423 C19H28O4
6 n-Hexadecanoic acid 21.226 256.424 C16H32O2
7 9,12-Octadecadienoic acid 24.382 280.452 C18H32O2
8 Z-(13,14-Epoxy) tetradec-11-en-1-o-1 acetate
24.507 268.391 C16H28O3
9 Octadecanoic acid 24.962 284.484 C18H36O2
10 Octadecane, 3-ethyl-5-(2-ethylbutyl) 27.918 366.707 C26H54
11 9,19-Cyclolanostane-3,7-diol 28.123 444.744 C30H52O2
12 3-Hydroxypropylpalmitate 30.064 314.503 C19H38O3
13 Octacosane 30.194 394.772 C28H58
14 Hexadecanoic acid, 2-hydroxy-1 (hydroxy methyl) ethyl ester
30.274 330.502 C19H38O4
15 Diisooctyl phthalate 30.549 390.564 C24H38O4
16 Sitosterol 31.100 414.718 C29H50O
17 Heptacosane 31.685 380.733 C27H56
18 1H-Indene, 1-hexadecyl-2,3-dihydro 33.330 342.601 C25H42
19 Hexacosane 33.536 366.707 C26H54
20 Octadecanoic acid 2,3 dihydroxy propyl ester
33.791 358.555 C21H42O4

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4.6 ANTIOXIDANT ACTIVITY
4.6.1 DPPH. RADICAL SCAVENGING ACTIVITY

The DPPH. radical scavenging activity is a sensitive antioxidant assay and is
independent of substrate polarity (Yamaguchi et al., 1998), this model is widely used to
evaluate antioxidant activities in a relatively short time compared with other methods.
As shown in Figure 11 the highest DPPH scavenging activity was observed in TIO2
nanoparticle (74.24%) wherease for chloroform extract of Caulerpa racemosa it was
70.95 at a a concentration of 500?g/ml. The scavenging activity of both the seaweed
extract and the TiO2 nanoparticle depends on their concentration. Ascorbic acid, which
is used as positive control showed 75% scavenging activity.

4.6.2 FERRIC ION REDUCING POWER (FRAP) ASSAY

The FRAP is another antioxidant assay that is measured using a reaction in which Fe 3+ is reduced to Fe2+. Figure 13 illustrates that the ferric reducing antioxidant
potential of the TiO2 was similar to that of the standard and comparatively higher than
the chloroform seaweed extract. The ferric ion reducing antioxidant potential (FRAP)
of the seaweed extract and the TIO2 nanoparticle was estimated from their ability to
reduce TPTZ-Fe (III) to TPTZ-Fe (II). This was determined by the amount of ferric
ions that are present, thus low ferrion ion concentration indicates higher reducing
power.. At a concentration of 200?g/ml both the seaweed extract and the TiO2
nanoparticle showed almost the same number of ferric ions.
83

Figure 12. DPPH radical scavenging activity of the synthesised TiO2 nanoparticle and the
chloroform seaweed extract
Figure 13 Ferric Reducing Antioxidant Power of the synthesised TiO2 nanoparticle
and the chloroform seaweed extract
84

4.6.3 NITRIC OXIDE RADICAL SCAVENGING ACTIVITY

Nitric oxide radical scavenging ability of the chloroform seaweed extract and
TiO2 nanoparticles along with the standard ascorbic acid was illustrated in the figure
14. This assay measures the conversion of reactive nitric oxide to nitite. The amount of
nitrite present is directly proportional to the amount of nitric oxide that is removed.
TiO2 exhibits higher nitric oxide scavenging activity when compared to the seaweed
extract. At 100?g/ml concentration the scavenging activity of TiO2 nanoparticle was
considerably more than that of the standard, ascorbic acid. The nitric oxide radical
scavenging activity is dose dependent in both the seaweed extract and TiO2
nanoparticles.

4.6.4 HYDROXYL RADICAL SCAVENGING ACTIVITY

This assay shows the ability of the seaweed extract and TiO2 nanoparticle to
inhibit hydroxyl radical-mediated deoxyribose degradation in an Fe3+ EDTA-ascorbic
acid and H2O2 reaction mixture. The generation of OH is detected by its ability to
degrade deoxyribose to form products which on heating with TBA forms a pink
coloured chromogen. Figure 15 illustrates that the percentage of hydroxyl scavenging
activity of the seaweed synthesised TiO2 nanoparticle was remarkably higher than that
of the chloroform seaweed extract. Further the scavenging activity was also found to be
dose dependent.

85

Figure 14 Nitric oxide radical scavenging activity of the synthesised TiO2 nanoparticle
and the chloroform seaweed extract

Figure 15 Hydroxyl scavenging activity of the synthesised TiO2 nanoparticle and the
chloroform seaweed extract
86

4.6.5 TOTAL REDUCING POWER ASSAY

The reducing power assay is often used to evaluate the ability of an antioxidant
to donate an electron (Yildirim et al., 2000). In this assay, the ability of the seaweed
extract and synthesised TiO2 nanoparticles to reduce Fe3+ to Fe2+ was determined.
The reducing power of the seaweed extract and the seaweed synthesised TiO2
nanoparticle was found to be slightly increasing with increase in the concentration.
Figure 16 showed the reducing activities of seaweed extract and the synthesised TiO2
nanoparticles in comparison with ascorbic acid as standard. The higher the absorbance
of the reaction mixture, the higher would be the reducing power.

4.7 INVITRO ANTICANCER ACTIVITY
4.7.1 CYTOTOXICITY ASSAY (MTT)

MTT assay was performed to determine the cytotoxic effect of the chloroform
extract and the synthesised TiO2 nanoparticle and the results are shown in figure 17.
The results of the experiments showed that the chloroform extract of the seaweed and
the TiO2 nanoparticle inhibits the cell viability in a dose dependent manner, thereby
exhibits strong cytotoxic effect on HCT-15 colon adenocarcinoma cells. A significant
inhibition of proliferation of HCT-15 cells was found after treatment with the chloroform extract and the synthesised TiO2 nanoparticle. IC50 value for the seaweed extract and the TiO2 nanoparticle was found to be 465 µg/ml and 440 µg/ml
respectively.

87

Figure 16 Total antioxidant activity of the synthesised TiO2 nanoparticle and the
chloroform seaweed extract
Figure 17 Cytotoxity assay (MTT) of the synthesised TiO2 nanoparticle and the
chloroform seaweed extract
88

4.7.2 FLUORESCENT ANALYSIS FOR APOPTOTIC DETECTION USING
DUAL STAINING

To determine whether the cytotoxicity of the seaweed extract and the
synthesised TiO2 nanoparticles was related to the induction of apoptosis,
morphological assay of cell death was investigated using AO/EB staining for
fluorescence microscopy. After HCT-15 cells were exposed to the seaweed extract and the synthesised TiO2 nanoparticles IC50 concentration for various time points, different morphological features were analyzed. Uniformly green live cells with normal
morphology were seen in the control group (Fig. 18A), where as orange apoptotic cells
are seen in the cells treated with seaweed extract (Fig. 18B) and synthesised TiO2
nanoparticles (Fig. 18B). The results suggested that both the seaweed extract and TiO2
nanoparticles was able to induce marked apoptotic morphology in HCT-15 cells.

4.8 INVIVO NANOTOXICITY IN ZEBRA FISH

Morphological responses of the gills, liver and brain to TiO2 nanoparticles
exposure were illustrated in Figure 19. The TiO2 nanoparticles exposure for 24h, 48hr,
72hr and 96hrs caused no mortality or significant morphological changes in the adult
zebrafish. There was no significant changes in the histopathology of the gills, liver and
brain of the nanoparticle exposed adult zebrafish.

89

A
B C Figure 18 AO/EB dual staining A. Control cells B. HCT-15 cells treated with
Chloroform seaweed extract C. HCT-15 cells treated with TiO2 nanoparticles

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Fig. 19 Effect of biologically synthesized TiO2 nanoparticles on Brain, Liver and Gill filament of zebrafsh after 24, 48, 72 and 96 hrs of nanoparticle exposure. Representative pictures of Brain, Liver and Gill filaments from 96 hrs control (C) and treated with TiO2 nanoparticles after 24 hrs (I), 48 hrs (II), 72 hrs (III), 96 hrs (IV).

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5. DISCUSSION

Marine organisms are rich source of bioactive compounds with remarkable
impact in the field of pharmaceutical, industrial and biotechnological product
developments. Biological entities from marine resources have typical nanostructures
such as seashells, pearls and fish bones, diatoms and sponges that are constructed with
nanostructured cover of silica and coral reefs made of calcium which are arranged in
significant architectures (Chinnappan et al, 2015). Marine species live in a stressful
habitat like extreme cold, medium to less light, and high pressure, as a consequence
they are able to produce unbelievable complex natural products, that possess significant
application.

Seaweeds are one such variety that belongs to a group of plants known as
marine algae. These plants form an important renewable resource in the marine
environment and have been a part of human civilization from time immemorial.
Reports on the uses of seaweeds have been cited as early as 2500 years ago in Chinese
literature (c.f. Tseng2). The long history of seaweed utilization for a variety of purposes
has led to the gradual realization that some of their constituents are more superior and
valuable in comparison to their terrestrial counterparts (Subba Rao and Vaibhav, 2006).

Seaweeds are considered as a source of bioactive compounds such as proteins,
lipids, carbohydrates, carotenoids, vitamins and many other secondary metabolites with
a wide range of biological activities are known to produce metallic nanoparticles due to
presence of varied reductants. Marine bio-nanotechnology has various applications in
nanomedicines, food industries, pharmaceuticals fabric industries, etc. Marine algae
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such as Sargassum wightii, Fucus vesiculosus, Gelidiella acerosa, Ulva fasciata,
Acanthophora spicifera, Chlorella pyrenoidusa, Kappaphycus alvarezii, Sargassum
myriocystum, Stoechospermum marginatum, Laminaria japonica and diatoms
(Navicula atomus, Diadesmis gallica, Stauroneis sp.) have been reported to synthesize
gold, silver, cadmium, silicon, germanium and lead nanoparticles (Asmathunisha and
Kathiresan, 2013; Ramakrishna et al, 2016). Scarano and Morelli (2003) synthesised
cadmium sulphide nanoparticles using Phaeodactylum tricornutum. Similarly Nayak et
al, (2006) reported gold nanoparticles using Rhizoclonium riparium, Navicula minima,
and Nitzschia obtusa. Phyconanotechnology has become a paramount in the research
field of nanoparticle biosynthesis in which algae is used as a bio-factory for synthesis
of metallic nanoparticles (Narayanan et al., 2011).

Caulerpa racemosa, a species of edible green macroscopic marine algae, a
seaweed in the family Caulerpaceae commonly known as sea grapes found in the
shallow seas. Caulerpa racemosa is an invasive species found in the coastal regions of
all over the world densely populated in the Mediterranean coast. India, a tropical south
asian country has a stretch of 7500 km coastline with highly diversified seaweed flora.
A total of 57 taxa belonging to 37 genera from 19 sampling sites from southern district
of Tamilnadu were recorded by Sahayaraj et al (2012). Caulerpales produces secondary
metabolites that are known to exhibit anti inflammatory, antinociceptive, anticancer,
antibacterial, antioxidant, antiproliferative, antiviral and antilarvicidal properties.

Caulerpenyne, one of the major algal metabolites, has been suggested to act as
chemical deterrent against herbivores, but several allelochemicals, neurotoxic and
cytotoxic properties have been also reported for this compound. Yet, to the best of our
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knowledge there is no report on the synthesis of nanoparticles using Caulerpa
racemosa. Thus, Caulerpa racemosa was considered a potent biological agent for the
synthesis of nanoparticles with novel properties and being investigated in this study. Of various other metals and metal oxides in nanoparticle synthesis, titanium di oxide (TiO2) have a wide spread application in various industries like biomedical, environment,
cosmetics, printing, paint, paper, electrical and food.

As Arthur C. Clarke stated, “Any sufficiently advanced technology is
indistinguishable from magic”. Nanobiotechnology, Nanobiology or
Bio-nanotechnology is a crosswalk of nanotechnology and biology. Nanoparticles are
particles sized less than 100 nm (European commission, 2011) and consist of different
biodegradable materials like natural or synthetic polymers, lipids or phospholipids,
even metals. Submicron particles possess a very high surface to volume ratios which
increases the dissolution rate of the particles. The global market for Nanotechnology
was worth $39.2 billion and it is estimated that this will rise to $90.5 billion by 2021
(McWilliams 2016). Nanoscaled particles are custom tailored by the use of designed
polymers for specific applications. Nanoparticles are essentially a varied form of basic
elements derived by altering their atomic and molecular properties of elements.

Metal based engineered nanoparticles are produced from iron, titanium and a
variety of other metals and their oxides. Recently engineered nano materials have
received attention for their positive impact in improving many sectors of economy,
including consumer products, pharmaceuticals, cosmetics, transportation, energy and
agriculture, etc., and are being increasingly produced for a wide range of applications.
Fabrication of nanoparticles by physical, chemical and hybrid methods is expensive
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and also toxic to the environment. Biological synthesis of nanoparticles such as
‘Biomimetic’, a technique that utilises plants, bacteria, fungi, yeast, actinomycetes and
algae for the synthesis of biocompatible metal and metal oxide nanoparticles reduces
the cost of downstream processing and increases the environmental safety. Thus, the
eco-friendly green synthesis of bio-nanoparticles became the target of the
scientific/research community (Ravinder Singh et al., 2015).

In the present study, we report the biological synthesis TiO2 nanoparticles using the aqueous extract of the seaweed, Caulerpa racemosa. The crude aqueous
extract of the seaweed was prepared by concoction and filtered through whattman no.1 filter paper. The filtrate was used to reduce titanium tetra isoporpoxide to TiO2 nanoparticle. The aqueous extract of the seaweed, Caulerpa racemosa reduced the titanium tetra isopropoxide to TiO2 nanoparticles, upon heating at 800C for 2 hr indicated by the change in colour from dark brown to light brown, which displays the synthesis of TiO2 nanoparticles.

The UV-Vis spectroscopy is considered as a crucial analytical tool for characterizing the optical properties of TiO2 nanoparticles. The biologically synthesized TiO2 nanoparticles had maximum absorption of 0.943 at 324nm that correlates to a band gap energy value of 3.8eV. A larger band gap energy means a
smaller particle size, due to addition of fewer molecular orbitals to the possible states of
the particle. Thus there is a possible shift towards shorter wavelengths, due to
absorption at higher energies (Bagheri et al., 2013). Our results are in concomitant with the biologically synthesized TiO2 nanoparticle using Hibiscus flower extract that showed a UV absorption at 324nm (Ganapathi Rao et al, 2014). While, the UV
95

absorption of bacterial synthesized TiO2 nanoparticles using Bacillus subtilis was found to be at 366nm (Kirthi et al, 2011). A broad absorption in the wavelength of 300-350nm was already reported in TiO2 nanoparticles (Das et al., 2010). Patra AK, 2011 documented absorption maxima at 328nm for as-synthesized mesoporous TiO2 nanoparticle. This suggests that the biologically synthesized TiO2 nanoparticles using Caulerpa racemosa might be mesoporous in nature.

Further the FESEM images revealed about the surface of TiO2 nanoparticles being porous and agglomerated. Thus, the size and shape of the nanoparticles was
determined by visualizing under transmission electron microscope (TEM). The particle size of the biologically synthesized TiO2 nanoparticles varies from 10nm – 50nm with an average particle size of 21.8nm based on measuring 15 individual nanoparticles. TEM images revealed polydispersed bimorphic TiO2 nanoparticles: nanorods and tetragonal shaped nanoparticles with irregular surface. Similarly polydispersed TiO2 nanoparticles were synthesized using Eclipta prostata leaf extract (Rajakumar G et al.,
2012), Halomonas elongata (Mojtaba et al., 2018) and Psidium guajava extract
(Santhoshkumar et al., 2014).

Different size and shapes of TiO2 were reported which depends on various factors, not limited to organism or biomolecules that facilitates the reduction. Spherical shaped TiO2 nanoparticles were synthesized by using Lactobacillus sp. (Jha et al., 2009) and Fusarium oxysporum (Bansal et al., 2005). Sundrarajan and Gowri (2011) reported
cubic shaped TiO2 nanoparticles synthesised from titanium isopropoxide solution using
nyctanthes arbor-tristis leaves extract. Two species of lactobacillus Streptococcus
thermophilus and Lactobacillus bulgaricus were used as templates to direct the
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formation of biomorphic TiO2 hollow spheres and tubes (Han et al., 2012). Hollow structures of nanotubes, nanohelixes, nanocables, twin spheres, chain spheres, etc are
synthesised using bacillus, spirillum, vibrio, fusiform bacteria, square bacteria,
star-shaped bacteria (Fan et al., 2009).

The selected area electron diffraction (SAED) pattern illustrates the diffraction spots for anatase phase of TiO2 nanoparticles with good crystallinity. The SAED pattern disclosed the dotted concentric Scherrer rings which substantiates the
polycrystalline non-spherical shape of biologically synthesized TiO 2 nanoparticles. Khade et al., 2015 reported similar pattern of concentric Scherrer rings in green synthesized anatase phase TiO2 nanoparticles. The EDAX spectrum analysis confirms the presence of major elemental components, Ti and O at 4.5 keV and 0.25 keV,
respectively which is due to its surface plasmon resonance. The atomic percentage of Ti and O was 17.94 and 65.33 that substantiate the presence of TiO2. The other elemental compounds (Sodium, Potassium, Magnesium, etc) presented in the study
represents the compounds present in the aqueous extract of Caulerpa recemosa coating the TiO2 nanoparticles.

The crystalline nature and phase structure of the biologically synthesized TiO2 nanoparticles were determined by X-ray diffraction analysis. The XRD pattern of the synthesized TiO2 nanoparticles revealed 6 major peaks that confirms the crystalline nature of the nanoparticles. The sharp peaks precisely suggests that the synthesized
particles are in nanovicinity. The XRD analysis disclosed distinct diffraction peaks at 25.320, 28.410, 37.870, 40.580, 48.010, 55.060 and 62.510 that matches with the miller
indices value at (101), (110), (004), (111), (200), (105) and (204) crystallographic plane
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respectively. The peaks at 25.320, 37.870, 48.010, 55.060 and 62.510 corresponds to the
anatase phase of TiO2 nanoparticles which is in agreement with the standard spectrum
of Joint committee on powder diffraction standards (JCPDS) card no. 21-1272 (anatase TiO2). Similarly peaks at 28.410 and 40.580 corresponds to the rutile phase of TiO2 nanoparticles which coincides with the standard spectrum of JCPDS card no. 21-1276 (Rutile TiO2). The 2? peak at 37.870 and 55.060 was obtained in TiO2 nanoparticles synthesised using Cinnamomum tamala that represents anatase phase (Feiping et al., 2017). The 2? peak at 25.320 confirms the TiO2 anatase structure (Antic et al., 2012) (Ba-Abbad et al., 2012). Nanoparticles synthesised using Hibiscus rosasinensis revealed diffraction peak at 620 that conforms rutile phase of TiO2 which was
proclaimed to be tetragonal in structure (Ganapathi Rao et al., 2014).

The intensity of XRD peaks of the synthesized nanoparticles are crystalline and
broad diffraction peaks indicate very small size crystallite. The average crystallite size of the TiO2 nanoparticle calculated using Scherrer’s equation was found to be 21nm that is in coherent with the TEM analysis data. The XRD pattern shows that the crystal phase of the TiO2 was a mixture of both anatase and rutile phase with high crystallinity in the mesoporous walls that is intelligible with the TEM image. Mixed rutile and
anatase phase of TiO2 nanoparticles were reported by Anbalagan et al., (2015) and
Santhoshkumar et al., (2014) using Azhadiracta indica and Psidium guajava respectively. On the contrary, the major peak of 2? is at 25.320 that correlates to the
(101) crystallographic plane of anatase phase of TiO2. This suggests that the structure
of TiO2 nanoparticle extensively resemble the anatase crystalline phase. Literal reports
reveals that the nanoparticles with both anatase and rutile phase was suggested to exhibit anatase phase due to the dominant peak at 27.810 (Kirthi et al., 2011).
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Further to identify the functional groups that are present in the TiO2, the nanoparticle was studied by Fourier transform infrared (FTIR) spectroscopy. The FTIR spectra of TiO2 nanoparticles displayed prominent bands at 3408 cm-1 (O-H stretch of phenol), 2926 cm-1 (dimer O-H of carboxylic acid) and 1630 cm-1 (CH=CH-R of alkenes). Other bands include 1402 cm-1, 1193 cm-1, 1109 cm-1, 991 cm-1, 660 cm-1 and 620 cm-1 that represents C-O stretch of carboxylic acid, C-O stretch of esters, Si-OR, CH=CH2 of alkenes, C-Br of alkyl halides and C-H of alkynes respectively. The FT-IR spectra of TiO2 nanoparticles contain a broad band between ~ 3300 cm-1 to ~ 3500 cm-1 is due to the hydroxyl (O-H) stretching mode and peak at 1630 cm-1 is due to (O-H)
bending vibrations of atmospheric water molecules gets adsorbed on sample during analysis. The spectra of TiO2 sample shows absorption bands between ~620 cm-1 and 660 cm-1 is a characteristic peaks due to O=Ti=O stretching vibration mode, thus
confirms the synthesis of TiO2 nanoparticles.

The peaks at 479?cm?1 and 652?cm?1 was reported to be in correlation with
the anatase phase of TiO2 nanoparticles (Karakitsou and Verykios, 1993). FTIR
spectrum reported in seaweeds, Kappaphycus alvarezii, (Sumayya and Murugan 2017)
Sargassum wightii, Acanthafora spicifera and Caulerpa racemosa (Deepak et al., 2018)
are in consistent with our reports. Phytochemicals viz hydroxyl, amino and carboxyl
groups, are suggested to be an effective metal reducing agents and as capping agents
which is evident from the coating on the metal nanoparticle (Mahdavi, 2013).
Krishnasamy et al., (2015) reported that the presence of O-H stretching and C=C group
indicates the presence of terpenoid compounds. Tripathy et al., (2010) also suggests
that the O-H stretching and C=C group are due to the presence of terpenoid compounds.
Thus the functional groups associated with the phytochemicals are suggested to be
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responsible for the bioreduction and capping of TiO2 nanoparticles. Further to investigate the phytochemicals involved in the biological synthesis of TiO2
nanoparticles, a detailed phytochemical profiling of the seaweed was performed.

For phytochemical profiling, 20g of the seaweed Caulerpa racemosa was
soaked in 150ml of chloroform and 150ml of dichloromethane for three days with mild
shaking. Both the extracts were filtered through whattman no.1 filter paper and the
filtrate was concentrated in a Rotary evaporator. Phytochemical screening of the
chloroform and dichloromethane extract was performed using standard procedures
(Kokate et al., 1986, Harborne et al., 1998). The qualitative analysis of the two
different extracts (chloroform and dichloromethane) of Caulerpa racemosa showed the
presence of phytochemical constituents such as terpenoids, saponins, glycosides,
cardiac glycosides and phenols. Azhagu Raj R et al., (2015) reported the presence of
reducing sugars, tannins, pholobatanins, saponins, coumarins, flavonoids, phenols,
xanthoproteins and carbohydrates in the methanolic extract of the seaweed Caulerpa
racemosa. However, the chloroform and dichloromethane extracts of Caulerpa
racemosa revealed the presence of other phytochemicals like terpenoids, cardiac
glycosides, glycosides. Yet, the chloroform extract exhibits higher concentration of
glycosides, cardiac glycosides and phenols compared to dichloromethane. Terpenoids
and phenols of the chloroform seaweed extract were further quantified. Total phenolic
content and total terpenoid content was calculated to be 7.84mg per g of catechol
equivalent and 11.5 mg per g respectively.

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Owing to this preliminary qualitative and quantitative analysis, the chloroform
extract of the seaweed was further analyzed using UV-Visible spectroscopy, FTIR and
GCMS. UV-visible spectroscopy has become a rapid, yet a powerful analytical tool for
the qualitative analysis of phytocomponents. The chloroform extract of the seaweed
Caulerpa racemosa was scanned in the wavelength range of 200nm – 1100nm. The
chloroform extract of the seaweed had absorption maxima of 0.571 at 262nm which
coincides with the UV-Vis spectrum peak of Leptadenia reticulata (Dhivya and
Kalaichelvi, 2017). Cerovic et al., (2002) explained the presence of simple phenols and
phenolic acids in the range of 235 to 305nm.

The FTIR spectra revealed the presence of phenol, alkanes amide, esters and alkyl halides. The strong absorption bands of FTIR spectra at 3457 cm-1 (O-H stretch of phenol), 1637 cm-1 (C=O stretch of amide) and 657 cm-1 (C-H bend of alkynes). Other bands of FTIR spectra includes 2970 cm-1 (C-H stretch of alkanes), 2069 cm-1 (N=C stretch), and 1109 cm-1 (C-O stretch of esters). The FT-IR spectra of chloroform seaweed extract contain a broad band between ~ 3300 cm-1 to ~ 3500 cm-1 is due to the
hydroxyl (O-H) stretching mode that might represent the phenolic compounds in the
seaweed. Silver nanoparticles synthesised using Pithophora oedogonia revealed the
presence of phytochemicals viz carbohydrate, tannins, terpenoids, saponins, steroid,
and proteins. The FTIR suggested that the possible compounds responsible for the
stabilisation and capping of these silver nanoparaticles were terpenoids, secondary
amide derivatives and long chain fatty acids (Narayan Sinha et al., 2015). In the
spherical silver nanoparticles synthesized using Sargassum plagiophyllum, Ulva
reticulata, Enteromorpha compressa, the major ligand involved was hydrogen bonded
alcoholic groups. Further, the phytochemical screening of the algal samples revealed
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the presence of alkaloids, amino acids, flavonoids, phenols, tannins and carbohydrates
(Dhanalakshmi et al., 2012). Chakraborty et al., (2009) hypothesized that the enzymes
secreted by the algal cells of Lyngbya majuscula, Spirulina subsalsa and Rhizoclonium
hieroglyphicum are responsible for the nanoparticle synthesis. Thus the phytochemicals
viz phenol and terpenoids of the seaweed were responsible for the bioreduction and stabilisation synthesised TiO2 nanoparticles.

The GCMS spectra of the seaweed chloroform extract demonstrated the
presence of around 20 compounds viz 2,4-di tert-butylphenol, 1-nonadecene,
2,4-Diphenyl-4-methyl-1-pentene, 3-Octadecene, Phthalic acid, butyl hept-4-yl ester,
n-Hexadecanoic acid, 9,12-Octadecadienoic acid, Z-(13,14-Epoxy)
tetradec-11-en-1-o-1 acetate, Octadecanoic acid, Octadecane, 3-ethyl-5-(2-ethylbutyl),
9,19-Cyclolanostane-3,7-diol, 3-Hydroxypropylpalmitate, Octacosane, Hexadecanoic
acid, 2-hydroxy-1 (hydroxy methyl) ethyl ester, Diisooctyl phthalate, Sitosterol,
Heptacosane, 1H-Indene, 1-hexadecyl-2,3-dihydro, Hexacosane and Octadecanoic acid
2,3 dihydroxy propyl ester. For the analysis of non-polar components, alcohols, acids,
volatile substances, alkaloids, phenols, esters, long chain and branched chain
hydrocarbons and other bioactive components, GC-MS studies are being used
extensively (Konovalova et al., 2013; Kalaivani et al., 2012; Praveenkumar et al., 2010;
Chibani et al., 2011; Sangeetha and Vijayalakshmi, 2011; Ghannadi and Dezfuly, 2011;
Venkataraman et al., 2012).

The FTIR bands and the GCMS spectra are compared and confirmed the
presence of the functional groups in the seaweed chloroform extract. The band at 3457 cm-1 (O-H stretch of phenol) affirms the presence of 2,4, di-tert butylphenol. Varsha et
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al., 2015 demonstrated that 2,4, di-tert butylphenol is an antioxidant, antifungal and a
very good protective agent against oxidative damage. The same author further
correlates the amount of total phenolic content to be directly proportional to the
antioxidant property of the compound.

1-nonadecene, an alkane hydrocarbon was confirmed by the band at 2970 cm-1 (C-H of alkanes). The band at 1637 cm-1 (C=O of amide) was correlated to the
n-hexadecanoic acid present in the seaweed extract. The C=O stretch of amide interrupts the amine NH2, so the band shifts to near 1637 cm-1. Sahaya Sathish et al., (2012) identified n-hexadecanoic acid, hexadecanoic acid, ethyl ester,
9,12-octadecadienoic acid (Z,Z)- and octadecanoic acid by GC-MS in the ethanolic leaf
extract of Vitex altissima. In that study, it was reported that n-hexadecanoic acid and
hexadecanoic acid, ethyl ester possess various biological activity like nematicide,
pesticide, hemolytic alpha 5- alpha reductase inhibitor, lubricant, antiandrogenic,
antioxidant, hypocholesterolemic; 9,12-octadecadienoic acid (Z,Z)- possess
hypocholestrolemic activity, antihistaminic, insectifuge, antiexzemic and antiacne
activity.

N-hexadecanoic acid, 9,12 octadecadienoic acid, octadecanoic acid,
Hexadecanoic acid, 2-hydroxy-1 (hydroxy methyl) ethyl ester, sitosterol and
octadecanoic acid 2,3 dihydroxypropyl ester are fatty acids that are confirmed by the peak at 1109 cm-1 that corresponds to the C-O stretch of esters. Octadecane, 3-ethyl-5-(2-ethylbutyl) is an alkane that corresponds to the peak at 2970.67cm-1.
Abubacker and Palaniyappan (2015) reported that Octadecane, 3-ethyl-5- (2-ethylbutyl)
possess invitro antifungal activity. The presence of Diisooctyl phthalate was affirmed
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by the FTIR band at 657 cm-1 which has C-H bend of alkynes, that indicates two
identical functional groups.

Duke ethnobotanical database (1992-1996) determined that hexadecanoic acid
2-hydroxy-1 (hydroxymethyl) ethyl ester possess antioxidant, anti inflammatory and
antihelminthic activity; sitosterol possess antimicrobial, anticancer, antiarthritic,
antiasthma, diuretic, anti inflammatory activity; and octadecanoic acid 2,3
dihydroxypropyl ester possess antimicrobial and anti inflammatory activity. Diisooctyl
phthalate might possess antimicrobial and antifouling properties (Sangeetha and
Vijayalakshmi, 2011)

1-nonadecene, and diisooctyl phthalate was proved to possess anticancer,
antioxidant and antimicrobial activity (Pravat et al., 2007). N-hexadecanoic acid which
is widely used in medicated oils for rheumatic symptoms was detected as an anti
inflamamatory agent (Aparna et al., 2012). Jones et al., 2002 confirmed that
N-hexadecanoic acid possess antioxidant and anti inflammatory activity;
9,12-octadecadienoic acid possess anti inflammatory and anti arthritic activity and
phthalic acid, an aromatic dicarboxylic acid to be a potent antihemorrhagic. The FTIR
and GCMS study thus confirmed that the phenol group is abundant in the seaweed chloroform extract. The FTIR of TiO2 nanoparticle and the seaweed extract thus confirms the presence of phytochemicals coated on the surface of the nanoparticle. Simultaneously the TiO2 nanoparticles biosynthesised using Psidium guajava extract was also found to possess antioxidant activity (Thirunavukkarasu et al., 2014). Further
the compounds identified by GCMS were known to possess strong antioxidant and anti
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inflammatory activity. The present finding thus assisted the research towards the
evaluation of the antioxidant activity of both the nanoparticle and the seaweed extract.

In biological systems, free radicals and reactive oxygen species are constantly
generated which interacts with other molecules present in a cell, which results in
oxidative stress and damage to tissues and biomolecules leading to various diseases,
especially degenerative diseases and extensive lysis. Reactive oxygen and nitrogen
species are two main sources of oxidative substances, including hydrogen peroxide,
superoxide, nitric oxide and peroxynitrite (ONOO?). These substances can damage
proteins, lipids and DNA. (Thanigaivel et al., 2016). Marine algal extracts have been
demonstrated to possess strong antioxidant properties (Yuan and Walsh, 2006). Marine
algae are rich in bioactive compounds which possess wide range of biological activity
that includes, antioxidant, antifungal, antibacterial, anticancer, anti inflammatory and
anti aging properties. (Kim et al., 2014; Fernando et al., 2016, 2017; Wang et al., 2017). Both the seaweed extract and the TiO2 nanoparticles were assessed by five spectrophotometric methods for their ability to scavenge the free radicals and reactive
oxygen species. Due to their oxidative stress, like hydrodynamics, air exposure and
intense UV radiations the seaweed naturally have high antioxidant potential. Similarly TiO2 nanoparticles was know to exhibit, a wide range of UV absorption, which corresponds to the antioxidant property. Both the seaweed extract and the TiO2 nanoparticle showed remarkable antioxidant activity in all the performed assays. Thirunavukkarasu et al., (2014) synthesized TiO2 nanoparticle using Psidium guajava and reported antioxidant property when compared to the leaf extract and the standard
ascorbic acid.

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DPPH radical scavenging was one of the extensive antioxidant screening
method that is used to assess the antioxidant potential of the plant extracts. The
decrease in absorbance indicates that the DPPH radical was scavenged by the hydrogen
donating ability of the antioxidant that is present in the sample. In our study, both the seaweed extract and the TiO2 nanoparticle exhibited effective DPPH scavenging activity which is visible from the yellow colour developed by DPPH upon absorption of
hydrogen from the antioxidant. The seaweed synthesised TiO2 nanoparticle have
higher free radical scavenging activity of 74%, when compared to the chloroform
seaweed extract alone (70%). The DPPH scavenging activity was found to increase in
dose- dependent manner in both the seaweed extract and the seaweed synthesised TiO2
nanoparticles. Novaczek et al., 2001, suggested that the radical scavenging activity of
Caulerpa racemosa could be due to the presence of folic acid, thiamine and ascorbic
acid. DPPH radical scavenging activity of crude ethanolic extract and ethyl acetate
fraction Caulerpa racemosa was reported to be less than 15% at 230?g/ml (Li et al.,
2012). Thus the chloroform extract of Caulerpa racemosa exhibits maximum DPPH
scavenging activity of 52% at 100?g/ml.

The reducing power of a compound may serve as a significant indicator for a
potential antioxidant. In the ferric reducing antioxidant power assay, the antioxidants in the seaweed synthesised TiO2 and the chloroform seaweed extract, reduced Fe 3+ to blue coloured Fe2+ that is measured spectrophotometrically. The reducing power of the seaweed synthesised TiO2 nanoparticle and the chloroform seaweed extract was similar to that of the standard ascorbic acid. This indicates that the antioxidant compounds
present in the seaweed extract, which is coated on the nanoparticles are potential
electron donors. The reducing power of both the chloroform seaweed extract and the
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seaweed synthesised TiO2 nanoparticles was similar and dose dependent. Phenolic
compounds are of potential source for antitumour, anti inflammatory, and antibacterial
properties (Novoa et al., 2011; Thomas and Kim, 2011; Wijesinghe and Jeon, 2012).

Nitric oxide generated from sodium nitroprusside (SNP) interacts with oxygen
to produce the nitrite ion (NO. ) The absorbance of the chromophore (purple azo dye)
formed during the diazotisation of nitrite ions with sulphanilamide and subsequent
coupling with naphthylethylene diaminedihydrochloride was measured at 546 nm. The
nitrite generated in the presence of the seaweed extract is directly proportional to the
nitric oxide generated by the SNP. The nitric oxide scavening activity of chloroform
seaweed extract and the seaweed synthesised TiO2 nanoparticles was dose dependent.
Both the chloroform seaweed extract and the seaweed synthesised TiO2 nanoparticles
significantly inhibited the accumulation of nitrite, which is a stable oxidation product of
nitric oxide that is liberated from SNP in the reaction medium. The toxicity of NO.
increases when it reacts with superoxide to form the peroxynitrite anion (.ONOO- ),
which is a potential strong oxidant that can decompose to produce .OH and NO2
(Pacher et al., 2007). The present study thus demonstrates that the TiO2 has a potent
nitric oxide scavenging activity than the seaweed extract alone.

Hydroxyl radicals are the major active oxygen species causing lipid
peroxidation that leads to enormous biological damage. These hydroxyl radicals were produced in this study by incubating ferric-EDTA with ascorbic acid and H2O2, and reacted with 2-deoxy-2-ribose to generate a malondialdehyde (MDA)-like product. The
generation of OH is detected by its ability to degrade deoxyribose to form products
which on heating with TBA forms a pink coloured chromogen. When the chloroform
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extract of the seaweed and TiO2 nanoparticle was added to the reaction mixture, they removed the hydroxyl radicals from the sugar and thus prevented the reaction. The hydroxyl scavenging activity of the seaweed synthesised TiO2 nanoparticle (36% at 500?g/ml) was remarkably higher than the chloroform seaweed extract (28% at
500?g/ml) and it was also found to be concentration dependent. Thus the study reveals that the seaweed synthesised TiO2 nanoparticles has a remarkable antioxidant potential. Li et al., 2012 reported that the hydroxyl scavenging activity of crude ethanolic extract
and ethyl acetate fraction Caulerpa racemosa was less than 15% at a concentration of 229?g/ml. The augmented antioxidant activity of the TiO2 nanoparticle might be due to the phytochemicals that are embedded on the surface of nanoparticle.

The reducing power assay is often used to evaluate the ability of an antioxidant
to donate an electron (Yildirim et al., 2000). In this assay, the ability of the seaweed
extract and synthesised TiO2 nanoparticles to reduce Fe3+ to Fe2+ was determined.
The presence of antioxidants in the seaweed extract and TiO2 resulted into reduction of
the ferric cyanide complex (Fe3+) to the ferrous cyanide form (Fe2+). In reducing
power assay, the reduction of the Fe3+ into Fe2+, was visible thereby changing the
solution into various shades from green to blue, depending on the reducing power of
the compounds. Strong reducing agents, however, formed Perl’s Prussian blue colour
and absorbed at 700?nm. Chloroform extract and TiO2 nanoparticles showed some
degree of electron donation. Reducing power increased with the increasing
concentration of the extract and the nanoparticles. The reducing power to reduce Fe3+
to Fe2+ of methanolic seaweed extracts were reported to be in a
concentration-dependent manner as reported by Vinayak et al., 2011. Interestingly, the
rate of reducing power of chloroform seaweed extract increased constantly with
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concentration wherease for TiO2 nanoparticles the increase in reducing power was
quite less. The reducing power of the standard, Ascorbic acid was found to be higher
than the other both. Shimada et al, 2009 reported that the reducing power of a
compound is related to its hydrogen-donating ability.

Our results revealed that the antioxidant activity was directly proportional to
the concentration of both the seaweed extract and TiO2 nanoparticles, which exhibits a
dose-dependent factor. Ganesan et al. (2008) investigated the reducing power as a
function of antioxidant activity and showed that increasing concentration increased the
reducing power. Kumaran and Karunakaran (2007) and Kuda et al. (2005) also reported
concentration dependent antioxidant activity in seaweeds. Duh (1998) suggested that
the antioxidant property might be associated to the presence of reductones (enediols)
which terminates the free radical chain reactions. The antioxidative activities detected
by various antioxidant assays reveal that both the seaweed extract and the TiO2
nanoparticles are strong antioxidants. This can be related to the presence of various
polyphenolic compounds and other organic acids and their hydrogen donating ability.
Phenolic compounds and flavonoids play an important role in exhibiting antioxidant
property which are associated to their electron donating ability (Sharma et al., 2012).
Many studies suggested that the bioactive compounds that are rich in polyphenols
possess significant antioxidant properties (Demla and Verma, 2012; Adithya et al.,
2012). Further these bioactive compounds are effective free radical scavengers that is
mainly due to their redox properties. Redox property play an important role in
adsorbing the free radicals and neutralizing them, thereby quenching decomposing
peroxides or singlet and triplet oxygen (Hasan et al., 2008). The chloroform seaweed
extract and the TiO2 nanoparticles might be an electron donors and could react with
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free radicals to convert them into more stable products and then terminate the free
radical chain reactions.

Previous reports suggests that the antioxidant activity of seaweed extract was
related to their polyphenol content (Monsuang et., 2009), which might be the cause of
the recorded inhibitory effects of the present study. Therefore, the present study
demonstrates that the chloroform seaweed extract have higher antioxidant potential
than any other solvent extract of Caulerpa racemosa reported elsewhere (Li et al., 2012; Chew et al., 2008; Mahendran and Saravanan, 2013). The seaweed synthesised TiO2 nanoparticles possess strong antioxidant property. This reducing power is due to the
presence of phenols and terpenoids of the seaweed that is coated on the surface of the TiO2 nanoparticle.

According to the World Health Organization (WHO), colorectal (colon),
mammary, lung, liver, stomach and esophageal are the most prevalent cancer types
around the globe (WHO, 2016). Antioxidants protect cells from damage caused by free
radicals, or unstable molecules and this is termed as oxidative stress, that is linked to
the damage in DNA which can contribute to the risk of certain cancers, as well as
diabetes, Alzheimer’s disease, and Parkinson’s disease.

Biofabrication of nanoparticles using seaweeds have various potential
application amongst cytotoxicity towards cancer cell line forms the base for
pharmaceutical application and environmental toxicology. Recently TiO2 nanoparticles
are reported to possess anticancer activity for several types of cancers viz MCF 7 cell
lines (Babji et al., 2016), A549 cell lines (Tedja et al., 2011), HeLa cell lines (Zhang et
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al., 2014). Marine algal composition includes: phycobiliproteins, polyphenols,
carotenoids, pigments, terpenes, phlorotannins, and polysaccharides (Lange et al., 2015;
Souza et al., 2012). Further they also contain minerals viz magnesium, iron, iodine,
zinc and calcium in addition to lipids, vitamins and fiber (Lange et al., 2015). Of these
bioactive compounds polysaccharides, terpenes and polyphenols reported to possess
anticancer activity Senthilkumar et al., 2013; Atashrazm et al., 2015; Peng et al., 2011).

In this direction, the present study is focused on the cytotoxic effect of biosynthesized TiO2 nanoparticles and chloroform seaweed extract. Further so as to determine the antiproliferative effect of TiO2 on cell proliferation of colon cancer cell lines (HCT-15), MTT assay was performed. The results of MTT assay revealed that both the TiO2 nanoparticles and the chloroform seaweed extract exhibited cytotoxicity on a dose dependent manner. The IC50 values of the chloroform seaweed extract and the TiO2 nanoparticles were calculated to be 465 µg/ml and 440 µg/ml respectively. Lotfian and Nemati, (2016) reported the use of invitro assay viz MTT assay on MCF-7 to evaluate the potential cytotoxicity of TiO2 nanoparticles. The results reveal that TiO2 nanoparticles significantly reduced the proliferation of MCF-7 cell is a dose dependent
manner. Garcias-Contreras et al., 2013 suggested that the effect of cytotoxicity may be
due to incorporation of TiO2 nanoparticles into mitochondria to reduce ATP synthesis
and nitrogenous base formation, and reaches the cell nucleus to create links by
interaction with DNA base pairs or binding to the groove, which finally induces
apoptotic cell death (Lopez et al., 2013).

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TiO 2 nanoparticles doped with Au and Pt were reported to kill human erythroleukemia tumor cell line K562 effectively (Lazau et al., 2007). Terpenes are
secondary metabolites and usually contain chlorine, bromine and iodine. These
compounds are secreated outside the cell by seaweeds as a defense against
environmental issues was known to possess anticancer activity. Halogenated
monoterpenes, present in the red seaweeds Plocamium cornutum and Plocamium suhrii,
showed antiproliferative activity when compared with cisplatin (anticancer drug)
(Antunes et al., 2011). Farideh et al., 2013, reported that the polypheno lrich marine
brown alga, Sargassum muticum, possess strong anticancer activity against ATCC
CCl-81 cell line. All these literature reports and the present study suggested that the
bioactive substances present in the seaweed and the TiO2 nanoparticles interact with
cancer associated receptors or special molecule, thereby triggering cancer cell death.

AO/EB double staining is an exquisite method to analyze the cell death upon
treatment with bioactive compound and nanoparticles. Being reported the seaweed
extract and the TiO2 nanoparticle inhibited HCT-15 cell proliferation in a dose
dependent manner, it was essential to determine whether the inhibition of cell
proliferation was due to apoptosis and not necrosis. When the cells are stained with
AO/EB, AO is a cationic dye, taken up by both viable and non viable cells and emits
green fluorescence if intercalated into double stranded DNA or red fluorescence if
bound to single stranded DNA. EB is taken up only by non viable cells and emits red
fluorescence by intercalating into DNA. Both the seaweed extract and TiO2
nanoparticles treated cells exhibits orange fluorescence which indicates the loss of
membrane integrity, membrane bulging, cellular shrinkage and nuclear fragmentation
which might be due to apoptosis. The findings revealed that the chloroform seaweed
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extract exhibited minimum apoptotic inducing effect when compared to TiO2
nanoparticles.
The present study was in correlation with the work done by Khoushika and
Brindha, 2018 who also reported that AO/EB dual staining showed minimum apoptotic
activity for hydroethanolic extract of the seaweed Turbinaria conoides when compared
to the ZnO-NPs. In the present study, the synthesized TiO2 nanoparticles was found to
exhibit cytotoxicity and apoptotic activity against HCT-15 cells, which suggests that
the TiO2 nanoparticles possess anticancer activity. TiO2 nanostructures are considered
as an ideal tumor targeted drug delivery system (Moosavi et al, 2015). Further
Qingning et al, 2009 also reported that titanium dioxide whiskers based delivery of
anticancer drugs holds a promise in drug delivery. Thus, TiO2 synthesised using
seaweed extract was found to be a promising anticancer drug with efficient drug
delivery system holds a promising future in cancer therapy.

Inspite of their imperative applications, adverse effects of nanoparticles are
likely to occur in very different scenarios. We are already exposed to large numbers of
ambient nanoparticles in environmental air pollution where the nanoparticles
component has been the focus of much research as one of the likely drivers of adverse
health effects. In addition, nano particles are being used in cosmetics, food additives
and even directly injected into the blood stream for diagnostic purposes. The enhanced
possibility of nanoparticle exposure and their toxic effects on consumers and
environment is also a thoughtful issue to be highlighted. The use of Zebra fish, globally
accepted animal model for toxicological studies invivo. It can be used during both adult
as well as embryonic stages. The reason behind zebra fishes being a popular animal
model is due to some exceptional set of characteristics such as small size, very high
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reproducibility, quick development, transparency of the embryo and acquiescent to
genetic as well as chemical screens. In addition to this Zebra fish allows transforming
the approach for toxicology testing through adoption of alternative systems which
provides major opportunities in reducing the animal use in the chemical safety
assessment and also they exhibit 75% similarity with the human genome that makes it a
feasible animal model. Hence, we have studied the nanotoxicity of biologically synthesized TiO2 nanoparticles using adult Zebra fish as invivo model.

The toxicity was tested in the Zebra fish upon chronic exposure to biologically synthesized TiO2 nanoparticles using Caulerpa racemosa. Although TiO2 nanoparticles were reported to be no/low toxic, does not cause acute lethality in Zebra fish, toxicity of biologically synthesized TiO2 nanoparticles using Caulerpa racemosa has not been studied earlier. To this point, we exposed the biologically synthesized TiO2 nanoparticles using Caulerpa racemosa and the titanium isopropoxide solution to Zebra fish for 96 hr, to study the effect of TiO2 nanoparticles toxicity in a time dependent manner. Specifically, zebrafish (5 groups) were exposed to 10 mg/L of TiO2 nanoparticles for 24 hr to 96 hr. Assuming the possible fluctuation of TiO2 nanoparticle over time in both concentrations and particle sizes, we transferred zebrafish every 24 h to a tank containing a new aliquot of TiO2 nanoparticles at appropriate concentrations (i.e., 10 mg/L) to ensure a relatively consistent TiO2 nanoparticles exposure throughout the study. Daily monitoring of the mortality and general health of the Zebrafish
revealed no apparent abnormalities, in concordance with Griffitt et al., (2009), that
TiO2 nanoparticles did not produce lethality during 48 hr exposures in Zebrafish at
concentrations up to 100 mg/L. The dynamic nature of the nanoparticles exposed in the
aquatic medium creates challenge for accurately measuring the exposure concentration
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in toxicity testing. The use of nominal or single dose of nanoparticles is insufficient to
accurately describe the exposure concentration (Griffitt et al., 2010). Wang et al., 2011,
reported 30% significant reduction in the cumulative number of Zebrafish egg was observed in the TiO2 nanoparticles treated group even at concentrations as low as 0.1 mgL-1 for 8, 10, 11, 12 and 13 weeks. While, embryos spawned from the females exposed to TiO2 nanoparticles for 8 weeks or longer showed an increase in mortality. On the contrary, our results showed no acute toxicity of the biologically synthesized TiO2 nanoparticles using Caulerpa racemosa.

The histopathology of the organs such as gill, lung and brain revealed that
there were no apparent morphological changes compared to the treatment control. The
exposure of the nanoparticles to gill is characterized by the cellular in the interlamellar
space. The gill filament following the TiO2 nanoparticle treatment did not show
significant changes. Earlier it was reported that nanotitania or nanosilver did not have
significant effect on gill filament at 48 hr exposure compared to other metal
nanoparticles (Griffitt et al., 2009).

The present study was carried out to synthesis and characterise TiO2
nanoparticles using the seaweed, Caulerpa racemosa. Further the phytochemical
profiling of the seaweed Caulerpa racemosa revealed the presence terpenoids, saponins,
glycosides, cardiac glycosides and phenol. The GC-MS analysis of the seaweed showed
20 bioactive compounds which possess various biological activity. The bioactive
compounds of the seaweed was found on the surface of the synthesised TiO2
nanoparticles. The chloroform seaweed extract and the synthesised TiO2
nanoparticles demonstrated strong antioxidant activity and anticancer activity. In
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addition, the toxicity of the synthesised TiO2 nanoparticles was assessed invivo using
zebrafish model. The histopathological section of the gill, brain and lung of the
zebrafish exposed to TiO2 nanoparticles revealed no significant morphological
changes.