PROJECT REPORT

On

PREPARATION AND CHARACTERIZATION OF NANOENCAPSULATED

FUCOXANTHIN: RELEASE KINETICS AND BIOAVAILABILITY

Submitted in partial fulfillment of the requirements

For the award of the degree
Of
MASTER OF TECHNOLOGY
In
FOOD AND NUTRITIONAL BIOTECHNOLOGY
By
R.M.Dhevya
Reg No: 1721210025

Under the guidance of

Mrs.Sasikala.S
Asst. Professor
Department of Food Process Engineering

FACULTY OF ENGINEERING AND TECHNOLOGY

DEPARTMENT OF FOOD PROCESS ENGINEERING
SCHOOL OF BIOENGINEERING
S.R.M UNIVERSITY
KATTANKULATHUR-603203

MAY 2014
 PROJECT REPORT

On

PREPARATION AND CHARACTERIZATION OF NANOENCAPSULATED

FUCOXANTHIN: RELEASE KINETICS AND BIOAVAILABILITY

Submitted in partial fulfillment of the requirements

For the award of the degree
Of
MASTER OF TECHNOLOGY
In
FOOD AND NUTRITIONAL BIOTECHNOLOGY
By
R.M.Dhevya
Reg No: 1721210025

Under the guidance of

Mrs.Sasikala.S
Asst. Professor
Department of Food Process Engineering

DEPARTMENT OF FOOD PROCESS ENGINEERING

SCHOOL OF BIOENGINEERING
FACULTY OF ENGINEERING AND TECHNOLOGY
S.R.M UNIVERSITY
KATTANKULATHUR-603203

MAY 2014
 ACKNOWLEDGEMENT

I am extremely thankful to Dr. M.Vairamani, Dean, School of Bioengineering, SRM

University for all the help needed to carry out my project successfully.

I am thankful to Mrs. Sasikala.S my project guide, who has guided and encouraged me
throughout my project work.

I express my sincere gratitude towards Mrs. K.A.Athmaselvi, Head of the Department,

Food Process Engineering, for her continuous guidance and support for the completion of the
dissertation.

I wish to express my deepest sense of respect and gratitude towards my external guide
Dr. V.Baskaran Senior Principal Scientist and Head, Department Of Molecular Nutrition, CSIR-
CFTRI, Mysore, for bringing out the best in me by guiding me thought out the project.

My acknowledgement will be incomplete without mentioning the cooperation of Mr.

Ravi Hindupur, Research Scholar, CSIR-CFTRI, who created a helpful and supportive
environment with his inspiration, advice and friendly support throughout the course of
investigation to make this project a valuable experience.

Above all I am deeply indebted to Almighty God for showering his abundant blessing on
me for successful completion of the project.

Last but not the least, I express my heartfelt gratitude to my family who have been the
source of my strength, achievements and inspiration at all stages of my life.
 DECLARATION

I do hereby declare that the project work entitled “PREPARATION AND

CHARACTERIZATION OF NANOENCAPSULATED FUCOXANTHIN: RELEASE
KINETICS AND BIOAVAILABILITY” for submission as dissertation towards partial
fulfillment of the requirement for the degree of Master of Technology in Food and Nutritional
Biotechnology at SRM University, is a record of bonafide work carried out by me, under the
supervision and guidance of Mrs.Sasikala.S, Assistant Professor, Department of food Process
Engineering, SRM University, Kattankulathur-603203. This project has not been submitted earlier
in part or full for the award of any degree, diploma, associate ship or fellowship .

Kattankulathur: R.M.Dhevya
Date: Reg No: 1721210025
 CERTIFICATE

This is to certify that the thesis entitled “PREPARATION AND CHARACTERIZATION OF

NANOENCAPSULATED FUCOXANTHIN: RELEASE KINETICS AND
BIOAVAILABILITY” submitted for the degree of MASTER OF TECHNOLOGY in FOOD AND
NUTRITIONAL BIOTECHNOLOGY is the bonafide work of MS. R M DHEVYA
(Reg.No.172120025), Faculty of Engineering & Technology, SRM University, Kattankulathur
Chennai, who carried out the thesis under our supervision. Certified further, that to the best of our
knowledge the work reported here is does not form part of any thesis or dissertation on the basis of
which a degree conferred on an earlier occasion on this or any other candidates.

PROJECT GUIDE HEAD OF THE DEPARTMENT

Mrs. Sasikala.S Mrs. K.A.Athmaselvi

Viva Voice Examination held on:

INTERNAL EXAMINER EXTERNAL EXAMINER

 TABLE OF CONTENTS

CHAPTER TITLE PAGE

NO. NO.
List of figures viii

List of flow charts ix

List of tables X

List of abbreviations xi

Abstract xii

1 Introduction 1

1.1 Carotenoids 2
1.2 Health Benefits of Carotenoids 2
1.3 Seaweed 3
1.4 Fucoxanthin 3
1.5 Biological Availability of Carotenoids 4
1.6 Nanoencapsulation 5
1.7 Benefits of Encapsulation 6
1.8 Scope of the Project 6

2 Review of literature 7

2.1 Carotenoids 9
2.2 Physical Properties of Carotenoids 10
2.3 Biochemistry of Carotenoids 11
2.4 Classification of Carotenoids 12
2.5 Fucoxanthin 12
2.6 Nanotechnology 16
2.7 Encapsulation Systems and Methods 19

3 Materials and methods 24

3.1 Materials 24
3.2 Methods 25

4 Results and Discussion 34

4.1 Extraction of Fucoxanthin 35

4.2 Fucoxanthin Concentration in Wakame 35
4.3 Fucoxanthin Separation by Thin Layer Chromatography 36
4.4 Quantification of Fucoxanthin by HPLC Method 37
4.5 Lipid Based Nanoencapsules Loaded with Fucoxanthin 38
4.6 Characterization Techniques 39
 4.7 Encapsulation Efficiency 42
4.8 In Vitro Release Kinetics 43
4.9 In Vitro Digestion 43

5 Summary and Conclusion 45

 LIST OF FIGURES

CHAPTER TITLE PAGE

NO. NO.
1 1 Structure of fucoxanthin 4

1.2 Scheme of dietary carotenoid absorption 5

2 2.1 Structure of Fucoxanthinol 14

2.2 Structure of Amaracioxanthin A 12

2.3 Schematic diagram of spray drier 21

3.1 Purification of fucoxanthin by column chromatography 26

3
3.2 Schematic representation of Nanoencapsules 28

4 4.1-Column chromatography for separation of FUCO 35

4.2 Absorption Spectrum of fucoxanthin 36

4.3 Separation of FUCO by Thin layer chromatography 37

4.4 HPLC peaks 38

(a) Standard FUCO
(b) Purified FUCO
4.5 Particle size for nanoencapsulated FUCO 39

4.6 SEM images of nanoencapsulated fucoxanthin 39

4.7 FT-IR spectra

(a) Chitosan 40
(b) Tripolypohosphate 41
(c) With lipid 41
(d) Without lipid

4.8 Encapsulation efficiency 42

4.9 Release kinetics of FUCO 43

4.10 In Vitro digestion 44

viii
 LIST OF FLOW-CHARTS
FLOW-CHART TITLE PAGE
NO. NO.

2.1 Types of Nanoparticles used as delivery system 18

3.1 Extraction and purification of fucoxanthin 26

3.2 Procedure for the preparation of nano-encapsulated fucoxanthin 29

3.3 Procedure for chart for the extraction of FUCO from the nano- 31
encapsules

3.4 Procedure for In Vitro Digestion 33

ix
 LIST OF TABLES

Table TITLE Page

No. No.

2.1 Health benefits of fucoxanthin 13

2.2 Related literatures on nanoencapsulation 18

3.1 List of chemicals used 24

3.2 List of Equipments used 24

x
 LIST OF SYMBOLS AND ABBREVATIONS
SYMBOLS ABBREVATIONS

DHA
Docosahexaenoic Acid
E.E
Encapsulation Efficiency
FT-IR
Fourier Transform Infra Red
FUCO
Fucoxanthin
gm
Gram
HPLC
High Performance Liquid Chromatography
LDL
Low Density Lipoprotein
mg
Milligram
ml
Milliliters
Nm
Nanometer
O.D
Optical Density
SEM
Scanning Electron Microscopy
TPP
Tripolyphosphate
V
Volume
W
Weight
µg
Microgram

xi
 ABSTRACT
Seaweeds belong to a group of plants known as algae, they are considered as a source of
bioactive compounds as they are able to produce a great variety of secondary metabolites
characterized by a broad spectrum of biological activities. Seaweeds are of nutritional interest as
they contain low calorie food but rich in vitamins, minerals and dietary fibers. Seaweed is a rich
source of carotenoids. Carotenoids are yellow to red isoprenoid polyene pigments; approximately
650 carotenoids are known from bacteria, fungi, plants and animals. Carotenoids, in general,
classified as carotenes or provitamin A carotenoids and xanthophylls or non-provitamin A
carotenoids. Among the xanthophyll carotenoid, fucoxanthin (FUCO), a marine carotenoid, in
specific, affords various physiological effects such as anti-obesity and diabetes, combat cancer,
anti-oxidation. FUCO is mainly found in brown algae (brown seaweeds) such as kelp, hijiki, and
wakame. The present study focused on the development of lipid based hybrid nanoencapsules
with an increased bioavailability, for this initially the FUCO was extracted from the brown algae
by Open Column Chromatography. The extracted FUCO was checked for purity by UV
Spectrophotometer and HPLC, and then the purified FUCO was used for the development of
nanoencapsules by ionic- gelation method. The developed nanoencapsulse were characterized by
using particle size analyzer, scanning electron microscopy and Fourier transform-infrared
spectrometer. Further, the encapsulation efficiency, release kinetics and in vitro digestion of
nanoencapsulated FUCO products were analyzed. From the structural analysis, it was found
that, the size of the nano-encapsulated product was and its shape was spherical. The
encapsulation efficiency of lipid based nano-encapsules is found to be higher than that of the
non-lipid based nano-encapsules. The encapsulated products were analyzed for its release
kinetics by in vitro release kinetics and the cumulative release profile of FUCO from CS-NGs
was 71%,Thus it can be concluded that, the burst effect was gradually reduced as the time
progressed from 0 till 24 h. Then the biological availability is analyzed by in vitro digestion
method, which proves that of FUCO in the CS-NGs with GL (57.6 %) was significantly higher
than the FUCO+GL (30.6%) and the control (17.6%). From this work, thus it can be concluded
that lipid based nano-encapsulation of FUCO not only helps in increasing the stability but also
render higher biological availability.
Keywords: FUCO, Nanoencapsulation, Release kinetics, Bioavailability.
INTRODUCTION

1
 CHAPTER 1
INTRODUCTION
1.1 Carotenoids:

Carotenoids are yellow to red isoprenoid polyene pigments, approximately 650

carotenoids are known from bacteria, fungi, plants and animals. They occur not only as free
forms but also as esters, glycosides, sulfates and carotenoproteins. Carotenoids consisting of
eight isoprenoid units in a molecule are called carotenes. The oxidized derivatives are called
xanthophylls. Carotenes and xanthophylls contain 40 carbon atoms in a molecule, but there
are some carotenoids that contain a fewer number of carbon atoms, which are called apo-
carotenoids. One of the main roles of carotenoids in organisms seems to relate to light
absorption functions such as photosynthesis, photoprotection, phototropism, photoreception
and camouflage effects for concealment from enemies. Carotenoids, in general, classified as
carotenes or provitamin A carotenoids and xanthophylls or non-provitamin A carotenoids.

1.2 Health Benefits of Carotenoids:

Carotenoids afford several health benefits. Provitamin A is a precursor of retinol (vitamin

A) which is an essential micronutrient. Most of the carotenoids are potent antioxidants.
Carotenoids are important for immune responses, gap junction communication and
carcinogen-metabolizing enzyme activity (Stahl et al., 1997; Wang, 1994). Carotenoids play
a protective role in cardiovascular diseases (Gaziano et al., 1995) and cancer (Van Poppel,
1996) particularly in cancer of lung (Dartigues et al., 1990; Knekt et al., 1990) and stomach
(Chen et al., 1992). Because of their antioxidant property, carotenoids have been suggested to
protect against coronary vascular disease.

1.2.3 Dietary Sources of Carotenoids:

The orange-colored fruits and vegetables including carrots, apricots, mangoes, squash,
and sweet potatoes contain significant amounts of beta-carotene, alpha-carotene, and beta-
cryptoxanthin. Green vegetables, especially spinach, kale, and collard greens, also contain
beta-carotene, and are the best sources of lutein. Lycopene is found in tomatoes, guava, and

2
pink grapefruit. Salmon, shellfish, milk, and egg yolks also provide carotenoids. The
contribution of spices to available carotenoids in the U.S. diet has increased steadily, making
spices a great choice for upping your carotenoid intake. Cayenne pepper and chili pepper are
worthy of special mention here. Exploring brown seaweeds as a source of fucoxanthin in
advisable.

1.3 Seaweed:

Seaweeds are habitually consumed in Asia, and are rich in vitamins and carotenoids.
However, although seaweeds are rich in several bioactive components, the bioavailability,
nutritional, and antioxidant property of those components have scarcely been investigated
and, their effects on reversing the nutritional deficiency induced biochemical and
physiological changes have not yet been studied well. Various types of brown algae, such as
Hijiki (Sargassum fusiforme), Kombu (Laminaria japonica), and Wakame (Undaria
pinnatifida), are staples in the diet of East Asians (Akira asai et al., 2004). Studies with
rodent models demonstrate the protective effect of dietary seaweeds against mammary,
intestinal and skin cancer. Moreover, cell culture studies have begun to elucidate the
mechanism underlying the potential anti-carcinogenic effects of seaweed extracts against
breast and colon cancer (Sangeetha et al., 2009). Scientific studies showed positive results in
belly fat reduction. The seaweed extract causes the liver to produce an omega-3 fat called
DHA. This healthy fat reduces the body’s levels of low-density lipoprotein or LDL “bad”
cholesterol. High LDL levels contribute to obesity and heart disease. The same healthy fat
DHA has been found to reduce insulin and blood sugar levels in animal studies.

1.4 Fucoxanthin:

Fucoxanthin (FUCO) is mainly found in brown algae (brown seaweeds) such as kelp,
hijiki, and wakame while, FUCO is found in higher concentrations in wakame. It is a type of
non-provitamin A carotenoid and belongs to xanthophylls. FUCO has the allene structure and
epoxide and hydroxyl groups as shown in the Fig.1. FUCO has been reported to have
activities to reduce the incidence of obesity and diabetes, combat cancer, prevent oxidation of
the body and is a major non-provitamin A carotenoid in brown algae (Haugan et al., 1992).

3
 Fig. 1 Structure of Fucoxanthin

1.5 Biological Availability of Carotenoids

Bioavailability is defined as “the fraction of an ingested nutrient that is available for

utilization in normal physiological functions for storage”. Dietary matrix or components
interfere with the rate of each of the absorption steps that will affect the overall
bioavailability of the ingested carotenoids (Figure1.2). Carotenoids are released from the
food matrix by heat, mechanical and enzymatic treatments during food processing, and in the
mouth, by mastication and the action of enzymes in the saliva. The released carotenoids
incorporate into the lipid phase, which is emulsified into small lipid droplets in the stomach.
From the lipid droplets, carotenoids are transferred to mixed micelles formed by the action of
bile salts, biliary phospholipids, dietary lipids and their hydrolysis products. However, the
less lipophilic xanthophylls can also be solubilized directly in mixed micelles. The mixed
micelles migrate to the brush border, where carotenoids are absorbed by the intestinal cells,
packed into chylomicrons and secreted to the lymphatic system. The uptake of carotenoids
from the intestinal lumen takes place by simple diffusion down a concentration gradient
through the brush border membrane into the cytoplasmof the enterocytes. However, some
reports have suggested the existence of carotenoid transport mediated by scavenger receptor
binding protein (SR-BI). The hairpin-like conformation of SR-BI external domain forms a
hydrophobic channel that may facilitate the uptake of carotenoids by the enterocytes, without
energy expenditure. (Yonekura and Nagao., 2007)

4
 Fig. 1.2 Scheme of Dietary Carotenoid Absorption

FUCO is sensitive to photo- and auto-oxidation and their bioavailability is low, so it is

necessary to provide a carrier as well as a coating substance to safeguard the molecule and to
improve its biological availability. Hence, the present study was aimed at investigating the
bioavailability and antioxidant property of micro and nanoencapsulated FUCO.

1.6 Nanoencapsulation:

Nanoencapsulation of drugs involves forming drug-loaded particles with diameters

ranging from 1 to 1000 nm. Nanoparticles are defined as solid, submicron-sized drug carriers
that may or may not be biodegradable (Couvreur, P et al., 1995). For nano-encapsulation,
physical methods include spray drying, centrifugal extrusion, vibrational nozzle; pan coating
and air suspension coating are used. Nanoparticles have been prepared using several methods
such as complex coacervation, solvent displacement method, spray-drying method. One of
the important methods for preparing nanoparticles is ionic gelation method. The ionic
gelation method is very simple and mild.

1.6.1 Ionotropic gelation Method:

Ionotropic gelation method consits of the ionic crosslinking of one component

with multivalent counter ions. The procedure is very simple and its is carried out in aqueous
medium, and does not invlove the use of organic solvents. The nanoparticles can be obtained

5
 by addition of a dilute acid solution, with stirring. In any case, the size of the particles is
strongly dependent on the concentration of both the ionic particles.

The techique was first reported in the year of 1997 by Calvo for the prepation of Citosan,
chitosan-poly(ethylene oxide) and chitosan- poly(ethylene oxide)- poly(propylene oxide)
nanoparticles. The size and surface charge of the particles cab be modified by varying te ratio
of chitoan and stabilizer. The main problematic aspects of the technique are time stability of
the collioidal dispersion which may require the addition of the stabilizers, and the need of
using very dilute solutions wich may be inconvenient when large amounts of nanaoparticles
are required.

1.7 Benefits of Encapsulation:

 Nanoencapsulated nutrients do not cling together, thus increasing their contact with
the absorptive cells of the digestive lining, which increases absorption.
 Spontaneous particle formation.
 Low energy input.
 High entrapment efficiency.
 High reproducibility.
 Superior handling of the active agent.
 Immobility of active agent in food processing systems.
 Improved stability in final product and during processing.
 Improved safety.
 Creation of visible and textural effect.
 Adjustable properties of active components.
 Off-taste masking.
 Controlled release.

1.8 Scope of the Project

The aim of the project is to prepare a chitosan-lipid based hybrid nanoencapsules loaded
with FUCO and to determine its in vitro release kinetic profile and it’s in vitro biological
availability.

6
Objectives:

 To extract and purify the carotenoid FUCO from marine algae (brown seaweeds).
 To prepare nanoencapsulated FUCO by ionic gelation method.
 To characterize the encapsulated products using Particle size analyzer, SEM, FTIR.
 To study the bioavailability, release kinetics and entrapment efficiency of the
encapsulated FUCO.

7
 REVIEW OF
LITERATURE

8
 CHAPTER 2
REVIEW OF LITERATURE
2.1 Carotenoids

Carotenoids are very sensitive to heat, oxidation, and light, due to their unsaturated
chemical structures,they are almost insoluble in water and only slightly oil soluble at room
temperature (about 0.2 g/L oil), but their solubility in oil increases greatly with increasing
temperature (Ax et al., 2001). It has been found that only a minor part of the carotenoids in
raw fruits or vegetables is absorbed in the intestines, probably due to the fact that carotenoids
in the nature exist as crystals or are bound in protein complexes. In contrast, carotenoids
dissolved in vegetable oils show a higher bioavailability (Parker RS., 1997).The various
sources of carotenoids are:

Higher plants

Carotenoids are accumulated in chloroplasts of all green plants as a mixture of α- and β-

carotene, β-cryptoxanthin, lutein, zeaxanthin, violaxanthin and neoxanthin. These pigments
are found as complexes formed by a noncovalent bonding with proteins. In green leaves,
carotenoids are free, nonesterified, and the composition depends on the plant and
developmental conditions. Some leaves of gymnosperms accumulate not very common
carotenoids in oily droplets, which are extraplastidial: rhodoxanthin in some members of the
families Cupressaceae and Taxaceae, and semi β-carotenone in young leaves of cycads. In
the reproductive tissues the following have been found: liliaxanthin in white lily and crocetin
in Crocus sp.stigmas.

Algae

Carotenoids are in the chloroplasts as complex mixtures that are characteristic of each
class; the exceptions Chlorophyta carotenoids, which have a tendency to accumulate the
pigments characteristics of higher plants. The red algae Rhodophyta have α- and β-carotene
and their hydroxylated derivatives. In the Pyrrophyta, the main pigments are peridinin,
dinoxanthin and fucoxanthin. Chrysophyta accumulates epoxy-, allenic-, and acetylenic-
carotenoids, and between them fucoxanthin and diadinoxanthin. Eutreptielanone has been
found in Euglenophyta. The principal carotenoids in Chloromonadophyta are diadinoxanthin,

9
heteroxanthin, and vaucheriaxanthin. Chrytophyta is characterized by their acetylenic
carotenoids, for example, alloxanthin, monadoxanthin and crocoxanthin. While the
Phaeophyta is characterized by its main pigment, fucoxanthin.

Bacteria

Photosynthetic bacteria synthesize approximately 80 different carotenoids. Usually,

the characteristics of the accumulated carotenoids are (1) most of carotenoids are aliphatic,
but in Chlorobiaceae and Chloroflexaceae some carotenoids have aromatic or β-rings; (2)
aldehydes with crossover conjugations and tertiary methoxy groups; (3) various classes of
carotenoids in each species; (4) all carotenoids are bound to the light-harvesting complexes
or reaction centres in membranal systems of bacterial cells.

Fungi

Carotenoid distribution in fungi, non-photosynthetic organisms, are apparently capricious,

but they usually accumulate carotenes, mono- and bi-cyclic carotenoids, and without
carotenoids with ε-rings. For example, plectaniaxanthin in Ascomycetes and canthaxanthin in
Cantharellus cinnabarinus has been found. Due to antioxidant properties of the carotenoids,
many investigations regarding their protective effects against cardiovascular diseases and
certain types of cancers, as well as other degenerative illnesses, have been carried out in the
last years (Stahl et al. 2004). A diet rich in carotenoids may also contribute to
photoprotection against UV radiation (Stahl et al. 2006). In vitro studies have shown that
carotenoids such as β-cryptoxanthin and lycopene stimulate bone formation and
mineralization. The results may be related to prevention of osteoporosis (Kim et al. 2003;
Yamaguchi and Uchiyama 2003; 2004; Yamaguchi et al. 2005).

2.2 Physical Properties of Carotenoids:

Carotenoids are very sensitive to heat, oxidation and light, die to their unsaturated
chemical structures. They are almost insoluble in water and only slightly oil soluble at room
temperature (about 0.2g/L), but their solubility in oil increases greatly with increasing
temperature (Ax et al. 2001).

10
 It has been found that only a minor part of the carotenoids in raw fruits or vegetables
is absorbed in the intestines, probably due to the fact that carotenoids in the nature exist as
crystals or are bound to protein complexes. In contrast, carotenoids dissolved in vegetable
oils show a higher bioavailability (Parker, 1997). Incorporation of carotenoids into micro and
nano structures may influence their solubility and crystallinity. After formulating carotenoids
into such particulate systems, they may be easily delivered into cellular compartments,
improving their bioavailability.

2.3 Biochemistry of Carotenoids:

Carotenoids, in general, are tetraterpenoids (8 molecules of isoprene units joined by

tail-to-tail linkage) whose order is inverted at the molecular centre. There are two metabolic
pathways of synthesizing terpenoids.

(i) Mevalonic acid pathway

Many organisms manufacture terpenoids through HMG-CoA reductase pathway, the
pathway that also produces cholesterol. The reaction takes place in cytosol. The pathway
was discovered in 1950s.

(ii) MEP/DOXP pathway

The 2-C-methyl-D-erythritol-4-phosphate/ 1-deoxy-D-xylulose-5-phosphate pathway
also known as non-mevalonate pathway or mevalonic-acid independent pathway, takes place
in plastids of the plants and apicomplexan protozoa, as well as in bacteria. It was discovered
in late 1980s.

Carotenoids are produced in plastids by the successive action of enzymes on linear

lycopene which initially leads to the formation of α-carotene and β-carotene in the α-branch
and β-branch respectively. Zeaxanthin is produced through the β-branch of the carotenoid
biosynthetic pathway (Cunningham & Gantt, 1998) by hydroxylation of β-C. The final steps
are catalyzed by an epoxidase that forms antheraxanthin and violaxanthin from zeaxanthin
(Ladygin, 2000).

11
2.4 Classification of Carotenoids:
Carotenoids are classified by their chemical structure as carotenes that are constituted
by carbon and hydrogen and oxycarotenoids or xanthophylls that have carbon, hydrogen and
additionally, oxygen. Also, carotenoids have been classified as primary or secondary.
Primary carotenoids group those compounds required by the plant in photosynthesis (β-
carotene, violaxanthin and neoxanthin) whereas secondary carotenoids are localized in the
fruits and flowers.

The most characteristic feature of all the carotenoids is presence of conjugated double
bond system which gives its chemical reactivity, light-absorbing properties. Amongst the
provitamin A carotenoids, β-carotene, α-carotene, γ-carotene and β-cryptoxanthin are
commonly present in fruits and vegetables. The common feature of the all provitamin A
carotenoids is the presence of one β-ionone ring attached to a polyene chain with conjugated
double bonds, which is identical to the structure of retinol.

The number of the double bonds present in the polyene chain and the functional groups
present determine the antioxidant property of the carotenoid. Nonprovitamin A carotenoids
include xanthophylls are more polar than β-carotene due to presence of oxygen containing
functional groups like hydroxy, ketone and epoxides on the β-ionone rings. Xanthophylls
provide protection against oxidative stress in the various chronic and degenerative diseases
like cancer, cardiovascular diseases, neurodegenerative diseases, etc. due to their antioxidant
activity.

2.5 Fucoxanthin:

Carotenoid considered for this work is fucoxanthin (FUCO), a major carotenoid found in
brown seaweed, has a unique structure including an allenic bond and a 5,6-monoepoxide in
the molecule. It is one of the most abundant carotenoids accounting for >10% of estimated
total natural production of carotenoids. In South East Asian countries, some seaweeds
containing FUCO are often used as a source of food. Among them Undaria pinnatifida
(Japanese name is Wakame) and Laminaria (Japanese name is Kombu) are the most popular
edible seaweeds in Japan (Hayato Maeda et al., 2008). In the present study, FUCO has been

12
extracted from Wakame with the classification Alariaceae, Laminariales, Phaeophyceae,
Heterokontophyta.

2.5.1 Wakame (Undaria pinnatifida) - Source of Fucoxanthin:

Carotenoids are not synthesized de novo in humans and they have to be supplemented in
diets. Carotenoids are one among the natural coloring agents. There is no chemical process
known to synthesize FUCO from commercially available raw materials. The better
alternative is to extract FUCO from its natural source and purify it economically. Wakame is
edible seaweed which is well known for its medicinal properties found widely around Japan
and Korea. It is thin and stringy seaweed, deep green in color. It is extremely low in calories
with only about five calories per serving with minimal fat. Although high in sodium, it’s a
good source of other minerals including magnesium, iodine, calcium, and iron. It’s also high
in vitamins A, C, E, and K as well as folate and riboflavin. It’s also a source of lignans,
which are thought to play a role in preventing certain types of cancer, particularly breast
cancer. As they many health benefits, wakame is considered as a best source of FUCO.

Table: 2.1 Health Benefits of Fucoxanthin

S.NO HEALTH LITERATURE

BENEFITS

Anti-obesity (Hayato Maeda et al., 2005) “Fucoxanthin from

1 edible seaweed, Undariapinnatifida, shows
antiobesity effect through UCP1 expression in
white adipose tissues”.

Anti-cancer (Kotake-Nara et al., 2001) ‘Carotenoids affect

2 proliferation of human prostate cancer cells’.

Anti-diabetic (Hayato Maeda et al., 2008) “Seaweed carotenoid,

3 fucoxanthin, as a multi-functional nutrient”
.
4 Antioxidant (Sangeetha Ravi Kumar et al., 2008)
“Fucoxanthin restrains oxidative stress induced by
retinol deficiency through modulation of Na+Ka+-
ATPase and antioxidant enzyme activities in
rats.”

13
 H3C
H3C CH3 O CH3
CH3 CH3 HO
O

O H3C
O CH3
HO CH3 CH3
CH3
Figure 2.1 Structure of Fucoxanthinol (FUOH)
H3C
H3C CH3 OH
CH3 CH3 HO

O H3C
O CH3
HO CH3 CH3
CH3

Figure 2.2 Structure of Amaracioxanthin (AAx)

2.5.2 Pharmacological Functions of Fucoxanthin:

(1) Anti-metabolic Syndrome Activity

There are believed to be nearly 860 million metabolic syndrome patients in six major
countries in the world. The number of obese people is increasing in Japan due to more
westernized and irregular dietary habits. Accumulated visceral fat increases free fatty acids in
the blood and triggers hyperlipidemia and insulin resistance. Various physiologically active
substances, namely adipocytokines, are secreted from visceral fat tissues. Fucoxanthin
activity to lower the serum leptin concentration is believed to be performed by reducing the
white adipose tissues. Thus, fucoxanthin is expected to reduce high blood glucose levels
caused by the accumulation of visceral fat (obesity).

(2) Skin Care and Skin-Whitening Activities

(i) Collagenase inhibitory activity
Collagen makes up 90% of the dermis of the skin. It is distributed throughout the dermis,
making the skin adequately elastic and strong. When collagenase is activated and collagen is
degraded, aging phenomena such as wrinkles and sagging occur. Fucoxanthin was confirmed
to inhibit the activation of collagenase thereby inhibiting the degradation of collagen.
(ii) Activity to promote collagen generation in normal human fibroblasts

14
 Collagen is essential to keep the skin in a beautiful condition. However, collagen
production declines after people reach the age of 20. Fucoxanthin was confirmed to promote
the collagen production in normal human fibroblasts.
(iii) Hyaluronidase inhibitory activity
Hyaluronic acid is widely distributed in the body including the skin, joint fluids, vitreous
bodies and ligaments. It performs various functions in the skin like bonding and protecting
cells, forming skin tissues, retaining moisture in tissues and maintaining elasticity of skin.
When the amount of hyaluronic acid declines, the skin becomes dehydrated and loses
firmness that result in wrinkles. Fucoxanthin inhibits hyaluronidase.
(iv) Elastase inhibitory activity
Elastin is a major protein making up the skin just like collagen and often found in skin.
When elastin is degraded, the skin becomes less elastic resulting in wrinkles. Fucoxanthin
inhibits elastase.
(v) Tryosinase Inhibitory Activity
Dullness, darkish tone and dark spots on the skin are caused by melanin. Dopaquinone is
produced out of tyrosine in the body due to actions of tyrosinase. Pure fucoxanthin purified
from kelp extract was confirmed to inhibit actions of tyrosinase.

2.5.3 Efficacy of Fucoxanthin as Drug:

It has been reported that FUCO causes cell growth inhibition of human neuroblastoma
GOTO cells, human leukemia cells, and prostate cancer cells (Masashi Hosokawa et al.,
2004). The antiproliferative effect of FUCO is stronger than the effects of β-carotene and
lycopene. It has also been reporeted that dietary FUCO suppressed weight gain of white
adipose tissue in an obese mouse model,KK-Ay (Hayato Maeda H et al., 2005). Because
most body fat is stored in white adipose tissue, decreasing white adipose tissue weight by
FUCO might be a very effective approach for preventing and/or alleviating obesity. These
findings indicate that diets containing FUCO might prevent obesity through the suppression
of adipocyte differentiation.

Because of these health benefits, it is appropriate to improve the FUCO bioavailability

through dietary means by providing protection to the molecule by means of
microencapsulation and nanoencapsulation.

15
 The above literature survey indicates that there is no systematic study with regard to
improve the bioavailability and absorption of nanoencapsulated carotenoids.

2.6 Nanotechnology:

Nanotechnology encompasses materials and devices with functional structures

between 1 and about 100 nanometers (10-9 m). Nanotechnology is a field of applied science
and technology covering a broad range of topics about materials in nano meter (10 -9 m)
dimension (Cristina Buzea et al., 2007) where its property is different from the bulk.
Nanoscience and nanotechnology are intrinsically interdisciplinary. Physicists, chemists,
biologists, materials scientists, engineers and other scientists join forces in interdisciplinary
teams studying how nature behaves at the scale of individual atoms and molecules and
working to integrate particles of tens of nanometers diameter into potential products.

The word “nano” refers to a Greek prefix meaning “dwarf” and depicts one billion (10–9)
of a unit. The first mention of some of the distinguishing concepts in Nanotechnology was in
“There’s a plenty of room at the bottom”, a talk given by Physicist Richard Feynman at an
American Physical Society meeting at Caltech on 1959 (Charles P Poole et al., 2003).
Feynman explored the possibility of manipulating material at the scale of individual atoms
and molecules. Norio Taniguchi first coined the word Nanotechnology in 1974.

2.6.1 Applications of Nanotechnology in Food Industry:

In today’s world, food materials are often considered not only a source of nutrients but
also as having to contribute to the health of consumers. Most of the nanoparticles used
traditionally belong to the group of colloids (i.e. emulsions, micelles, mono-and bi-layers)
(Fendler, J.H., 2001). For food applications, nanotechnology can be applied by two different
approaches, either ‘bottom-up’ or ‘top-down’. The top-down approach is achieved basically
by means of a physical processing of the food materials, such as grinding and milling. For
example, dry-milling technology can be used to obtain wheat flour of fine size that has a high
water-binding capacity (Degant, O. and Schwechten, D., 2002). By contrast, self- assembly
and self-organization are concepts derived from biology that have inspired a bottom-up food
nanotechnology. The organization of casein micelles or starch and the folding of globular

16
proteins and protein aggregates are examples of self-assembly structures that create stable
entities. (Dickinson, E. and Van Vliet, T., Eds 2003).

The application of nanotechnology in the food industry offers many potential benefits for
both consumers and manufacturers. The ultrafine dimensions of nanoparticles, and
consequently their very large surface area, enable them to function more effectively than
conventional macro-scale structures in many applications.

Although nanotechnology applications for the food sector are relatively recent, there have
been rapid developments in this area in recent years. The main developments so far been
aimed at altering the texture of food components, encapsulating food components or
additives, developing new tastes and sensations, controlling the release of flavors, and
increasing the bioavailability of nutritional components. Broadly, the currently known and
projected applications of nanotechnology for the food sector fall into the following main
categories:

 Where food ingredients have been processed or formulated to form nanostructures.

 Where nano-sized, nanoencapsulated or engineered nanoparticles additives have
been used in food.
 Where nonmaterials have been incorporated to develop improved, “active”, or
“intelligent” materials for food packaging.
 Where nanotechnology-based devices and materials have been used (Quasim
Chaudhry et al., 2008).

It has been reported that nanoparticles are already naturally present in food, given that many
food and feed ingredients are comprised of endogenous proteins, carbohydrates and fats with
sizes extending from large biopolymers (macromolecules) down to the nanoscale. The
principal areas in the food sector where nanotechnology has potential for use are in
encapsulation and emulsion formation, in food contact materials and sensor development,
and some applications of the technology are close to utilization.

One of the many benefits of nanotechnology is the ability to alter or enhance the release
of molecules from food matrices. Increased bioavailability through delivery of
nutraceuticals,bioactive,and inorganic materials promises to be one of the major applications
17
of nanotechnology in the food industry. (Bouwmeester et al.,2007) reported the types of
nanoparticles used as delivery systems which have been classified in flow chart

2.1 Types of Nanoparticles used as delivery system

Lipid based capsules

Nanoliposomes
ENCAPSULATED NANOPARTICLES
Archaesomes

Nanocochleates
Micelles

Polymer based capsules

Nanospheres

Nanocapsules

Polymersomes

Micelles

Table 2.2 Related literatures on nanoencapsulation

S.No Drug/Substrates Polymer Used References

1 Lysozyme Chitosan- TPP Qian-ying Deng et

al., 2006

2 Capsaicin Acacia – Gelatin Fubao Xing et al.,

2004

3 Ovalbumin Chitosan-sodium Olga Borges et al.,

alginate 2005

4 Lutein HPMC Haugan,JAet.al 1994

18
 5 Beta carotene Casein-Dextrin El-Gorabet.al,
1975

7 Retinol Chitosan Dong-Gon Kim et

al., 2006

8 Vitamin D Sodium caseinate Semo et al., 2007

9 Lutein Chitin Biagini et al.,

2008

2.7 Encapsulation Systems and Methods:

Encapsulation is generally achieved in a two-stage process. In the first stage, the core
material is dispersed within a dense solution of the wall material; the second stage consists of
drying or cooling of emulsion (Madene et al., 2006; Minemoto et al., 1997).

2.7.1. Chemical Processes:

(i) Coacervation:
Often regarded as original method of encapsulation (Madene et al., 2006). It consists of
formation of emulsion, followed by physical or chemical precipitation of the continuous
phase around the dispersed phase, which contains the sensitive material, thus forming
microcapsules (Vieira, 1998). It’s a very promising encapsulation method due to high
encapsulation efficiency and good controlled release possibilities based on mechanical stress
and temperature.
(ii) Co-crystallization:
In this process, supersaturated sugar syrup is held at high temperature, where
crystallization becomes spontaneous. If the core material is dispersed in the syrup, the
small crystals are formed which incorporate the sensitive compounds, either by inclusion
or entrapment or simultaneous crystallization ( Madene et al; 2006). It has great
advantage of higher wall and also a good flowability and dispersion properties ( Madene
et al., 2006).

19
 (iii) Molecular inclusion:
A sensitive molecule fitted into and surrounded by the structural network of another
molecule (Godshall, 1997, Madene et al., 2006). The most documented case of such
process is the use of cyclodextrins for encapsulation of flavors (Madene et al., 2006;).
Cyclodextrins are the result of cleavage of starch, followed by joining of edges of the
resulting polymer. This is the action of cyclodextrin glucosyltransferase enzyme (Madene
et al., 2006). It’s a circular molecule with hydrophobic inner cavity, which is large
enough to partially or fully include polar molecules, such as most of the flavors (Madene
et al., 2006; Pegg and Shahidi et al., 1999).

(iv) Ionotropic gelation method:

Ionotropic gelation method consits of the ionic crosslinking of one component with
multivalent counter ions.The procedure is very simple and its is carried out in aqueous
medium,and does not involve the use of organic solvents.The nanoparticles can be
obtained by addition of a dilute acid solution, with stirring. In any case, the size of the
particles is strongly dependent on the concentration of both the ionic particles.

Mechanical Processes

(i) Spray-drying:
It is the most widespread method for large-scale encapsulation procedures, due to the
relatively low costs, possibility of continuous processing, wide variety of possible matrix
materials, and overall good retention and stability properties of the product (Reneccius,
1989,Madene et al., 2006). The principle is to dry emulsion, solution or suspension, where
sensitive molecule is dispersed in a solution containing the matrix. The emulsion is then
spray dried i.e., atomized into a chamber where hot air is flowing at high velocity and the
small droplets are quickly dehydrated, thus ensuring low particle temperature, despite their
contact with hot air (Gharsallaoui, A et al(2007).
Although there is heat involved in the process which may cause loss of volatiles,
temperatures reached are low, which allows the method to be applied to some volatile core
materials, (Madene et al., 2006). For example, spray drying encapsulation of carotenoid
pigments, in gelatin or starch with sucrose, provides a protective effect against degradation

20
(Robert et al., 2003, cit. in Fuchs et al., 2005). The formation of capsules is based on the
changes in physical and chemical properties of matrix and the core material, input flow rate
of the drier, drying temperature and the nature of the encapsulating support. Since the
emulsion is sprayed through nozzle, the encapsulating material must be highly soluble and
have low viscosity when in solution, which limits the choice of the wall material (Madene et
al., 2006).This drying process is liable to leave some of the dispersed droplets uncoated, thus
not encapsulating some of the core material, which will remain on the capsules surface.
The spray-drying method often results in very fine powders, usually in the range of 10-
100μm in diameter. Consequently, the material’s specific surface is high, which results in
quicker dehydration of the amorphous matrix. To prevent this, the powder particle size can
be increased by agglomeration, using the fluidized bed process (Madene et al., 2006; Roos,
1995). The schematic diagram of the spray-drier is as shown in the figure 2.3.

Fig. 2.3 Schematic diagram of the spray-drier

(ii) Solvent evaporation:

Emulsification/solvent evaporation involves two steps. The first step requires

emulsificationof the polymer solution into an aqueous phase .During the second step polymer
solvent is evaporated, inducing polymer precipitation as nanospheres (Catarina Pinto Reis et
al., 2006).

(iii) Freeze-drying:
Encapsulation by freeze-drying involves emulsion formation and drying of the continuous
phase by sublimation known as lyophilization (Minemoto et al., 1997). This method is

21
extremely useful when encapsulating thermo sensitive substances, since it operates on very
low temperatures, unlike spray-drying. However, freeze-drying tends to be unattractive,
unless for very high value foods, due to long processing time, and very high costs, upto 50
times higher than that of spray-drying.( Madene et al., 2006).

(iv) Spray-chilling/cooling:
Spray-chilling/cooling is very similar in principle to spray-drying. The core material is
dispersed (emulsified) in a liquefied wall material, and is then atomized through cooling
medium, which solidifies the matrix. One essential difference from spray-drying is that no
water is withdrawn, which means that the core material must be dispersed in a melt before
processing,( Madene et al., 2006).Since it doesn’t involve high temperatures, it is especially
suitable for heat sensitive core materials. However, this method may require special
handling and storage conditions in any stage of the process (Madene et al., 2006).

(v) Extrusion:
Encapsulation by extrusion is accomplished by adding the sensitive compound to a low-
moisture carbohydrate melt, which is then extruded through small orifices under pressure.
The extruded material is then immediately immersed in a cold isopropanol bath, where it
attains its glassy state, and is dried in hot air (Reineccuis, 1994).

22
MATERIALS AND
METHODS

23
 CHAPTER 3
MATERIALS AND METHODS
3.1 Materials:

Seaweed (Undaria pinnatifida) was collected from the Mandapam coast of Tamil Nadu.
It was dried and powdered.

3.1.1 Chemicals:

Table 3.1 List of Chemicals used

S.No Materials Supplier

1 Methanol, Acetonitrile, S.D. Fine Chemicals

Dichloromethane & Hexane (Mumbai, India), [HPLC
Grade]

2 Acetonitrile, Hexane, Methanol, Sisco Research laboratories

Ethyl acetate, Dichloromethane (Mumbai, India) [HPLC
& Anhydrous sodium sulphate Grade]

3 Pepsin, bile salts, pancreatin HiMedia, (Mumbai, India)

3.1.2 Equipments:

Table 3.2 List of Equipments used

S.No Equipments Manufacturer

1 HPLC Shimadzu model series with

SPD-10AVP detector

2 UV Spectophotometer Shimadzu,Japan

3 CPS disc centrifuge nanoparticles CPS

analyszer instrument,Inc.Florida,USA

4 SEM(Scanning Electron Leo-435 Vp electron

24
 Microscope) microscope

5 FTIR Thermo Nicolet,Nexus,USA

3.2 Methods:

3.2.1 Extraction of Fucoxanthin from Brown Algae Wakame (Undaria pinnatifida):

Fucoxanthin (FUCO) was extracted and purified from the Indian brown seaweed,
Undaria pinnatifida by the procedure previously described by (Haugan et al., 1992) with
slight modification. In brief, U. pinnatifida (50 g) was washed with fresh water and dried at
38 ± 2°C in a drier. The dried seaweed was ground to a fine powder using a mixer grinder.
FUCO was extracted by homogenization with ice-cold acetone (4 times). For each extraction,
the conical flask containing the seaweed was shaken at 100 strokes/min at 4°C for 2 hours.
The pooled extract was filtered using Whatman no.1 filter paper, evaporated to dryness using
rotary evaporator at 30°C and re-dissolved in methanol (100 mL). The lower methanol: water
phase was subjected to extraction thrice by diethyl ether (200 mL).

3.2.2 Open Column Chromatography for Purification of Fucoxanthin:

FUCO was isolated by open column chromatography (45 cm × 3 cm), on an activated

silica gel (mesh size 60-120) column equilibrated with hexane. Chlorophylls and carotenoids
other than FUCO were eluted with (300 mL) followed by 250 mL of hexane: acetone (9:1,
v/v). The FUCO rich fraction was eluted with 200 mL of hexane: acetone (7:3).the extract
was evaporated to dryness using a flash evaporator at 30°C. The residue was re-dissolved in
methanol (3 mL) and the concentration was checked by spectrophotometer. The purity of
FUCO eluted was checked by HPLC against the reference standard. The peak identity,
spectrum, absorption maxima (max) and concentrations of FUCO were confirmed by HPLC.

25
 Fucoxanthin

Other carotenoids

Chlorophyll

Figure 3.1 Purification of fucoxanthin by column chromatography

3.1 Flow chart for extraction and purification of fucoxanthin

Seaweed powder

Homogenization with cold acetone (4 times)

Extract was kept in a shaker at 100 strokes/min at

4°C for 2 hours

Extracts were pooled, filtered and evaporated to dryness by rotary

evaporator at 30 °C and re-dissolved in methanol

Extract was partitioned in methanol: water: hexane (10: 1:10, v/v/v),

washed several times with hexane

Lower methanol: water phase was subjected to extraction thrice by

diethyl ether

26
 Diethyl ether phase containing FUCO was evaporated to dryness by flash
evaporation at 30°C and re-dissolved in 5mL hexane.

Purification by OCC

Elution with hexane: acetone (9:1) to remove chlorophylls

Elution with hexane: acetone (7:3) to obtain fucoxanthin

Concentration of fucoxanthin

Spectrophotometer, HPLC and LC-MS analysis

3.2.3 Quantification of Fucoxanthin:

3.2.3.1 Spectrophotometric Method:

The purified FUCO was analyzed by spectrophotometer to find the concentration of

FUCO.

The concentration was calculated by using the following formula.

O.D × Dilution Factor

Extinction Coefficient of the solvent used

3.2.3.2 Thin Layer Chromatography:

The purified FUCO was run against standard FUCO to determine its presence.

The Retention Factor (RF) was calculated by using the following Formula

= Distance travelled by the solute

Distance travelled by the solvent

27
3.2.3.3 HPLC Method
The purified FUCO was separated on a C-30 column (1:445nm, 8nm) the mobile phase
used, Acetonitrile: methanol: water (60:35:5, v/v/v) with 0.1% ammonium acetate was used
as a mobile phase for the separation of carotenoids. The volume of sample injected for HPLC
analysis was 20 L. An isocratic condition was maintained at a flow rate of 1 mL/min. All
the carotenoids were monitored at 446 nm with UV-visible detector (Shimadzu, Japan). The
peak identities and max of carotenoids were confirmed by their retention time and
characteristic spectra of standard chromatograms, recorded with a Shimadzu model series
equipped with SPD-10AVP detector. They were quantified from their peak areas in relation
to the respective reference standards.

3.2.4 Preparation of Nanoencapusles by Ionic-Gelation Method:

The FUCO was encapsulated with Chitosan-TPP, along with glycolipid. Initially,
Chitosan is dissolved with 1% acetic acid and then glycolipid was added to it and allowed to
stir in a magnetic stirrer until an emulsion is formed. FUCO is dissolved in 0.03% TPP and it
is added to the emulsion drop by drop for the formation of nanoencapusles.

Fig: 3.2 Schematic representation of Nanoencapsulse

28
 3.2 Flow Chart for Preparation of Nanoencapsules

Take a known amount of Chitosan and dissolve it in 1% acetic acid by using a magnetic
stirrer.

Add Glycolipid to the sample.

Dissolve the sample till it appears to be an emulsion.

Fucoxanthin was added in methanol with Sodium tripolyphosphate.

Stir it for 3-4hrs for the formation of nanogels, store it at 4°C

3.2.6 Characterization Techniques:

3.2.6.1 Particle Size Analysis:

Principle:

The CPS disc centrifuge measures particle size distributions using sedimentation, a well-
known and reliable method of particle size analysis. Particles settle in a fluid under a
gravitational field according to stokes’ law. Sedimentation velocity increases as the square of
the particle diameter, so particles that differ in size by only a few percent settle at
significantly different rates. The actual size range that can be measured depends on the
difference in density between the particles being measured and the density of the fluid in
which the analysis is run.

Procedure:

The nanoencapsules size were analyzed using CPS disc centrifuge nanoparticles analyser
(CPS Instruments, Inc. Florida, USA) operating at a speed of 10,000 rpm for 20 minutes and
sample volume of 300uL. All the analyses were performed against a known calibration
standard (PVC) and the particles in the solution were analysed by size distribution graph.

29
3.2.6.2 Scanning Electron Microscope (SEM):

Principle:

A scanning electron microscope is a type of electron microscope that images a

sample by scanning it with a higher energy beam of electrons in a raster scan pattern. The
electrons interact with the atoms that make up the sample producing signals that contain
information about the sample’s surface topography, composition and other properties such as
electrical conductivity.

Procedure:

Nanoencapsulated FUCO was carefully mounted on an aluminum stub using double stick
carbon tape. Samples were then introduced into the chamber of the sputter coater and coated
with a very thin film of gold/palladium before SEM examination. To know the structure of
nanoencapsules, the SEM model used was Leo-435 VP electron microscope.

3.2.6.3 Fourier Transform Infrared Spectroscopy (FTIR):

Fourier transform infrared spectroscopy is a technique which is used to obtain an infrared

spectrum of absorption, emission, photoconductivity or Raman scattering of a solid, liquid or
gas. An FT-IR spectrometer simultaneously collects spectral data in a wide spectral range.
The term Fourier transform infrared spectroscopy originates from the fact that a Fourier
transform is required to convert the raw data into the actual spectrum.

The infrared source emits a broad band of different wavelength of infrared radiation. The IR
radiation goes through an interferometer that modulates the infrared radiation. The
interferometer performs an optical inverse Fourier transform on the entering IR radiation.
The modulated IR beam passes through the sample where it is absorbed to various extents at
different wavelengths by the various molecules present. Finally a detector detects the
intensity of the IR beam. The detected signal is digitized and Fourier transformed by the
computer to get the IR spectrum of the sample.

30
Procedure:

The functional groups present in the nanoencapsules and FUCO were analyzed using the
Fourier transform infrared spectroscopy FTIR. (Thermo Nicolet, NEXUS, TM, USA).
Samples were lyophilized, gently mixed with 300 mg of KBr powder and compressed into
discs at a force of 10KN for 2 min using a manual tablet presser.

3.2.7 Encapsulation Efficiency:

FUCO may exist in two forms, adsorbed on the surface and embedded in the nanosphere,
i.e. nanoencapsules. Thus, the encapsulation efficiency is defined as the ratio of the mass
embedded to the total amount of FUCO in the product and it is calculated as per the formula:

Encapsulation Efficiency(%) =Total amount of FUCO-Total amount of FUCO in supernatant

Total amount of FUCO

Procedure:

Preparations of nanoencapsules with and without lipid by ionic-gelation method and the
samples are taken and centrifuged at 10000 rpm for 30 min at 4°C.Then the FUCO sample is
extracted from the nanoencapsules by Baskaran et al., 2003 method. The extracted FUCO
was analyzed using HPLC.

3.3 Flow chart for the extraction of FUCO from the nanoencapsulse

Take the supernatant

Add Methanol (vortex)

Add Hexane

(Vortex and centrifuge at 2000rpm for 3min)

Discard the upper layer

Repeat hexane extraction for 2 times

(Hexane: Methanol: water-10:10:1)

31
 Discard Hexane layer

Evaporate the solvent

Re-dissolve in 0.1ml of pure Methanol

Read in UV Spectrometry and HPLC

3.2.8 In Vitro Release Kinetics

Release kinetics determines the amount of FUCO released from the nanoencapusles
when kept under a constant temperature and stirring over a period of time. Nanoencapusles
are prepared according to the method described elsewhere in the text. Then they are kept in
a stopper tube and placed in a water bath at 37 °C and the samples are taken at constant
intervals (2,4,6,8,12 and 24 hr) for the further analysis. Then the collected samples are
extracted as described by the procedure elsewhere in the text and analyzed in UV
Spectrophotometer and HPLC 446mn.

3.2.9 In Vitro Digestion:

To determine the role of lipid in the nanoencapsulated FUCO in vitro, purified FUCO
was encapsulated by ionotrophic gelation method to form nanoencapsulation as described
by the procedure elsewhere in the text. The schematic representation of the experimental
protocol describing the in vitro digestion of FUCO is shown in flow chart 3.4. In Brief, in
a 15-ml screw cap test tube, nanoencapsulated FUCO was added. The samples were
subjected to in vitro digestion simulating the gastric and small intestinal phase of digestion
according to the method proposed by Garret et al. with slight modification.3 ml of 0.5%
pepsin in phosphate buffer (3.6 mmol/L CaCl2, 1.4 mmol/L MgCl2.6H2O, 49 mmol/L
NaCl, 12 mmol/L KCl, 6.4 mmol/L KH2PO4) was added to the nanoencapsulated FUCO.
The pH was adjusted to 2.02 with 2 mol/L HCl. The tubes were screw capped under a
stream of nitrogen and incubated for 1 hr at 37°C in a shaking water bath at 120
strokes/min. On cooling, the pH was raised to 5.0 with 1 mol/L NaHCO 3 followed by the
addition of 16 mL 0.1 mol/L NaHCO3 0.32gm pancreatin in 20ml of 0.1mol/L 16 g/L and
0.125 gm of bile extract in 4ml of 0.1mol/L.The pH of the digesta was further adjusted to
7.5 by 1N NaOH. The test tubes were blanketed with a stream of nitrogen and subjected to

32
incubation at 37°C with shaking at 120 strokes/min for 2 hrs(intestinal phase). After
incubation, an aliquot of digesta (1mL) was withdrawn from each sample, centrifuged (Z
360 K, BHG Hermle Gosheim) at 12,000×g at 4°C for 120 min to separate the aqueous
fraction and this fraction was used for quantification of FUCO by HPLC.

Flow Chart 3.4 Procedure for in vitro digestion

Seaweed (Undaria Purified Nanoencapsulated product

pinnatifida) fucoxanthin containing fucoxanthin

Crude extract Column purification In vitro digestion

HPLC- analysis Pepsin (pH 2.02)

Incubated at 37°C for 1 hr

Gastric Digestion

Bile extracts (pH 7.5) Small

Pancreatin extract (pH intestine
7.5) Incubated at 37°C Digestion
for 2 hrs

Digesta-analyzed for
fucoxanthin content by
HPLC

33
RESULTS AND
DISCUSSIONS

34
 CHAPTER 4

RESULTS AND DISCUSSIONS

4.1 Extraction of Fucoxantin

4.1.2 Open Column Chromatography:

The extracted carotenoids were purified by Open Column Chromatography (OCC) on an

activated silica column to verify its purity. The purity of fucoxanthin (FUCO) eluted by OCC
was clarified by HPLC (Figure 4.2), and it was found to be 95%. The OCC method proposed
in this study is relatively simple and provides purified FUCO. This value was almost consistent
with the results of Sugawara et al (2002), who have purified FUCO from wakame using OCC on
alumina column and have reported the purity as 91- 93%

Fig 4.1-Column chromatography for separation of FUCO

4.2 Fucoxanthin Concentration in Wakame

4.2.1 Quantification of Fucoxanthin by Spectrophotometric Method:

The amount of FUCO extracted from seaweed wakame was identified using the
spectrophotometer method. The level of FUCO was calculated as:

% FUCO Conc. = 0.794 * 400

1660

35
 = 0.1914g/100ml *10

= 1.914mg/ml * 20(total volume of FUCO)

% FUCO Conc. = 38.289 mg/100g of dry weight

The concentration of FUCO in Wakame was found to be 38.289 mg/100mg. The FUCO was
identified with the help of its characteristic spectra and its λmax (446nm) given in Figure 4.2.

Fucoxanthin, λmax = 446nm

Absorbance

Wavelength (nm)

Figure 4.2 Absorption Spectrum of fucoxanthin

According to Kanami Mori et.al (2004), the amount of FUCO extracted from Korean
wakame was almost similar to that of the other species. The amount of FUCO present in the
edible black sea algae was about 0.36 mg/g determined by Galina.N et.al (2005). It is
therefore evident that studies on the carotenoid composition of seaweeds of the Indian sub-
continent are scarce and the amount of FUCO is almost similar irrespective of the
geographical location.

4.3 Fucoxanthin Separation by Thin Layer Chromatography

Purified FUCO from open column chromatatography was separated on TLC sheet.
Hexane: Acetone (7:3) allowed of the separation of the FUCO from the other carotenoids.
FUCO was detected as orange color band having a RF value about 3.5 similar to that of the
Standard FUCO (Gaurav Rajauria et.al 2013)

36
 Fig 4.3 Separation of FUCO by Thin layer chromatography

4.4 Quantification of Fucoxanthin by HPLC Method:

The HPLC chromatogram of column-purified FUCO is shown in Figure 4.3.The HPLC

chromatograms of purified all-trans-fucoxanthin showed one major peak with a retention
time of 31.7min for standard and 31.0 min for purified FUCO. The cis form of FUCO is very
less so it doesn’t appear in the chromatogram, xiajoun yan et.al (1999).The purity of standard
peak is > 87% and the purified FUCO is > 91%.

Sangeetha Ravi Kumar (2011) screened 18 Indian marine algae and found that
Fucoxanthin, was found in 11 algae and was higher in D. dichotoma, S. tenerrium, S.
cristaefolium, P. tetrastomatica, C. sertularciocles, U. pinafida and Palmari .

37
 (a)

(b)

Fig: 4.4 HPLC peaks (a) Standard FUCO (b) Purified FUCO

4.5 Lipid Based Nanoencapsulses Loaded with Fucoxanthin:

The commercially available chitosan and Tripolyphosphate has been used for the
development of nanoencapsules and the glycolipid is used to enhance the penetration of
nanoencapsules through the lipid barrier of intestine. Thus the nanoencapsules prepared
doesn’t only have higher stability but also have higher bioavailability.
Chitosan is a kind of hydrophilic polysaccharides, which not only is degradable and
nontoxic, but also shows excellent mucoadhesive and permeation enhancing effect across
biological surfaces Lian-Yan Wang et.al (2005). Márcia R. de Moura et.al (2009). CS-TPP
nanoparticles have an improved barrier and mechanical properties.

38
 4.6 Characterization Techniques:

4.6.1 Particle Size Analysis:

Fig: 4.5 Particle size of nanoencapsules

The particle size of chitosan-lipid loaded with FUCO is around 550 nm as shown by
particle size analyser.

4.6.2 Scanning Electron Microscope:

Figure 4.6 shows the SEM images of the nanoencapsulated fucoxanthin synthesized by
solvent evaporation method. The size of the particles is small and found to range between
200- 400 nm. The nanoparticles were found to be spherical in shape. From the figure, it can
be seen that particles are uniformly distributed over the surface.

Figure 4.6 SEM images of nanoencapsulated fucoxanthin

39
 4.6.3 Fourier Transform Infrared Spectrometer (FTIR):

From the spectrum 4.7a , it is evident that peaks such as hydroxyl, amino and C-N groups
near 1080 cm-1, NH 2 plane band is formed near in 1623 cm-1 confirming the presence of
chitosan, OH stretches are found in the range of 2920-3412 cm-1 as seen in the chitosan
nanoparticles.

From fig 47.7 b and c FTIR reveals 1246 cm-1,1736 cm-1 and 1926.8 cm-1 corresponds
to C-O (carbonyl) stretching (carboxylic acid/acetate), C-O vibration stretch (acetate) and
C=C=C (allene group) confirms FUCO functional groups. The absorption band at 3510 cm-1
attributed to –NH group in chitosan was broadened by the physical interactions with TPP. the
–NH2 bending vibration was observed at 1630 cm-1 in place of 1590 cm-1 due to the
interactions of TPP ions with –NH3 + ions of chitosan .There is not much variation between
the with lipid and without lipid binding efficiency.

10 0 c h2

95

90

85

80

75

667.2
70

900.0
3741.8

1256.0
%T

65
1320.6
1424.0

60
1623.5

55
1378.9

1154.0

1080.1

50
1660.8
2880.0
2920.1

45

40
3407.4

35

40 00 35 00 30 00 25 00 20 00 15 00 10 00 50 0

c m-1

Fig: 4.7a FT-IR of chitosan spectra

40
 %T

%T

64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98

40 00
42
44
46
48
50
52
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98

40 00
3 70 0.8
3 65 6.0
3 72 8.2

C H TTP FU G O w i th ou t G L
C H TTP FU C O w i th G L

3 52 4.0

35 00
3 59 7.0
3 45 0.3

35 00
3 40 9.4

30 00
30 00
2 88 7.5
2 92 8.0

2 85 2.0

2 61 5.3

25 00
25 00

41
c m-1
c m-1

20 00
20 00

1 93 1.8

1 73 7.5

1 66 4.0 1 73 5.3
Fig:4.7b FT-IR spectra of with lipid

Fig: 4.7c FT-IR spectra without lipid

1 60 9.8
1 52 9.0

15 00
15 00

1 42 4.0
1 46 0.0
1 37 8.4
1 36 7.2

1 19 8.8
1 16 4.0
1 11 2.0 1 20 0.1
1 15 6.2
1 11 1.4
1 03 0.2
1 02 8.0

10 00
1 00 4.0
10 00

1 00 1.9

7 87 .5
7 95 .6
6 95 .4 7 24 .0
6 94 .9
6 41 .0
6 44 .0
5 89 .1
5 51 .7 5 89 .8

50 0
5 52 .0
50 0

5 02 .5
4 33 .2
4.7 Encapsulation Efficiency:

The average entrapment efficiency between the lipid and without lipid based
nanoencapsules is quiet good around 85.4%, the encapsulation efficiency of lipid based
nanoencapsules is 90.9% and without lipid based nanoencapsules is 80%, thus it can be
concluded the lipid based nanoencapsules is more efficient than that of the without lipid
nanoencapsules.

According to Diego T. et.al (2010) Encapsulation efficiency of the nanoencapsules is

quiet good than that of the microencapsulation technique. Wan Ajun et.al (2009), the
encapsulation efficiency is affected greatly by TPP pH value. Quan Gan et.al (2007), there is
a better encapsulation with higher molecular weight, lower chitosan concentration, lower
chitosan/TPP mass ratio.
Encapsulation efficiency (%)

100

80

60

40

20

0
L

L)
G

(-G
+
es

es
ul

ul
ps

ps
ca

ca
en

en
O

O
C

C
FU

FU

Fig: 4.8 Encapsulation efficiency

42
4.8 In Vitro Release Kinectics:

0.10 Fig: 4.9

Rmax
Release profile (%) 0.08

0.06

0.04

0.02

0.00
0 5 10 15 20 25
Time (h)
Release kinetics of FUCO

The release of FUCO from the CS-NGs dispersed in glycolipid indicated a rapid burst
release following the zero order rate kinetics up to 15 h followed by continuous and almost
reaching sustainable release pattern over 24 h (Fig. 4.9) Results showed that there was
initially rapid burst of 17.3% FUCO (within 2 h) on incubation when the FUCO
concentration is 0.1 mg. Over 24 h, the cumulative release profile of FUCO from CS-NGs
was 71%. The Rmax is the time at which the maximum release of FUCO from chitosan
nanoparticles occurs. This phenomenon shows the FUCO’s interaction (-OH and acetate
functional groups) with the amino (–NH3+) groups of the polymeric CS. The burst effect was
gradually reduced as the time progressed from 0 till 24 h.

4.9 In Vitro Digestion:

In vitro bioavailability studies (simulated gastric and intestinal digestion) illustrated that
the percent micellization of FUCO in the CS-NGs with GL (57.6 %) (Fig.) was significantly
higher than the FUCO+GL (30.6%) and the control (17.6%). This clearly demonstrates the
FUCO entrapped in CS-TPP-NGs which is dispersed in GL is having more bioavailability

43
than the FUCO+GL which is in non-encapsulated indicating that bioavailability depends on
the size of micelles

Fig: 4.10 In vitro digestion showing chitosan nanogels with FUCO+ GL (57.6%) higher
micellerization than that of the FUCO + GL (30.6%)
CHAPTER 5 and the control (17.6 %).

44
SUMMERY &
CONCLUSIONS

45
 CHAPTER 5

SUMMERY & CONCLUSIONS

 In this study procedure for isolation and purification of fucoxanthin (FUCO) from
marine algae wakame (Undaria pinnatifida) was optimized using a suitable solvent
system, column chromatography and HPLC techniques.

 The purity of silica column purified FUCO was 91-93%.

 The purified FUCO was nanoencapsulated with chitosan-tripolyphosphate polymers

using ionotropic gelation technique. .

 The encapsulated products were characterized using particle size analyzer, Scanning
Electron Microscopy (SEM) and Fourier Transform Infrared Spectrometer (FTIR).

 The functional groups present in the nanoencapsulated product were analyzed using
Fourier Transform Infrared Spectrometer.

 The shape of the nanoencapsulated FUCO was determined by SEM and it is found to
be spherical.

 The particle size of the nanoencapsulated products was found to be in the range
between 550 nm.

 The encapsulation efficiency of the nanoencapsules of chitosan-lipid based

nanoencapsules was 90.9% and the without lipid based system was 80.5%, thus the
lipid based nanoencapsulse have higher entrapment when compared with the without
lipid based nanoencapsules.

46
 From the release profile of FUCO it was observed that there is a initial rapid burst of
17.3% FUCO (within 2 h) and over a period of 24 h, the cumulative release profile of
FUCO from CS-NGs was 71%,Thus it can be concluded that, the burst effect was
gradually reduced as the time progressed from 0 till 24 h.

 In vitro bioavailability studies illustrates that the percent micellization of FUCO in

the CS-NGs with GL (57.6 %) was significantly higher than the FUCO+GL (30.6%)
and the control (17.6%).

47
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