1
Acknowledgement
Firstly, thanks to Allah s. w. t because giving me success for my final
year project.
I wish to express my gratitude to individuals who have helped me with
creating this. This would have never come to light without their massive efforts
and help.
I'm deeply grateful to my supervisor, " Dr- Ayman Salah El-deen Al-Husini
"who has advice me and always helping to complete of my final year project . I
consider myself very fortunate for being able to work with a very considerate
and encouraging lecture like him .
Also I'd like to thank" Dr-Ibrahim Mohey" head chemistry department
Port said university for his contributions.I would like to express my deepest
thanks to all doctors and demonstrators who taught me during the four years.
I would have never forget the contributions ,encouraging and supporting of
family , fiancé and friends. Their contributions were very essential to me ….
2
*** Contents **** :-
*Page.No*
1. List of figures ……………………………………………………….
5
2. Abbreviations ……………………………………………………....
6
3. Introduction …………………………………………………………
7:8
4. chemistry of chitosan ………………………………………….
9:11
5. Production of chitosan ……………………………………….... 11:22
6. History of chitosan ……………………………………………… 22:23
7. Properties of Chitosan ………………………………………… 23:24
8. Degradation ………………………………………………………..
24:25
9. Molecular Weight ………………………………………………..
25:26
10. Solvent Properties………………………………………………
26:27
11. Degree of Deacetylation ………………………………………
27:29
12. Solubility of chitosan …………………………………………. 29:33
13. It’s application ………………………………………………….
33:34
14. Water Treatment Applications ……………………………. 34:36
15. Medical & pharmacutical Applications ………………… 36:38
16. Orthopedics …………………………………………………………
38
17. Tissue Engineering ……………………………………………… 38:40
18. Wound Healing ……………………………………………………
3
40
19. Drug Delivery……………………………………………………… 40:41
20. Surgical Adhesion……………………………………………….
41:42
21. Hemostatic Agent ……………………………………………….. 42:43
22. other bio-medical applications ……………………………..
43
23. Biotech nological Applications …………………………….
44
24. Cell-Stimulater ……………………………………………………
44
25. Fat-Net……………………………………………………………….
44:45
26. several potential clinical applications …………………. 46:47
27. Administering Chitosan ………...……………………………
47
28. Potential industrial use ……………………………………….
48
29. Agricultural & Horticultural use ………………………..
48:49
30. Applied as seed coating agents …………………………….
49:50
31. Applied as foliar treatment agents ………………………
50:52
32. Applied as soil amendment ………………………………….
52
33.Applications of chitosan derivatives …………………….
52
34. Application of glycol chitosan for entrapment of protein
molecules…………………………………………………………….
35. Amphiphilic derivatives of glycol chitosan …………..
36. Glycol
chitosan-coated
52:53
53
MRI(Magnetic-Resonance-Imaging)
agent safer effective in detecting breast cancer….….. 53-54
4
37. Cosmetics ………………………………………………………….... 54:57
38. References………………………………………………………..... 58:64
** List of figures** :
*Page.No*
Fig (1) : chemical structure of chitosan ……………………….
9
Fig (2): Deactylation of chitin to chitosan ……………………..
9
Fig(3) : chitin & chitosan manufacturing process …………..
11
Fig(4) : preparation of chitin & chitosan ……………………….
15
Fig(5) :
sweet
Fungus Gongronella butleri USDB 0201 was grown on
potato
pieces
in
a
tray-type
solid
fermentor………………………………………………………………….
substrate
17
Fig(6) : Extraction of chitosan from mycelia of fungus G.butleri
grown
in
solid
…………………………………………………………………………………..
substrate
18
Fig(7) : Extraction of chitosan and glucan from the AIM suspended
in 0.35M acetic acid by treatment with Termamyl Type LS (Nwe &
Stevens, 2002 and Nweetal.,2008) ………………………………..
21
Fig (8): types of methods of extraction of chitosan
from fungi ……………………………………………………………………
22
Fig (9) : Chemical structure of CS-g-CMC biomaterials …….
36
Fig(10) : cell scaffold interaction ……………………………………
39
5
Abbreviations :-
 (SSF) : Solid Substrate Fermentation.
 (SMF) : Submerged Fermentation .
 (M.W) : Molecular Weight.
 (HPLC): High Performance Liqud Chromatography.
 (D.D) : Degree of Deacetylation.
 (CS-g-CMC): Chitosan-g Carboxy Methyl Cellulose .
 (CD)
: Cyclodextrin .
 (HDL) : High-Density Lipoprotein.
 (LDL) : Low Density Lipoprotein.

(ABA) : Abscisic Acid .
 (GC)
: Glycol chitosan .
 (BSA) : Bovine Serum Albumin.
6
1- Introduction :-
A polymer is a chemical compound or mixture of compounds consisting of
repeating structural units created through a process of polymerization.
(1)
The term
derives from the ancient Greek word πολύς (polus, meaning "many, much") and μέρος
(meros, meaning "parts"), and refers to a molecule whose structure is composed of
multiple repeating units, from which originates a characteristic of high relative
molecular mass and attendant properties. (2)
Hence, the terms polymer and polymeric material encompass very large, broad
classes of compounds, both natural and synthetic, with a wide variety of properties.
Because of the extraordinary range of properties of polymeric materials
(3)
.
they play
an essential and ubiquitous roles in everyday life. (4)
Over the last years, many attempts have been made to replace petrochemical
products by renewable, biosourced components. Abundant naturally occurring
polymers – as starch, collagen, gelatin, alginate, cellulose and chitin – represent
attractive candidates as they could reduce the actual dependence on fossil fuels, and
consequently have a positive environmental impact.
In this respect, chitosan is a quite unique bio-based polymer: its intrinsic
properties are so singular and valuable that chitosan possesses noactual petrochemical
equivalent. Consequently,the inherent characteristics of chitosan make it exploitable
directly for itself. Chitosan, a natural and linear biopolyaminosaccharide, has received
7
much attention as a functional biopolymer with applications in pharmaceuticals, food,
cosmetics and medicines.(5)
Chitosan is soluble in aqueous acids because of the protonation of amino
groups, but it is insoluble in water and most of organic solvent so as to restrict the
applications.
Nevertheless, chitosan application is limited by its solubility in aqueous
solution An elegant way to improve or to impart new properties to chitosan is the
chemical modification of the chain,generally by grafting of functional groups, without
modification of the initial skeleton in order to conserve the original properties. The
functionalization is carried out on the primary amine group, generally by
quaternization, or on the hydroxyl group.
Chitosan is a safe and friendly substance for the human organism; therefore, it
has become of great interest not only as an underutilized resource, but also as a new
functional material of high potential in various fields.
Some unique properties make chitosan an excellent material for the
development of new industrial applications and recent progress in chitosan material is
quite noteworthy. In this review, we mainly take a closer look at various chitosan
Applications.
8
2- Chemistry of Chitosan:-
Fig (1) : chemical structure of chitosan
It
is
a
hetero
polymer
consists
of
β(1-4)
2-acetamido-2-deoxy-β-
Dglucopyranose(N-acetyl glucosamine) and 2-amino-2-deoxy-β-D-glucopyranose(Dglucosamine) units, randomly or block distributed throughout the biopolymer. The
chain distribution is dependent on the processing method used to derive the
biopolymer (Dodane and Vilivalam, 1998; Kumar, 2000; Khor and Lim,2003). It is
the N-deacetylated derivative of chitin, but the N-deacetylation is almost never
complete (Kumar, 2000; Santos et al., 2005).
Fig (2): Deactylation of chitin to chitosan
9
Chitin and chitosan are names that do not strictly refer to a fixed stiochiometry.
Chemically, chitin is known as poly-N-acetyl glucosamine, homo polymer of b-(1fi4)linked-acetyl-D-glucosamine (Muzzarelli, 1997). (6) and in accordance to this proposed
name, the difference between chitin and chitosan is that the degree of deacetylation in
chitin is very little, while deacetylation in chitosan occurred to an extent but still not
enough to be called polyglucosamine (Muzzarelli, 1973). Chitosan has one primary
amine and two free hydroxyl groups for each monomer with a unit formula of
C6H11O4N. This natural biopolymer is a glucosaminoglycan and is composed of two
common sugars, glucosamine and N-acetylglucosamine, both of which are
constituents of mammalian tissues(Khan, 2001; Snyman et al., 2002). Chitosan is the
second abundant polysaccharide next to cellulose (Duarte et al.,2002; Sinha et al.,
2004), but it is the most abundant natural amino polysaccharide and is estimated to be
produced annually almost as much as cellulose (Kumar, 2000).
Chitosan can be chemically considered as analogues of cellulose, in which the
hydroxyl at carbon-2 has been replaced by acetamido or amino groups (Krajewska,
2004). As a point of difference from other abundant polysaccharides, chitin and
chitosan contain nitrogen in addition to carbon, hydrogen and oxygen. Chitin and
chitosan are of commercial interest due to their high percentage of nitrogen (6.89%)
compared to synthetically substituted cellulose (1.25%) (Muzzarelli and Muzzarelli,
1998; Kumar, 2000).
As most of the present-day polymers are synthetic materials, their
biocompatibility and biodegradability are much more limited than those of natural
polymers such as cellulose, chitin, chitosan and their derivatives. However, these
naturally abundant materials also exhibit a limitation in their reactivity and
processability. Chitosan is recommended as suitable functional material, because this
10
natural polymer has excellent properties such as biocompatibility, biodegradability,
non-toxicity and adsorption properties. Recently, much attention has been given to
chitosan as a potential polysaccharide source (Kumar, 2000).
Chitosan can be degraded by soil microorganisms and water microorganisms.
This makes chitosan environmental friendly. This was acknowledged by the US
Environmental Protection Agency when it exempted chitosan from tolerance level
testing (Hennen, 1996). Chitin and chitosan are obtained from the shells of
crustaceans such as crabs,prawns, lobsters and shrimps, the exoskeletons of insects,
and the cell walls of fungi such as aspergillus and mucor where it provides strength
and stability (Dodane and Vilivalam, 1998; Kumar, 2000; Khor and Lim, 2003;
Krajewska,2004; Sinha et al., 2004; Qin et al., 2006). (7)
3 - Production of Chitosan:-
Fig(3):chitin & chitosan manufacturing process
11
Chitin and chitosan are found as supporting materials in many aquatic
organisms (shells of shrimps and crabs and bone plates of squids and cuttlefishes) (8),
in many insects (mosquitoes, cockroaches, honey bees, silkworms, Drosophila
melanogaster,Extatosoma tiaratum and Sipyloidea sipylus), in terrestrial crustaceans
(Armadillidium vulgare, Porcellio scaber), in nematode, in mushrooms (Agaricus
bisporus, Auricularia auriculajudae, Lentinula edodes, Trametes versicolor,
Armillaria mellea, Pleurotus ostreatus, Pleurotus sajo-caju and Pleurotus eryngii)
and in some of microorganisms (yeast, fungus, and algae) (Carlberg,1982; Nemtsev et
al., 2004; Veronico et al., 2001; Paulino et al., 2006; Moussian et al., 2005;Tauber,
2005; Hild et al., 2008; Anantaraman & Ravindranath, 1976; Pochanavanich
&Suntornsuk, 2002; Mario et al., 2008; Yen & Mau, 2007 cited in Nwe et al., 2010).(9)
Crab and shrimp shell wastes are currently utilized as the major industrial
source of biomass for the large-scale production of chitin and chitosan. Processing
wastes from marine food factories help to recycle the wastes and make the derivatives
or by-products for use in other fields. These crustacean shell wastes are composed of
protein, inorganic salts, chitin and lipids as main structural components. Therefore,
extraction of chitin and chitosan was mainly employed by stepwise chemical methods
(Kim and Rajapakse, 2005).
The production of chitosan from fungal mycelia has a lot of advantages over
crustacean chitosans such as the degree of acetylation, molecular weight, viscosity and
charge distribution of the fungal chitosan are more stable than that of crustacean
chitosans; the production of chitosan by fungus in a bioreactor at a technical scale
offers also additional opportunities to obtain identical material throughout the year
and to obtain chitosans with a radioactive label or with specific changes in its
polymeric composition; and the fungal chitosan is free of heavy metal contents such
12
as nickel, copper (Tan et al., 1996, Arcidiacono & Kaplan, 1992
(10)
, Nwe & Stevens,
2002a). Moreover the production of chitosan from fungal mycelia give medium-low
molecular weight chitosans (1–12 × 104 Da), whereas the molecular weight of
chitosans obtained from crustacean sources is high (about 1.5 ×106Da) (Nwe &
stevens, 2002). Chitosan with a medium-low molecular weight has been used as a
powder in cholesterol absorption (Ikeda et al., 1993) and as thread or membrane in
many medical-technical applications.
For these reasons, there is an increasing interest in the production of fungal
chitosan. However, so far, the extraction of high yield pure chitosan production from
fungal cell wall material has not been accomplished upto 2001 (Stevens, 2001).
*Steps :
In the first stage, chitin production was associated with food industries such as
shrimp canning. In the second stage, the production of chitosan was associated with
fermentation processes, similar to those for the production of citric acid from
Aspergillus niger, Mucor Rouxii, and Streptomyces, which involved alkali treatment
yielding chitosan. Briefly, shells were ground to smaller sizes and minerals, mainly
calcium carbonate, were removed by extraction (demineralization, decalcification)
with dilute hydrochloric acid followed by stirring at ambient temperature.
The protein was extracted (deproteinisation) from the residual material by
treatment with dilute aqueous sodium hydroxide and thereby prevents contamination
of chitin products from proteins. The resulting chitin was deacetylated in 40 - 45%
sodium hydroxide at 120ºC for 1- 3 hours with exclusion of oxygen, and followed by
purification procedures to form chitosan with a cationic nature. The alkali removed
13
the protein and the deacetylated chitin simultaneously. Depending on the alkali
concentration, some soluble glycans would be removed. In the deacetylation process,
some of the acetyl groups were removed from the molecular chain of chitin.
This shortened the chain lengths of the chitin molecule, eventually leaving
behind a polymer with a complete amino group called chitosan. This treatment
produces 70% of deacetylated chitosan (Kumar, 2000; Khan, 2001; Krajewska, 2004;
Kim and Rajapakse, 2005). Methods based on alkaline treatments were employed to
achieve N-deacetylation, as Nacetyl groups cannot be removed by acidic reagents as
effectively as with alkaline treatment. However, partial deacetylation could occur
under this harsh treatment (Muzzarelli, 1973).
The extent of deacetylation mainly depends upon alkali concentration, time and
temperature employed throughout the process. For example, increasing temperature or
strength of sodium hydroxide solution can remove acetyl groups, resulting in a range
of chitosan molecules with different physicochemical properties and applications
(Khan, 2001).
According to Kumar (2000), to produce 1 kg of 70% deacetylated chitosan
from shrimp shells, 6.3 kg of HCl and 1.8 kg of NaOH are required in addition to
nitrogen, water (1.4 tons). Commercially, chitosan is available in the form of dry
flakes, solution and fine powder (Duarte et al., 2002; Sinha et al, 2004). The
hydrolysis of chitin with concentrated acids under drastic conditions produces
relatively pure D-glucosamine (Kumar, 2000).
In India, the Central Institute of Fisheries Technology, Kerala, initiated research
on chitin and chitosan. From their investigation, they found that dry prawn waste
14
contained 23% and dry squilla contained 15% chitin. Chitin and chitosan are now
produced commercially in India, Japan, Poland, Norway and Australia (Kumar, 2000).
It is likely that future sources of chitin and chitosan will come from biotechnology
innovation, especially when medical applications are the focus (Khor and Lim, 2003).
Thus, production and utilization of chitosan constitutes an economically attractive
means of crustacean shell wastes disposals, which is sought worldwide.
Fig(4) : preparation of chitin & chitosan
15
Investigation of a method to produce high
quality and quantity of fungal chitosan :**Growth of fungus and extraction of chitosan by traditional method:
Chitosan is a substantial component of cell wall of certain fungi, particularly
those belonging to the class Zygomycetes (Bartniki-Garcia, 1968). (11) Tan et al., 1996
evaluated the yield of chitosan from several Zygomycetes fungi including Absidia,
Gongronella, Mucor and Rhizopus and concluded that G.butleri gave the highest yield
of chitosan.
At the same time, Crestini et al., 1996 reported that the yield of chitosan
produced from Lentinus edodes grown in solid state fermentation, 6.18 g/kg was
higher than that in submerged fermentation, 0.12 g/l. In 1998, fungus Gongronella
butleri was selected to produce chitosan in our research .
Firstly, a comparison was made between the yield of chitosan from fungal
mycelia grown in solid substrate fermentation (SSF) and in submerged fermentation
(SMF) using various nitrogen sources. The Termamyl assayed extraction method was
not discovered yet at that time. The chitosan was extracted using vacuum filtration and
β-glucanase treatment method. It was observed that the yield of chitosan obtained
from fungal mycelia grown in SSF (3.7 g chitosan/kg of solid substrate) was higher
than that in SMF (0.6 g chitosan/L of fermentation medium) due to the low amount of
mycelia produced in SMF (Nwe et al.,2002). Based on the results obtained from our
research and Crestini et al., 1996, solid substrate fermentation was selected as the best
fermentation method to produce chitosan by fungus Gongronella butleri . (12)
16
Fig. 5. Fungus Gongronella butleri USDB 0201
was grown on sweet potato pieces in a traytype solid substrate fermentor.
Sweet potato pieces were used as solid support and as carbon source. The dried
fungal mycelia were used to extract chitosan. The history of the development of
chitosan extraction procedure by enzymatic method started with the work of Mr. Su
Ching Tan from the National University of Singapore, Singapore. In his method,
mycelia were treated with 1 M NaOH and the resultant alkaline insoluble material
(AIM) was treated with 0.35 M acetic acid at 25oC for 2 h (Tan et al., 1996).
The yield of chitosan extracted from fungal mycelia grown in solid substrate
fermentation was 2-3 g/100 g of mycelia. An effective chitosan extraction procedure
is essential for an economical production of fungal chitosan.). Most methods used
17
1 M NaOH to remove protein and other cell wall materials and then the chitosan was
extracted with 2 % acetic acid. The yield of chitosan produced from the fungal
mycelia treated in this way is very low. The extraction procedure for high yield
production of pure chitosan from the fungal cell wall material has not yet been
accomplished
up
to
2001
(Stevens,
2001).
(
13
)
Fig. 6. Extraction of chitosan from mycelia of
fungus G.butleri grown in solid substrate
chitin/chitosan occurs in two forms, as free aminoglucoside and covalently
bonded to β-glucan (Bartnicki-Garcia, 1968; Gooday, 1995; Robson, 1999; Wessels et
al., 1990). In 1990,Wessels et al. proposed that initially chitin and β-glucan chains
accumulate individually in the fungal cell wall and thereafter form the inter polymer
linkage. The formation of the chitin/chitosan–glucan complex chains results in a rigid
cross-linked network in the cell wall (Gooday, 1995; Robson, 1999) and causes a
18
considerable problem for the extraction of intact chitosan and glucan. It does not break
down easily under mild extraction condition (Muzzarelli et al., 1980).
Under the above mentioned conditions only free chitosan, that is chitosan
unbounded to other cell wall components is extracted (Nwe & Stevens, 2002). (14)
Chitosan bounded to insoluble cell wall components will not be extracted. To extract
the high quality and quantity of chitosan and glucan from cell wall of fungi, the bond
between chitosan and glucan in cell wall of fungi must be investigated.
Most of the researchers are trying to find the linkage between the chitosan and
glucan in the fungal cell wall by digestion with glucanase, chitinase and amylase. In
1979, Sietsma and Wessels reported that 90%of β-glucan obtained from the chitinglucan complex by digestion with (1- 3)-β-glucanase and N-acetyl-glucosamine,
lysine and/or citrulline were identified as products after digestion with chitinase.
Therefore they proposed that the bridge linking the glucan chain with the chitin
contains lysine, citrulline, glucose and N-acetyl-glucosamine. Similar evidence was
obtained by Gopal et al., 1984 for the degradation of chitin-glucan complex by (1,3)β- and (1,6)-β-glucanase, and subsequently by chitinase.
Carbohydrate expressed as glucose and N-acetyl-glucosamine monomers was
detectable in equivalent amounts in the hydrolysate. The residue after chitinase
treatment was further treated with α- amylase but additional release of glucose could
not be detected colorimetrically. Surarit et al., 1988 suggested a glycosidic linkage
between position 6 of N-acetyl-glucosamine in chitin and position 1 of glucose in β(1-6)-glucan in the cell wall of Candida albicans.
19
In 1990, Wessels et al., proposed that a direct link between free amino groups
in the glucosaminoglycan and the reducing end of the glucan chains forming the interpolymer linkages in the chitin-glucan complex. Up to 1992, no cleare evidence of the
identity of the chemical link between the chitosan and glucan polymer chains had
been uncovered (Roberts, 1992).
After 1995, Kollar et al., 1995 and Fontaine et al. 2000 digested the cell wall of
Saccharomyces cerevisae and Aspergillus fumigatus with 1%SDS and 1 M NaOH
respectively and the insoluble fractions were digested with (1,3)-β-endoglucanase and
chitinase.
The soluble fractions were analyzed. Based on their results, they concluded that
the terminal reducing residue of a chitin chain is attached to the non-reducing end of a
β-(1,3)-glucan chain by a β-1,4 linkage. An insoluble residue was remained at the end
of both extraction processes. Importantly, cell wall matrix must be broken down far
enough in order to study the linkage between the chitosan and glucan in the fungal cell
wall and to extract total chitosan (i.e., freechitosan plus chitosan bounded to glucan).
In 1994, Muzzarelli et al., reported that the chitosan-glucan complex can be split by
25 % NaOH
20
Fig.7.Extraction of chitosan and glucan from
the AIM suspended in 0.35M acetic acid by
treatment with Termamyl Type LS (Nwe &
Stevens, 2002 and Nwe et al., 2008)
21
(15)
Fig (8): types of methods of extraction of
chitosan from fungi
4 - History of Chitosan:The human use of chitosan can be traced back to 1811 when chitin, the source from
which it is derived, was first discovered by Braconnot, a professor of natural history in
France. According to historians, while Braconnot conducted research on
mushrooms,he isolated
what was
later to be called chitin.
Some 20 years later, an article on insects was published, which noted that a
similar substance was present in the structure of insects and plants. The author called
this substance "chitin." Basically, the name chitin came from the Greek word meaning
"tunic" or "envelope". In 1843, Lassaigne showed the presence of nitrogen in chitin.
After the discovery of chitin came chitosan. It was first observed by Rouget during his
22
experiments on chitin. Rouget observed that the compound of chitin could be
manipulated through chemical and temperature treatments and become soluble. In
1878, Ledderhose showed chitin to be made of glucosamine and acetic acid. It was not
actually
until
1894
that
Hoppe-Seyler
named
this
supstance
"chitosan."
By the early 20th century, a great deal of research had been performed on the subject
of chitosan. It involved the sources of chitin, specifically crab shells and fungi. It was
the work of Rammelberg in the 1930s that led to confirming chitosan in these sources.
Experts determined (by hydrolyzing chitin) that chitin is a polysaccharide of
glucosamine.
The 1950s arrived, and x-ray analysis advanced the study of chitosan in fungi.
Only recently has technology proved reliable in identifying the presence of chitin and
cellulose in cell walls. The first book on chitosan was published (in 1951) 140 years
after Braconnot made his initial
observations.
In the early 1960s, chitosan was studied for its ability to bind with the red blood
cells. The substance was considered a hemostatic agent. For three decades now,
chitosan has been used at water purification plants for detoxifying water. It gets spread
over the surface of the water, where it absorbs grease, oils, and other potential toxins.
Today, it is famous as a dietary supplement that is good for weight loss. In Japan
and Europe it has been marketed for weight loss for about 20 years. Many people call
it the "fat blocker". It is this recent use for chitosan that brought the otherwise
mundane substance to public attention.(16)
23
5 - Properties of Chitosan: -
The properties of chitosan are greatly affected by the conditions under which it
is processed, because it is the process conditions that control the amount of
deacetylation that occurs. The degree of deacetylation controls the amount of free
amino groups in the polymer chain. The free amino groups give chitosan its positive
charge. The amino group along with the hydroxyl group gives chitosan it functionality
which allows it to be a highly reactive polysaccharide.
Chitosan’s positive charge allows it to have many electrostatic interactions with
negatively charged molecules. The processing conditions as well as the amount of
functional groups created by deacetylation allow for side group attachment, which
then effects crystallinity which directly relates to chitosan’s ability to solubilize in
acidic aqueous solutions, which is an important aspect of chitosan’s processability.(17)
(18) (19)
Chitosan has many physicochemical (reactive OH and NH2 groups) and
biological (biocompatible, biodegradable) properties that make it an attractive material
for use in various applications. These properties include: biodegradability, lack of
toxicity, anti-fungal effects, wound healing acceleration, and immune system
stimulation.(20) (21) (22) Because of chitosan’s biological and chemical properties it has
the ability to bind to particular materials including cholesterols, fats, proteins, metal
ions, and even tumor cells. This allows chitosan to be used as a chelating agent in
various application. (23)
24
5.1-Degradation:
Chitosan can be degraded through several means and because its degradation
rate is inversely proportional to the degree of crystallinity and consequently the
amount of deacetylation, its degradation rate is able to be engineered by controlling
the amount of deacetylation that occurs during processing. At temperatures above 280
ºC thermal degradation occurs and polymer chains rapidly break down. Enzymatic
degradation is the leading means of controlling the break down of chitosan.
A wide array of hydrolytic enzymes, such as lysozyme, which is the primary
enzyme responsible for degradation of chitosan and is found in the lyphoid systems of
humans and animals, can be used to naturally degrade chitosan. Within the body the
degradation of chitosan leads to the release of aminosugars, which can be easily
processed and released through the metabolic system. Degradation is an important
property to understand so that processing and end applications can be designed
accordingly .
5.2 - Molecular Weight: Chitosan usually refers to a family of polymers that are characterized by the
number of sugar units per polymer molecule (n), which defines its molecular weight
(Dodane and Vilivalam, 1998).
The physico-chemical properties, which include viscosity, solubility, adsorption
on solids, elasticity, and tear strength, are dependant on the molecular weight of the
polymer concerned (Khan, 2001).
25
Chitosan has received much attention as a functional biopolymer for diverse
applications. These functions have been revealed to be dependent not only upon their
chemical structure but also the molecular size (Qin et al., 2003).
Crystal size and morphological character of its prepared film can be affected by
the molecular weight of chitosan. It was shown that crystallinity of membrane
increased with a decrease in chitosan molecular weight (Khan, 2001). It has been
reported that the molecular weight of chitosan products is dependant on the
deacetylation process and would decrease as the time of deacetylation increased
(Francis and Matthew, 2000). Depending on the source and preparation procedure, the
average molecular weight of chitosan may range from 50 to 1000kDa (Francis and
Matthew, 2000), 3.8 to 2000 kDa (Sinha et al., 2004) or 50 to 2000kDa (Chenite et
al., 2001).
Chitosan molecular weight distributions have been obtained using HPLC
technique . (24) In addition, the weight-average molecular weight (Mw) of chitosan has
been determined by light scattering.
(25)
Viscometry is a simple and rapid method for the determination of molecular
weight. The charged nature of chitosan in acid solvents and chitosan’s propensity to
form aggregation complexes require care when applying these constants. Furthermore,
converting chitin into chitosan lowers the molecular weight, changes the degree of
deacetylation, and thereby alters the charge distribution, which in turn influences the
agglomeration. The weight-average molecular weight of chitin is 1.03�106 to
2.5�106, but the N-deacetylation reaction reduces this to 1�105 to 5�105.
26
(26)
5.3- Solvent Properties:Chitin and chitosan degrade before melting, which is typical for
polysaccharides with extensive hydrogen bonding. This makes it necessary to dissolve
chitin and chitosan in an appropriate solvent system to impart functionality. For each
solvent system, polymer concentration, pH, counter-ion concentration and temperature
effects on the solution viscosity must be known. For example, at pH value below 4,
most of the amino groups of chitosan are supposed to be protonated, and since this
effect promotes electrostatic repelling between charged groups of the same sign, it
leads to enhanced swelling of the polymer network (Nystrom et al., 1999). While
Muzzarelli and Muzzarelli (1998) reported that at pH 5.2, an unstable structure is
generated.
As a general rule, the maximum amount of polymer is dissolved in a given
solvent towards a homogeneous solution. A coagulant is required for polymer
regeneration or solidification. The nature of the coagulant is also highly dependent on
the solvent and solution properties as well as the polymer used . (27) (28)
Water-soluble chitin, however, can be prepared by either homogeneous
deacetylation of chitin
(29)
or homogeneous N-acetylation of chitosan. (30, 31) Water
solubility is obtained only when the deacetylation degrees of chitin is about 0.5.
It should be emphasized that the water soluble chitin is obtained by
homogeneous reaction instead of heterogeneous reaction. The former treatment gives
a random copolymer of N-acetyl-Dglucosamine and Dglucosamine units, whereas the
latter one ihrandom copolymer was almost amorphous, but the block copolymer was
highly crystalline, although the degree of deacetylation of the two polymers is the
27
same. Kurita et al. concluded that the water solubility was attributed to the greatly
enhanced hydrophilicity resulting from the random distribution of acetyl groups and
the destruction of the tight crystalline structure of chitin.
5.4- Degree of Deacetylation:Chitosan is a semi-crystalline polymer and the degree of crystallinity is a
function of the degree of deacetylation. Crystallinity is maximum for both chitin (i.e.
0% deacetylated) and fully deacetylated (i.e. 100%) chitosan (Francis and Matthew,
2000). Despite those specific chemical designations, the names ‘chitin’ and ‘chitosan’
actually correspond to a family of polymers varying in the acetyl content measured by
the degree of deacetylation (DD) (Duarte et al., 2002). Chitosan is the universally
accepted non-toxic N-deacetylated derivative of chitin, where chitin is N-deacetylated
to such an extent that it becomes soluble in dilute aqueous acids (Kumar, 2000).
The process of deacetylation involves the removal of acetyl groups from the
molecular chain of chitin, leaving behind a complete amino group (-NH2). Chitosan
versatility depends mainly on this high degree of chemically reactive amino groups.
To increase the amine group content of chitosan and higher deacetylation, chitosan
(for example, DD > 90%) is subjected to repeated alkaline treatment (Wan et al.,
2003). Increasing either the temperature or strength of the alkaline solution can also
enhance the removal of acetyl groups from chitin (Khan, 2001).
An important parameter to examine closely is the degree of deacetylation in
chitosan, it is the ratio of 2-acetamido-2-deoxy-D-glucopyranose to 2-amino-2deoxy-D-glucopyranose structural units (Kumar, 2000).
28
The degree of deacetylation of chitosan, which determines the content of free
amino groups,can be employed to differentiate between chitin and chitosan. When the
number of 2-amino-2-deoxy-D-glucopyranose units is more than 50%, the biopolymer
is termed chitosan. Conversely, when the number of 2-acetamido-2-deoxyDglucopyranose units is higher, the term chitin is used (Nystrom et al., 1999;
Brugnerotto et al., 2001; Khor and Lim, 2003).
According to Kumar (2000), chitosan is the fully or partially N-deacetylated
derivative of chitin with typical degree of deacetylation of more than 0.35. While
Khan (2001) reported that chitin with DD of 75% and above is generally known as
chitosan but Montembault et al. (2005) reported that chitosan has the DD of 60% and
above.
Tommeraas et al. (2002) reported that the commercially available chitosan
usually has the range between 0 to 0.3 fraction of N-acetyl unit. This ratio has a
striking effect on the performance of chitosan in many of its applications. It has been
reported that DD has prominent roles in the biochemical significance of chitosan. The
DD of chitosan has been shown to correlate with its solubility in acidic solution and
the crystallinity of its membrane (Khan, 2001; Duarte et al., 2002).
Conversion of chitin into chitosan increases DD, and thereby alters the charge
distribution of chitosan molecules (Francis and Matthew, 2000). It is known that the
charge density along the chain increases with an increase in the DD, and that chain
flexibility of chitosan molecules can be manipulated by changing the DD (Nystrom et
al., 1999). Commercially available chitosan has degree of deacetylation ranging from
50 to 90% (Francis and Matthew, 2000), 66 to 95% (Sinha et al., 2004) or 40 to 98%
(Chenite et al., 2001).
29
5.5 - Solubility of chitosan : Chitosan is a semi-crystalline polymer, a weak base, which is insoluble in
water,alkali or aqueous solution above pH 7, and common organic solvents due to its
stable and rigid crystalline structure. Chitosan is normally polydispersed and has the
ability to dissolve in certain inorganic and organic acids such as hydrochloric acid,
phosphoric acid, lactic acid, propionic acid, succinic acid, acetic acid, tartaric acid,
citric acid and formic acid at certain pH values after prolonged stirring (Muzzarelli,
1973; Sugimoto et al., 1998; Francis and Matthew, 2000; Zong et al., 2000; Khan,
2001; Krajewska, 2004; Perez-Orozco et al., 2004; Chung et al., 2005; Qin et al.,
2006). Nitric acid could dissolve chitosan, but after dissolution, white gelatinous
precipitate would occur (Muzzarelli, 1973).
Sulphuric acid does not dissolve chitosan because it would react with chitosan
to form chitosan sulphate, which is a white crystalline solid (Muzzarelli, 1973). The
solubility of chitosan also depends on the pKa of these acids and their concentrations.
Investigation of chitosan dissolution characteristics revealed that its dissolution rate
varied according to the type of acid used (Sugimoto et al., 1998; Khan, 2001; PerezOrozco et al., 2004).
Chitosan behaves as a sphere in aqueous acetic acid solution or as an expanded
random coil in urea (Muzzarelli and Muzzarelli, 1998; Perez-Orozco et al., 2004). A
mixture of dimethylformamide and dinitrogen tetroxide at a ratio of 3:1 has been
reported to be a good solvent for chitosan (Muzzarelli, 1973; Khan, 2001).
30
Upon neutralization with an excess NaOH, the ionic strength of the solution
increases and therefore the size of the aggregates decreases due to compaction of the
macromolecular coils.
The free amino groups form intermolecular hydrogen bonds with the oxygen of
the adjacent chains. At pH value greater than 6.5, which is approximately the pKa of
the amino group in chitosan, the size of the aggregates increases and phase separation
occurs. The polymer coagulates and can be recovered as an amorphous solid
(Muzzarelli and Muzzarelli, 1998; Nystrom et al., 1999).
The uniqueness of chitosan depends on the distribution of the acetyl groups
remained along the chain but mostly depends on the free amino (-NH2) groups which
is important in forming conformational features through intra and / or intermolecular
hydrogen bonding. This makes it soluble in acidic solutions below pH of
approximately 6.5 and thereby overcoming associative forces between chains. Amino
groups make chitosan a cationic polyelectrolyte (pKa ≈ 6.5), one of the few found in
nature.
In contrast, other polysaccharides are either neutral or negatively charged. The
basicity gives chitosan singular properties: chitosan is protonated upon dissolution in
aqueous acidic medium at pH < 6.5, but when dissolved possesses high positive
charge on –NH3+ groups and the resultant soluble polysaccharide is positively
charged. As a result, it adheres to negatively charged surfaces. Chitosan aggregates
with polyanionic compounds, and chelates heavy metal ions. Both the solubility in
acidic solution and aggregation with polyanions impart chitosan with excellent gelforming properties (Brugnerotto et al., 2001; Chenite et al., 2001; Khan, 2001; Yang
et al., 2002; Krajewska, 2004; Santos et al., 2005; Qin et al., 2006).
31
Even though chitosan is known to have important functional activities, the poor
solubility of chitosan is the major limiting factor in its utilization. This interferes with
the biomedical application of chitosan, especially at the physiological pH value (7.4)
where chitosan is insoluble and ineffective as an absorption enhancer (Snyman et al.,
2002). Hence, improving the solubility of chitosan is crucial if this plentiful resource
is to be utilized across a wide pH range.
Despite this limitation, various applications of chitosan and modified chitosan
have been reported (Kubota et al., 2000; Kumar, 2000; Chen and Park, 2003; Kim and
Rajapakse, 2005).
Chitosan possesses distinct chemical and biological properties. In its linear
polyglucosamine chains of high molecular weight, chitosan has reactive primary
amino and hydroxyl groups, amenable to chemical modification and provide a
mechanism for side group attachment using a variety of mild reaction conditions
(Francis and Matthew, 2000;Krajewska, 2004).
Modification of chitosan provides a powerful means to promote new biological
activities and to modify its mechanical properties. The general effect of addition of a
side chain is to disrupt the crystal structure of the material and hence increase the
amorphous fraction. This modification generates a material with lower stiffness and
often altered solubility (Francis and Matthew, 2000).
Various studies were conducted to make water-soluble derivatives of chitosan
by chemical modification techniques, such as PEG-grafting (Ouchi et al., 1998;
Sugimoto et al., 1998; Gorochovceva and Makuska, 2004), sulfonation (Francis and
Matthew, 2000), partial N-acetylation (Kubota et al., 2000), N-acetylation (Kumar,
32
2000; Francis and Matthew, 2000), chitosan carrying phosphonic and alkyl groups
(Ramos et al., 2003), hydroxypropyl chitosan (Xie et al., 2002), branching with
oligosaccharides (Tommeraas et al., 2002), chitosan-saccharide derivatives (Yang et
al., 2002; Chung et al., 2005), O-succinyl-chitosan (Zhang et al., 2003) quaternisation
(Snyman et al., 2002) and carboxymethylation chitosan (Chen and Park, 2003).
New interest has recently emerged on partially hydrolyzed chitosan where
molecular weight of chitosan decreases which in turn makes it readily soluble in water
due to their shorter chain lengths and free amino groups in D-glucosamine units
(Ilyina et al., 1999; Qin et al., 2003). The low viscosity and greater solubility of such
chitosan at neutral pH have attracted the interest of many researchers to utilize
chitosan in its lower molecular weight form (Kim and Rajapakse, 2005).
6- It’s application:Due
to
chitosan’s
many
attractive
properties
such
as
reactivity,
biodegradability, natural origin, abundance, etc., it has many areas of application
including: waste and water treatment, medical, biotechnological areas, and
fabrications.(32)
Chitosan is a versatile biopolymer and therefore its derivatives have shown
various functional properties, which make them possible to be used in many fields
including, food, cosmetics, biomedicine, agriculture, environmental protection,
wastewater management and fibre industries (Duarte et al., 2002; Kim and Rajapakse,
2005).
33
Chitosan has been found to have an LD50 of over 16 grams/day/kg body weight
in mice (Hennen, 1996). To put these data in context, chitosan was compared to
common sugars, it appears that chitosan was less toxic than these substances. Mice are
not men.
For safety purposes, the data gathered in mice were divided by 12 to get the
human equivalent. The relative LD50 in humans then would be 1.33 grams/day/kg.
Given that an average person weighs 150 or 70 kg, this means that the toxic amount
for a person would be greater than 90 grams per day. Conservatively, one could feel
very safe with the level below 10%, or 9 grams per day. Clinical studies have used 3-6
grams per day of chitosan with no adverse effects (Hennen, 1996).
6.1-Water Treatment Applications : Chitosan’s functional groups and natural chelating properties make chitosan
useful in wastewater treatment by allowing for the binding and removal of metal ions
such as copper, lead, mercury, and uranium from wastewater. It can also be utilized to
remove dyes and other negatively charged solids from wastewater streams and
processing outlets.
Chitosan grafted with poly(acrylonitrile) has been further modified to yield
amidoximated chitosan
(33)
a derivative having a higher adsorption for Cu2+, Mn2+,
and Pb2+, compared to cross-linked chitosan. The adsorption capacity had a linear
dependence on pH in cases of Cu2+ and Pb2+. However, a slight decrease in the
adsorption capacity was observed in case of Zn2+ and Cd2+.(34)
34
Chitosan has been modified with different mono as well as disaccharides.
( 35 )
Others
have also reported the metal uptake abilities of macrocyclic diamine
derivative of chitosan. The polymer has high metal uptake abilities, and the selectivity
property for the metal ions was improved by the incorporation of azacrown ether
groups in the chitosan.
The selectivity for adsorption of metal ions on polymer was found to be
Ag+>Co2+>Cr3+. These results reveal that the new type chitosan-crown ethers will
have wide ranging applications for the separation and concentration of heavy metal
ions in environmental analysis.
In addition, a novel type cellulosebased ion exchanger, chitosan-g
carboxymethylcellulose (CS-g-CMC) (Fig. 10), has been successfully prepared by
thermal graft copolymerization for removal heavy metal ions from aqueous solutions.
The adsorption properties of the grafted copolymer relied on pH value, CS content and
reaction temperature. The high adsorption selectivity and good kinetic properties of
metal ions indicated that the novel CS-g-CMC ion exchanger could be used to remove
the heavy metal ions from aqueous solution . (36)
Cyclodextrin (CD) containing polymers, due to their ability to form host– guest
complexes, are compound of interest in many applications; from the stabilization and
the controlled release of active components in formulation to extraction and separation
processes. The chitosan grafted with �-CD derivatives have ability to form complexes
with a variety of other appropriate compounds, to develop novel sorbent materials . (37,
38 , 39 , 40)
35
Fig (9) :((Chemical structure of CS-g-CMC
biomaterials))
6.2- Medical & pharmacutical Applications : A wide variety of medical applications for chitosan and chitosan derivatives
have been reported over the last three decades (Kumar, 2000). Chitosan has been
considered for pharmaceutical formulation and drug delivery applications in which
attention has been focused on its absorption-enhancing, controlled release and
bioadhesive properties (Dodane and Vilivalam, 1998).
Indeed, chitosan is known for being biocompatible allowing its use in various
medical applications such as topical and ocular applications, implantation or injection.
Moreover, chitosan is metabolized by certain human enzymes, especially lysozyme,
and is considered as biodegradable. Due to its positive charges at physiological pH,
chitosan is also bioadhesive, which increases retention at the site of application
36
(Berger et al., 2004). Chitosan has been used extensively to prepare microspheres for
oral and intra-nasal delivery.
Chitosan polymer has also been proposed as a soluble carrier for parenteral
drug delivery (Gomez and Duncan, 1997). Chitosan is a versatile carrier for
biologically active species and drugs due to the presence of free amino groups as well
as its low toxicity. Gomez and Duncan (1997) reported that chitosan polymers when
used as soluble polymeric carriers for intravenous administration have the potential to
induce cellular toxicity. There are many studies showing that chitosan accelerates
wound healing in many clinical cases. It was reported that chitosan granules could
enhance regeneration of normal skin in open wounds. It has been suggested that
chitosan may be used to inhibit fibroplasias in wound healing and to promote tissue
growth and differentiation in tissue culture (Kumar, 2000).
Chitosan is used as raw material for man-made fibres, filament, powder,
granule, sponge, and composite with cotton or polyester in most studies. Medical
product made of chitosan is useful as absorbable sutures and wound-dressing
materials. It appears that chitosan, having structural characteristics similar to
glycosamino glycans and could be considered for developing such substratum for skin
replacement (Kumar, 2000; Kweon et al., 2003). Chitosan could also inhibit the
growth of tumor cells by exerting immuno-enhancing effects. Results of some related
studies suggested that, the observed antitumor activities were not due to direct killing
of tumor cells, but might be due to increased production of lymphocytes, leading to
manifestation of antitumor effect through proliferation of cytolytic T-lymphocytes
(Qin et al., 2002; Kim and Rajapakse, 2005).
37
Chitosan has also been shown to have antacid and antiulcer activities, which
prevent or weaken drug irritation in the stomach. The anti-ulcer activity is due to its
capacity to bind free gastric acid and to a significant ability to act as demulcent. Also,
chitosan matrix formulations appear to float and gradually swell in an acidic medium
(Muzzarelli, 1973; Kumar, 2000; Falk et al., 2004).
Due to chitosan’s ability to function in many forms it has many areas of interest
within the medical industry including: orthopedic, tissue engineering, wound healing,
drug delivery, and surgical adhesion ,etc.
6.2.1-Orthopedics:Chitosan’s functional groups allow it to interact with many materials, which
allow it to be used in conjunction with materials such as hydroxyapatite, or other
calciumbased minerals to form composites that have multiple applications within the
orthopedic and periodontal industries. These calcium-chitosan composites can be used
as a coating in conjunction with joint prostheses. As the chitosan is degraded, new
bone can be deposited adjacent to the prosthesis to stabilize the implant within bone.
An additional use for chitosan in orthopedics includes a direct replacement of bone or
hard tissue. It is also a natural bioadhesive used to improve bone cement which is used
to secure implants as well as to fill bone cavities .
6.2.2-Tissue Engineering:
Chitosan’s ability to be manufactured in many forms such as fibers, filaments,
films, sponges, gels, and composites make it easily engineered for particular end
applications or for use within a particular area of the body or in conjunction with a
38
certain tissue. In this respect, there are three majors to be considered for the success of
tissue regeneration: cells, scaffold, and cell scaffold interaction shown in Fig(11).
Fig(10) : cell scaffold interaction
Chitosan can be used to make three-dimensional scaffolds that act as an
artificial extracellular matrix, which can be resorbed by the body over time as new
tissue is formed and a natural extracellular matrix is formed helping to further
integrate new tissue into the body. In addition, chitosan is used in this application due
to its biocompatibility, ability to have an engineered degradation rate, antimicrobial
activity, ability to interact with other materials to form composites, and its ability to
interact with and encourage cellular attachment and growth . ( 41 , 42 , 43 )
39
Its mechanical properties can be enhanced or reduced to closely resemble the
properties of the tissue it is replacing; for example it can be made to support hard
tissues such as bones or cartilage or soft tissues such as muscles and blood vessels. It
also has the ability to attract glycosaminoglycans which enables chitosan to collect
growth factors which enhances cell attachment and proliferation .
6.2.3- Wound Healing :Chitosan enhances the functions of cells that immerge during the inflammatory
response, while accelerating the migration of these cells to the injured area .
(44, 45)
These cells such as macrophages kill microorganisms, remove dead cells, and
stimulate the other immune system cells, which improve overall healing by reducing
the opportunity for infection. Chitosan’s positive charge allows for electrostatic
interactions with glycosaminoglycans, which attract growth factors that enhance cell
growth.
Its cationic nature also allows it to associate with anions that are connected
with the bacterial cell wall, which retards the bacteria’s ability synthesize .Several
chitosan’s possible material forms can be used independently, such as hydrogels,
while others can be used in conjunction with traditional bandages to provide a wound
protection from the outside elements, while maintaining a moist environment that
promotes healthy healing.
A bandage material that does not require removal due to its ability to safely
biodegrade within the body is an additional potential use that makes chitosan an
appealing wound healing material choice .
40
6.2.4 - Drug Delivery : The use of polymers such as chitosan to deliver drugs to their appropriate
location within a biological system is an area of great interest. Chitosan is able to be
degraded within a biological system over time, and furthermore that degradation rate
is easily engineered based on the amount of deacetylation that occurs during
processing. This allows drugs to be released into the body in a controlled manor to be
as effective as possible. The free amine group that gives chitosan a positive charge is
imperative to drug delivery for it is this charge that permits it to interact with
negatively charged drugs, polymers, and bioactive molecules. This is also the feature
that enables chitosan to adhere to mucous membranes making it especially useful for
drug delivery via the respiratory system . (46)
Its ability to be used in various forms such as gels, copolymers, etc. is another
characteristic that makes chitosan an attractive material for drug transport. It can form
colloidal particles and entrap negatively charged molecules through several means
such as chemical and ionic crosslinking. Chitosan’s versatility along with its other
biological properties including biocompatibility begets a material well suited for drug
delivery . (47)
6.2.5 - Surgical Adhesion :Biological adhesives are used for tissue adhesion, hemostasis, and sealing of the
leakage of air and body fluids during surgical procedures. An adhesion is the
formation of fibrous tissue that causes internal organs to be bound together in an
unnatural fashion. These adhesions often occur during pelvic, abdominal or
gynecological surgeries such as hysterectomies, cesarean sections, colectomies, and
hernia repairs .
(48)
41
After these procedures are completed and the body is attempting to heal its self
through normal wound healing responses, swelling occur causing organs to be in
closer proximity to one another than under normal internal conditions. Another
component of natural wound healing is for the body to deposit fibrin to help repair
damaged or injured tissues.
This type of tissue formation can lead to infertility when adhesions twist ovaries
and or tubes resulting in the blocking of the egg to the uterus. A photocrosslinkable
chitosan to which both azide and lactose moieties were introduced (Az-CH-LA) was
prepared as a biological adhesive for soft tissues and its effectiveness was compared
with that of fibrin glue .
(49)
A cytocompatible chitosan solution that is space-filling,
gels within minutes, and adheres to cartilage and bone in situ was developed . (50)
6.2.6- Hemostatic agent:
Chitosan's properties allow it to rapidly clot blood, and has recently gained
approval in the United States and Europe for use in bandages and other hemostatic
agents. Chitosan hemostatic products have been shown in testing by the U.S. Marine
Corps to quickly stop bleeding and to reduce blood loss, and result in 100% survival
of otherwise lethal arterial wounds in swine. (51)
Chitosan hemostatic products reduce blood loss in comparison to gauze
dressings and increase patient survival.
(52)
Chitosan hemostatic products have been
sold to the U.S. Army and are currently used by the UK military. Both the US and UK
have already used the bandages on the battlefields of Iraq and Afghanistan.
Chitosan is hypoallergenic and has natural antibacterial properties, which
further support its use in field bandages.( 53 ) Chitosan hemostatic agents are often
42
chitosan salts made from mixing chitosan with an organic acid (such as succinic or
lactic acid).
( 54 )
The hemostatic agent works by an interaction between the cell
membrane of erythrocytes (negative charge) and the protonated chitosan (positive
charge) leading to involvement of platelets and rapid thrombus formation. (55)
The chitosan salts can be mixed with other materials to make them more
absorbent (such as mixing with alginate), or to vary the rate of solubility and
bioabsorbability of the chitosan salt.
The chitosan salts are biocompatible and biodegradable making them useful as
absorbable haemostats. The protonated chitosan is broken down by lysozyme in the
body to glucosamine and the conjugate base of the acid (such as lactate or succinate)
are substances naturally found in the body. The chitosan salt may be placed on an
absorbable backing. The absorbable backing may be synthetic (for instance made
from existing absorbable suture materials e.g. Tephaflex polymer) or natural (e.g.
cellulose or gelled/solidified honey).
6.2.7 – other bio-medical applications: It also inhibits LDL cholesterol and boosts HDL cholesterol.
 reduces blood levels of uric acid.
 helps prevent constipation.
 acts as an antacid.
 helps prevent irritable bowel syndrome.
 enhances calcium to strengthen bones.
 inhibits plaque/tooth decay.
 offers anti-tumor action.
 Weight loss.
43

may be helpful in kidney failure.
 may inhibit the expected rise in blood pressure after a high-salt meal.
 stimulate the immune system and prevent cancer.
6.3 - Biotechnological Applications:-
6.3.1-Cell-Stimulater :Soyabeans were coated with a thin layer of depolymerized chitin,
carboxymethyl (CM)-chitin and hydroxyethyl (HE)-chitin, and the seeds were
cultured in the field. It was observed that the seed chitinase increased 1.5– 2.0-fold,
the seed germination rate increased by 6%, the pod number increased by 9%, the plant
dry weight increased by 8%, and the crop yield also increased by 10–12% over the
control. (56)
Dressing with chitin films, sponges and fibres enhanced chitinase activity in
tree-bark tissues around wounds up to four-fold over the control. The chitin films,
which were implanted in or used to dress the tree-bark tissues, were digested within 4
to 24 weeks thereafter.
The fate of N-acetyl-D-glucosamine in plant tissue is unknown. Phenylalanine
ammonia-lyase was stimulated by treatment with chitin, and lignin formation in the
plant increased. As a result, wound healing was increased. (57)
6.3.2 - Fat-Net : Many supplements can help in the fat reduction process, including pyruvate and
chitosan. Pyruvate, found in red apples, some types of cheese, and red wine,
stimulates fat loss and boosts exercise performance.
44
Chitosan attaches itself to fat in the stomach before it is digested, thus trapping
the fat and preventing its absorption by the digestive tract. Fat in turn binds to the
chitosan fibre, forming a mass which the body cannot absorb, and which is eliminated
by the body. In some sort, it creates a "grease ball" from this excess fat, which is too
large to be absorbed by the body. It thus becomes an inert substance and is excreted
in the stool. Besides from the obvious effect that fat is not absorbed into the body
with the presence of chitosan, it goes a lot deeper in benefiting weight loss.
Chitosan is a 100% natural and acts as a super fiber. Thus, supplementing the
diet with chitosan, is part of creating a cleansing process which is said to be extremely
vital to weight loss. Take note that these are again the simple declarations made by the
producers of chitosan and are supported with no background studies or thus medical
proof of any sort. Another stated advantage of chitosan comes from the idea that the
chitosan-bound
fat leaves
the intestinal
tract
without ever entering
the
bloodstream. Exactly how this process takes place is not clear and seems to be
somewhat far fetched. If this indeed is possible, then the point is made that there
would be no caloric value and no matter how much chitosan a person takes, the caloric
count remains zero.
The producers of chitosan-based products also try to claim that since a person
taking chitosan continues to eat some sort of fats and is able to continue eating these
types of food, the body does not crave such fattening foods nor is it starving or feeling
any added sense of hunger. By supplementing chitosan into one's diet, there is less fat
that the body accumulates. With less fat entering the body, the body turns to
previously stored body fat to burn up. This shifts the energy source from your diet to
your stored body fat and results in a net reduction in that fat - and in your weight.
45
Obviously, the allegations are extremely pleasing, now whether or not they
actually could be a reality is another side to these claims. Chitosan fiber differs from
other fibers in that it possesses a positive ionic charge, which gives it the ability to
bond chemically with the negatively charged lipids, fats and bile acids .
(58 , 59 )
** Chitosan is currently under research for several potential
clinical applications:

As a soluble dietary fiber, it increases gastrointestinal lumen viscosity and
slows down the emptying of the stomach.

It alters bile acid composition, increasing the excretion of sterols and reducing
the digestibility of ileal fats .It is unclear how chitosan does this, but the
currently favored hypotheses involve the increase of intestinal viscosity or bile
acid-binding capacity.

Chitosan is relatively insoluble in water, but can be dissolved by dilute acids,
which would make it a highly-viscous dietary fiber. Such fibers might inhibit
the uptake of dietary lipids by increasing the thickness of the boundary layer of
the intestinal lumen, which has been observed in animal experiments.

Having very few acetyl groups, chitosan contains cationic groups. This may
cause chitosan to have bile acid-binding capacity, which causes mixed micelles
to be entrapped or disintegrated in the duodenum and ileum. This would
interrupt bile acid circulation, causing reduced lipid absorption and increased
sterol excretion, which has also been observed in animal experiments.

Increase
The
benefits
of
chitosan
in
weight
The benefits of chitosan in weight loss can be greatly boosted by the simple
additions to go along with the chitosan supplements. First, M.D. Labs have
followed current research and clinical studies and have determined that by
adding Vitamin C, one can enhance the absorption of lipids. The producers
46
claim that increased appetite suppression is obtained by the addition of citric
acid, which boosts the swelling action of chitosan. They claim that this addition
could so much as double the effectiveness of chitosan. (Your Health Store Chitosan) Thus, many of the products on the market today which contain
chitosan as a chief substance, also include the addition of some sort of vitamin
C supplement. One such product is the Fat Zapper, which contains 250 mg of
chitosan, 240 mg of high quality grapefruit fiber, and 10 mg of vitamin C per
capsule.
Administering Chitosan :The instructions in which to properly administer the supplementation of
chitosan are quite simple. The most common advice is to follow some sort of simple
plan such as the one that follows: Take one or two capsules ten minutes before each
of your three daily meals.
Here the chitosan will supposedly help bind excess fat from the meal or snack
which has just been consumed. It also serves as an added fiber intake, which aides in
digestion, soothing the stomach lining and speeding up the process of elimination of
undigested fats and wastes.
Also, drink about six to eight glasses of water daily. This is common in any
situation where there is an addition of fiber or a need for faster digestion. Also, it is
extremely advisable to include moderate exercise.
Simple exercises which contain any type of sustained aerobic activity can
greatly enhance the results and the time in which physical results will appear. These
steps are fairly easy and create a setting in which one's body, with the added chitosan,
can actively work to reduce the amount of body fat.
47
6.3.3 -Potential industrial use: Scientists have recently developed a polyurethane coating that heals its own
scratches when exposed to sunlight, offering the promise of scratch-free cars and other
products. The self-healing coating uses chitosan incorporated into traditional polymer
materials, such as those used in coatings on cars, to protect paint. When a scratch
damages the chemical structure, the chitosan responds to ultraviolet light by forming
chemical chains that begin bonding with other materials in the substance, eventually
smoothing the scratch. The process can take less than an hour.
Marek W. Urban, a scientist working on this project, said the polymer can only
repair itself in the same spot once, and would not work after repeated scratches.
Whether this technology can be applied to industrial materials, however, depends on a
number of factors (long-term persistence of "healability", stiffness and heat resistance
of coating, knowledge of the exact mechanism of healing, etc.) not present initial
studies; further investigation into these factors can potentially take decades to rectify.
6.4 - Agricultural & Horticultural use : Chitosan is used primarily as a natural seed treatment and plant growth
enhancer, and as a substance that boosts the ability of plants to defend against fungal
infections..
Chitosan is effective for shelf-life extension of fruits, vegetables, meat and fish
products. As a viscous soluble dietary fiber, it can also be used in functional food
products. The main applications for chitosan in foods today are as a natural antimicrobial agent and as an edible food coating agent.
48
Both chitin and chitosan have demonstrated antiviral, antibacterial, and
antifungal properties, and have been explored for many agricultural uses. They have
been utilized to control disease or reduce their spread, to chelate nutrient and minerals,
preventing pathogens from accessing them, or to enhance plant innate defenses.
When used to enhance plant defenses, chitin and chitosan induce host defense
responses in both monocotyledons and dicotyledons. These responses include
lignifications
( 60 )
, ion flux variations, cytoplasmic acidification, membrane
depolarization and protein phosphorylation
,phytoalexin biosynthesis
(63)
(61)
, chitinase and glucanase activation
(62)
, generation of reactive oxygen species, biosynthesis of
jasmonic acid (64), and the expression of unique early responsive and defense-related
genes .
In addition, chitosan was reported to induce callose formation
(65)
, proteinase
inhibitors, and phytoalexin biosynthesis in many dicot species. The response to chitin,
chitosan, and derived oligosaccharides varies with their acetylation degree.
6.4.1 - Applied as seed coating agents : Guan et al.
(66)
examined the use of chitosan to prime maize seeds. Although
chitosan had no significant effect on germination under low temperatures, it enhanced
germination index, reduced the mean germination time, and increased shoot height,
root length, and shoot and root dry weights in two tested maize lines. In both tested
lines, chitosan induced a decline in malonyldialdehyde content, altered the relative
permeability of the plasma membrane and increased the concentrations of soluble
sugars and proline, and of peroxidase and catalase activities.
49
In other studies, seed priming with chitosan improved the vigor of maize
seedlings . (67) It was also reported to increase wheat seed resistance to certain diseases
and improve their quality and/or their ability to germinate .
Similarly, peanut seeds soaked in chitosan were reported to exhibit an increased
rate of germination and energy, lipase activity, and gibberellic acid and indole acetic
acid levels .
(68)
Ruan and Xue
(69)
showed that rice seed coating with chitosan may
accelerate their germination and improve their tolerance to stress conditions. In carrot,
seed coating helps restrain further development of Sclerotinia rot. Chitosan has also
been extensively utilized as a seed treatment to control F. oxysporum in many host
species . (70)
6.4.2- Applied as foliar treatment agents: Foliar application of chitosan has been reported in many systems and for
several purposes. For instance, foliar application of a chitosan pentamer affected the
net photosynthetic rate of soybean and maize one day after application .
(71)
This
correlated with increases in stomatal conductance and transpiration rate.
Chitosan foliar application did not have any effect on the intercellular CO 2
concentration. the observed effect on the net photosynthetic rate is, in general,
common in maize and soybean after foliar application of high molecular weight
chitosan. Foliar applications of these oligomers did not, on the other hand, affect
maize or soybean height, root length, leaf area, or total dry mass.
Bittelli et al. suggested that chitosan might be an effective anti-transpiring to
preserve water resources use in agriculture. In their investigation, they examined the
potential of foliar applications of chitosan on pepper plants transpiration in the growth
room and in the field. In both experiments, the authors monitored plant water use
50
directly and indirectly. The plant biomass and yield were determined to calculate
biomass-to-water ratios and the differences in canopy resistance between control and
chitosan-treated plants were analyzed. Using scanning electron microscopy and
histochemical analyses, stomata were shown to close in response to treatment with
chitosan, resulting in a decrease in transpiration. Reduced water use of pepper plants
upon treatment with chitosan was estimated at 26–43%, while there was no change in
biomass production or yield . (72)
Iriti et al.
(73)
unveiled some of the aspects through which chitosan was able to
reduce transpiration in bean plants after being used as a foliar spray. The authors
showed that this activity was likely occurring thanks to the increase in abscisic acid
(ABA) content in the treated leaves. Using scanning electron microscopy and other
histocytochemistry techniques, the authors showed that upon treatment and increase in
ABA content, a partial stomatal closure occurred and led, among others, to a decrease
in conductance for water vapor and in the over all transpiration rate. Interestingly, the
authors revealed a new chitosan anti-transpirant mechanism in bean plants that was
not described by their commercial supplier Vapor Gard ®, and in which a formation of
a thin anti-transpirant film at the surface of the leaves was much more efficient than
stomatal closure.
This difference in mechanisms also suggested an important consideration for
the environmental conditions under which chitosan is applied as shown by the authors
but may also depends on the intrinsic properties of the tested plant species.
Chitosan has also been extensively utilized as a foliar treatment to control the
growth, spread and development of many diseases involving viruses, bacteria, fungi
and pests .It has also been used to increase yield and tuber quality of micropropagated greenhouse-grown potatoes .
51
Similarly, Faoro et al.
(74)
showed that the use of chitosan applied as a foliar
spray on barley reduced locally and systemically the infection by powdery mildew
pathogen Blumeria graminis f. sp. hordei.
6.4.3 - Applied as soil amendment: Chitosan utilized as a soil amendment was shown to control Fusarium wilts in
many plant species . Applied at an optimal concentration, this biomaterial is able to
induce a delay in disease development, leading to a reduced plant wilting .Similar
results were reported in forest nurseries suffering from F. acuminatum and
Cylindrocladium floridanum infections. These infections were dramatically reduced
upon the use of chitosan as soil amendment .
Aspergillus flavus was also completely inhibited in field-grown corn and peanut
after soil treatment with chitosan .Part of the effect observed by chitosan on the
reduction of soilborne pathogens comes from the fact that it enhances plant defense
responses. The other part is linked to the fact that this biopolymer is composed of
polysaccharides that stimulate the activity of beneficial microorganisms in the soil
such as Bacillus, fluorescent Pseudomonas, actinomycetes, mycorrhiza and
rhizobacteria .(
75 )
This alters the microbial equilibrium in the rhizosphere
disadvantaging plant pathogens. Beneficial organisms, on the other hand, are able to
outcompete them through mechanisms such as parasitism, antibiosis, and induced
resistance . (76)
52
6.5- Applications of chitosan derivatives: 6.5.1- Application of glycol chitosan for
entrapment of protein molecules:Glycol chitosan (GC) is a commercially-available derivative of chitosan that
exhibits complete solubility in water at any pH. It was chosen as a facilitator to
immobilise bovine serum albumin (BSA) on the surface of iron oxide magnetic
nanoparticles using layer-by-layer deposition approach.
(77)
An alternate deposition of
GC and BSA on the surface of magnetic nanoparticles from aqueous solutions (pH
7.4) resulted in formation of multilayered nano-coatings. It was established that the
structure and biological activity of BSA deposited on the surface of magnetic
nanoparticles remains unaltered.
6.5.2-Amphiphilic derivatives of glycol
chitosan: GC was also modified chemically to yield amphiphilic polymers capable to
self-assemble in aqueous solutions and enhance aqueous solubility of poorly soluble
drugs.
6.5.3 - Glycol chitosan-coated MRI(MagneticResonance-Imaging) agent safer effective in
detecting breast cancer: By using glycol chitosan - a sugar-based polymer that reacts to acids - the
engineers allowed the nanocarriers to remain neutral when near healthy tissue, but to
53
become ionized in low pH. The change in charge that occurs in the vicinity of acidic
tumors causes the nanocarriers to be attracted to and retained at those sites.
This approach has another benefit: the more malignant a tumor is, the more it
disrupts surrounding blood vessels and the more acidic its environment becomes. This
means that the glycol chitosan-coated is a good detector of malignancy, opening up
treatment options above and beyond diagnosis
6.6 – Cosmetics :Chitosan has different molecular weights and degree of deacetylation, which is
tailored for use in different cosmetic fields such as skin care, deodorants and hair care.
Chitosan has been noted for its application as a film-forming agent and
hydrating agent in cosmetics in view of its durable moisturizing effect on the skin. By
reducing the trans-epidermal water loss, it increases water-binding capacity and skin
moisture. It also improves the sensorial parameters and the dermatological
compatibility of formulations (Dodane and Vilivalam, 1998; Muzzarelli and
Muzzarelli, 1998; Klingels et al., 1999).
The film-forming ability of chitosan assists in imparting a pleasant feeling of
smoothness to the skin and in protecting it from adverse environmental conditions and
consequences of the use of detergents.
Chitosan was found to be superior to hyaluronic acid as far as lasting hydrating
effects are concerned (Muzzarelli and Muzzarelli, 1998).Klingels et al. (1999)
reported that chitosan is a multifunctional active ingredient for the skin with additional
advantages in sun protection and lip care. Sunscreen products must care for the skin to
54
prevent drying out and should exhibit prolonged water-resistance. Complete water
resistance is not achievable.
However, water resistance can be increased by addition of hydrophobic waxes /
oils, film formers or cationic polymers. For this reason, Klingels et al. (1999)
conducted a test to determine whether chitosan as a cationic polymer could also
increase the water resistance of UV filters in a sunscreen formulation. It was found
that chitosan significantly increased water resistance. The chitosan film improved the
adhesion of the UV filters and thus protected them against washing off. The protection
provided by the chitosan containing formulation was thus correspondingly enhanced
(Klingels et al., 1999).
Lipstick is one of the most widely used decorative cosmetics. Lip-care sticks
are also used by many consumers. The main components of lipstick are waxes and
oils. However, lip-care ingredients have also been increasingly used such as UV
filters, and lipophilic agents (allantoin, bisabolol and vitamin E). Hydrophilic
substances, such as moisturizers, are rarely used because the stick compound itself is
generally occlusive and retains moisture. Lip-care sticks are mainly used to treat
brittle, chapped lips.
Chitosan activates various skin cell types and acts on the wound-healing
process. Klingels et al. (1999) obtained results from a test with fibroblasts which
revealed positive effects of chitosan with regards to improved cell adhesion. These
properties support the use of chitosan as an active lip-care ingredient and not just to
protect the lips against drying out. The addition of chitosan makes lips softer and also
supports longterm colour adhesion (Klingels et al., 1999) Klingels et al. (1999)
55
reported that chitosan is also suitable as a “deo-active” component in deodorants and
as a styling polymer in hair cosmetics.
With its antimicrobial properties (Klingels et al., 1999; Kumar, 2000), it may be
an added advantage to use chitosan in deodorants since it inhibits the activity of
enzyme-producing bacteria. It is suitable for maintaining the spray ability of
deodorant containing chitosan and has the antimicrobial effects at the same time.
Various tests were carried out to assess the effectiveness of chitosan against odourproducing bacteria.
A study was conducted to provide information on the compatibility and
sensorial properties of a formulation and not just on its deodorizing effect. The
deodorizing effect and skin compatibility of the chitosan formulation were judged to
be better when compared with triclosan.
This result can be explained by the additional effects of chitosan, such as
improved skin compatibility. In another comparative study, the fragrance adhesion
and intensity of perfume oil in a brand-name deodorant formulation with and without
added chitosan were assessed by a perfume expert and an expert laboratory panel.
The chitosan containing formulation was rated much more highly by both
groups. Chitosan retains the perfume fragrance for a longer period and with greater
intensity and at the same time masks the odour of perspiration over a longer period of
time.
56
Chitosan can be used as a sole deodorizing componentand may also be
combined with other commercially available deodorizing agents (Klingels et al.,
1999).
Chitosan is the only natural cationic gum that becomes viscous on being
neutralized with acid. These materials are used in creams, lotions and permanent
waving lotions and nail lacquers (Kumar, 2000; Krajewska, 2004).
Chitosan has been suggested as emulsifiers in cosmetics and pharmaceuticals.
Modifying chitosan by introducing the phosphoric and alkyl groups onto its structure
resulted in the presence of hydrophobic and hydrophilic group that controls solubility
properties.
In many cases, emulsion stabilization is achieved by the addition of specially
designed polymers, which have hydrophilic and hydrophobic segments. For this
reason, chitosan has been suggested as an emulsifier because it is absorbed at the
interfacial surface, thus stabilizing the emulsion (Ramos et al., 2003).
57
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