firstly, thanks to - m5zn web viewdeeply grateful to my supervisor, " dr- ayman salah el-deen...

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

Im 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

2

Also Id like to thank Dr-Ibrahim Mohey head chemistry department Port said university for his contributionsI 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 fianceacute and friends Their contributions were very essential to me hellip

Contents - PageNo1 List of figures helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 5

2 Abbreviations helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 6

3 Introduction helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 78

4 chemistry of chitosan helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 911

5 Production of chitosan helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 1122

6 History of chitosan helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 2223

7 Properties of Chitosan helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 2324

8 Degradation helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 24253

9 Molecular Weight helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 2526

10 Solvent Propertieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 2627

11 Degree of Deacetylation helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 2729

12 Solubility of chitosan helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 2933

13 Itrsquos application helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 3334

14 Water Treatment Applications helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 3436

15 Medical amp pharmacutical Applications helliphelliphelliphelliphelliphelliphellip 3638

16 Orthopedics helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 38

17 Tissue Engineering helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 3840

18 Wound Healing helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 40

19 Drug Deliveryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 4041

20 Surgical Adhesionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 4142

21 Hemostatic Agent helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 4243

22 other bio-medical applications helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 43

23 Biotech nological Applications helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 44

24 Cell-Stimulater helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 44

25 Fat-Nethelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 4445

26 several potential clinical applications helliphelliphelliphelliphelliphelliphellip 4647

27 Administering Chitosan helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 47

4

28 Potential industrial use helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 48

29 Agricultural amp Horticultural use helliphelliphelliphelliphelliphelliphelliphelliphellip 4849

30 Applied as seed coating agents helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 4950

31 Applied as foliar treatment agents helliphelliphelliphelliphelliphelliphelliphelliphellip 5052

32 Applied as soil amendment helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 52

33Applications of chitosan derivatives helliphelliphelliphelliphelliphelliphelliphellip 52

34 Application of glycol chitosan for entrapment of protein

moleculeshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 5253

35 Amphiphilic derivatives of glycol chitosan helliphelliphelliphellip 53

36 Glycol chitosan-coated MRI(Magnetic-Resonance-Imaging) agent

safer effective in detecting breast cancerhelliphellip 53-54

37 Cosmetics helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 5457

38 Referenceshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 5864

List of figures PageNo Fig (1) chemical structure of chitosan helliphelliphelliphelliphelliphelliphelliphelliphellip 9

Fig (2) Deactylation of chitin to chitosan helliphelliphelliphelliphelliphelliphelliphellip 9

Fig(3) chitin amp chitosan manufacturing process helliphelliphelliphellip 11

Fig(4) preparation of chitin amp chitosan helliphelliphelliphelliphelliphelliphelliphelliphellip 15

5

Fig(5) Fungus Gongronella butleri USDB 0201 was grown on sweet

potato pieces in a tray-type solid substrate

fermentorhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip

17

Fig(6) Extraction of chitosan from mycelia of fungus Gbutleri

grown in solid substrate

helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip

18

Fig(7) Extraction of chitosan and glucan from the AIM suspended

in 035M acetic acid by treatment with Termamyl Type LS (Nwe amp

Stevens 2002 and Nweetal2008) helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 21

Fig (8) types of methods of extraction of chitosan

from fungi helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip

22

Fig (9) Chemical structure of CS-g-CMC biomaterials helliphellip 36

Fig(10) cell scaffold interaction helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip 39

6

Abbreviations -

(SSF) Solid Substrate Fermentation

(SMF) Submerged Fermentation

(MW) Molecular Weight

(HPLC) High Performance Liqud Chromatography

(DD) 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

7

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 ndash as starch collagen gelatin alginate cellulose and chitin ndash 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 Consequentlythe inherent characteristics of chitosan make it exploitable

directly for itself Chitosan a natural and linear biopolyaminosaccharide has received

8

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 chaingenerally 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

9

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(D-glucosamine) 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 Lim2003) 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 chitosan10

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 al2002

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 (689) compared to synthetically substituted

cellulose (125) (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

11

However these naturally abundant materials also exhibit a limitation in their

reactivity and processability Chitosan is recommended as suitable functional

material because this 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 crabsprawns 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 Krajewska2004 Sinha et

al 2004 Qin et al 2006) (7)

3 - Production of Chitosan-

12

Fig(3)chitin amp chitosan manufacturing process 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 melanogasterExtatosoma 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) (Carlberg1982 Nemtsev et al 2004 Veronico et al

2001 Paulino et al 2006 Moussian et al 2005Tauber 2005 Hild et al

2008 Anantaraman amp Ravindranath 1976 Pochanavanich ampSuntornsuk 2002

Mario et al 2008 Yen amp 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

13

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 as nickel copper (Tan et al 1996 Arcidiacono amp

Kaplan 1992 (10) Nwe amp Stevens 2002a) Moreover the production of chitosan

from fungal mycelia give medium-low molecular weight chitosans (1ndash12 times 104

Da) whereas the molecular weight of chitosans obtained from crustacean

sources is high (about 15 times106Da) (Nwe amp 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)

14

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 120ordmC for 1- 3 hours with

exclusion of oxygen and followed by purification procedures to form chitosan

with a cationic nature The alkali removed 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

15

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 63 kg of HCl and 18 kg of NaOH are required in

addition to nitrogen water (14 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 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

16

Fig(4) preparation of chitin amp chitosan

17

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 Gbutleri 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 618 gkg was

higher than that in submerged fermentation 012 gl 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 (37 g chitosankg of solid substrate) was higher

than that in SMF (06 g chitosanL of fermentation medium) due to the low amount of

mycelia produced in SMF (Nwe et al2002) 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)

18

Fig 5 Fungus Gongronella butleri USDB 0201 was grown on sweet potato pieces in a tray-

type 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 035 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 g100 g of mycelia An effective chitosan extraction procedure

is essential for an economical production of fungal chitosan) Most methods used

19

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 Gbutleri grown in solid substrate

chitinchitosan 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 1990Wessels 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 chitinchitosanndashglucan complex chains results in a rigid

cross-linked network in the cell wall (Gooday 1995 Robson 1999) and causes a

considerable problem for the extraction of intact chitosan and glucan It does not break

down easily under mild extraction condition (Muzzarelli et al 1980) 20

Under the above mentioned conditions only free chitosan that is chitosan

unbounded to other cell wall components is extracted (Nwe amp 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 90of β-glucan obtained from the chitin-

glucan complex by digestion with (1- 3)-β-glucanase and N-acetyl-glucosamine

lysine andor 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 (13)-

β- and (16)-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

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 inter-

polymer linkages in the chitin-glucan complex Up to 1992 no cleare evidence of the

21

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 1SDS and 1 M NaOH

respectively and the insoluble fractions were digested with (13)-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-(13)-glucan chain by a β-14 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 (ie freechitosan plus chitosan bounded to glucan)

In 1994 Muzzarelli et al reported that the chitosan-glucan complex can be split by

25 NaOH

22

Fig7Extraction of chitosan and glucan from the AIM suspended in 035M acetic acid by

treatment with Termamyl Type LS (Nwe amp Stevens 2002 and Nwe et al 2008) (15)

23

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

mushroomshe 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

experiments on chitin Rouget observed that the compound of chitin could be

24

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)

5 - Properties of Chitosan -

25

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

Chitosanrsquos 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 chitosanrsquos ability to solubilize in

acidic aqueous solutions which is an important aspect of chitosanrsquos 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 chitosanrsquos 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)

51-Degradation Chitosan can be degraded through several means and because its degradation

rate is inversely proportional to the degree of crystallinity and consequently the

26

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

ordmC 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

52 - 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)

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)

27

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) 38 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 chitosanrsquos 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 1031048577106 to

251048577106 but the N-deacetylation reaction reduces this to 11048577105 to 51048577105 (26)

53- Solvent Properties-Chitin and chitosan degrade before melting which is typical for

polysaccharides with extensive hydrogen bonding This makes it necessary to dissolve

28

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 52 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 05

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

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

29

54- 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 (ie

0 deacetylated) and fully deacetylated (ie 100) chitosan (Francis and Matthew

2000) Despite those specific chemical designations the names lsquochitinrsquo and lsquochitosanrsquo

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 gt 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-2-

deoxy-D-glucopyranose structural units (Kumar 2000)

30

The degree of deacetylation of chitosan which determines the content of free

amino groupscan 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-deoxy-

Dglucopyranose 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 035 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 03 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)

31

55 - Solubility of chitosan -

Chitosan is a semi-crystalline polymer a weak base which is insoluble in

wateralkali 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 Perez-

Orozco 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 31 has been

reported to be a good solvent for chitosan (Muzzarelli 1973 Khan 2001)

32

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 65 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 65 and thereby overcoming associative forces between chains Amino

groups make chitosan a cationic polyelectrolyte (pKa asymp 65) 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 lt 65 but when dissolved possesses high positive

charge on ndashNH3+ 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 gel-

33

forming 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)

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 (74)

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 2000Krajewska 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)

34

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

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 rsquos application- Due to chitosanrsquos 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

35

wastewater management and fibre industries (Duarte et al 2002 Kim and Rajapakse

2005)

Chitosan has been found to have an LD50 of over 16 gramsdaykg 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 133 gramsdaykg

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)

61-Water Treatment Applications - Chitosanrsquos 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)

36

Chitosan has been modified with different mono as well as disaccharides

Others (35) 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+gtCo2+gtCr3+ 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 hostndash 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 1048577-CD derivatives have ability to form complexes

with a variety of other appropriate compounds to develop novel sorbent materials (37

38 39 40)

37

Fig (9) ((Chemical structure of CS-g-CMC biomaterials))

62- Medical amp 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

38

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

(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)

39

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 chitosanrsquos 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

621-Orthopedics-Chitosanrsquos 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

622-Tissue Engineering Chitosanrsquos 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

40

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 interactionChitosan 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 )

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

41

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

623- 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 Chitosanrsquos 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 bacteriarsquos ability synthesize Several

chitosanrsquos 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

624 - Drug Delivery -

42

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 Chitosanrsquos versatility along with its other

biological properties including biocompatibility begets a material well suited for drug

delivery (47)

625 - 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)

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

43

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 photo cross linkable

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)

626- Hemostatic agent

Chitosans 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 US 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 US 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

chitosan salts made from mixing chitosan with an organic acid (such as succinic or

44

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 eg Tephaflex polymer) or natural (eg

cellulose or gelledsolidified honey)

627 ndash 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 plaquetooth decay

offers anti-tumor action

Weight loss

may be helpful in kidney failure

45

may inhibit the expected rise in blood pressure after a high-salt meal

stimulate the immune system and prevent cancer

63 - Biotechnological Applications - 631-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 15ndash 20-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 10ndash12 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)

632 - 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

46

stimulates fat loss and boosts exercise performance

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 ones diet there is less fat

that the body accumulates With less fat entering the body the body turns to

47

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

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

48

additions to go along with the chitosan supplements First MD 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

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

49

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 ones body with the added chitosan

can actively work to reduce the amount of body fat

633 -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

64 - Agricultural amp Horticultural use -

50

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 anti-

microbial agent and as an edible food coating agent

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 (61) chitinase and glucanase activation (62)

phytoalexin biosynthesis (63) 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

641 - Applied as seed coating agents -

51

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

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 andor 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)

642- 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 CO2

concentration the observed effect on the net photosynthetic rate is in general

52

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

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 26ndash43 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 Gardreg and in which a formation of

a thin anti-transpirant film at the surface of the leaves was much more efficient than

stomatal closure

53

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 micro-

propagated greenhouse-grown potatoes

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

643 - 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

54

disadvantaging plant pathogens Beneficial organisms on the other hand are able to

outcompete them through mechanisms such as parasitism antibiosis and induced

resistance (76)

65- Applications of chitosan derivatives -

651- 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

74) 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

652- 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

653 - Glycol chitosan-coated MRI(Magnetic-Resonance-Imaging) agent

55

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

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

66 ndash 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

56

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 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

57

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)

reported that chitosan is also suitable as a ldquodeo-activerdquo 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 odour-

producing 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

58

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)

References

59

1() httpwwwmerriam-webstercomdictionarypolymer

2() httpgoldbookiupacorgM03667html accessed 7 October 2012

3() Painter Paul C Coleman Michael M (1997) Fundamentals of polymer science an introductory text Lancaster Pa Technomic Pub Co p 1 ISBN 1-56676-559-5

4() McCrum N G Buckley C P Bucknall C B (1997) Principles of polymer engineering Oxford New York Oxford University Press p 1 ISBN 0-19-856526-7

5() Majeti NV Ravi K A review of chitin and chitosan applications React amp Funct Polym 2000461-27

6() Muzzarelli RAA 1997 Human enzymatic activities related to thetherapeutic administration of chitin derivatives Cell Mol Life Sci53 131ndash140

7() Fang Fengab Yu Liuc Binyuan Zhaoc Keao Huc- Characterization of half N-acetylated chitosan powders and Films-Procedia Engineering 27 (2012) 718 ndash 732

8() httpwwwfrance-chitinecomfabehtml

9( ) Production of Fungal Chitosan by Enzymatic Method and Applications in Plant Tissue Culture and Tissue Engineering 11 Years of Our Progress Present Situation and Future Prospects - Nitar Nwe Tetsuya Furuike and Hiroshi Tamura

10 () Arcidiacono S Kaplan D L (1992) Molecular weight distribution of chitosan isolated from Mucor rouxii under different culture and processing conditions Biotechnology and Bioengineering 39 281ndash6

11( ) Bartnicki-Garcia S (1968) Cell wall chemistry morphogenesis and taxonomy of fungi Annual Review of Microbiology 22 87ndash108

12( ) Crestini C Kovac B amp Giovannozzi-Sermanni G (1996) Production and isolation of chitosan by submerged and solid state fermentation from Lentinus edodes Biotechnology and Bioengineering 50 207ndash10

13 ( ) Nwe N Chandrkrachang S amp Stevens W F (2001) Fungal chitosan from solid state fermentation Proceeding of 8th International Conference on Chitin and Chitosan and 4th Asia Pacific Chitin and Chitosan Symposium pp 482-484 ISBN4-906464-43-0 Yamaguchi

14( ) Nwe N Chandrkrachang S Stevens W F Maw T Tan T K Khor E amp Wong S M (2002) Production of fungal chitosan by solid state and submerged fermentation Carbohydrate Polymers 49 235-237

15( ) Nwe N Stevens W F Tokura S amp Tamura H (2008) Characterization of chitin and chitosan-glucan complex extracted from cell wall of fungus Gongronella butleri USDB 0201 by enzymatic method Enzyme and Microbial Technology 42 242- 251

16( ) Article Originally Published On httpwwwarticlesnatchcom

17() Dvir T Tsur-Gang O Cohen S ldquoDesignerrdquo Scaffolds for Tissue Engineering and Regeneration Isr J Chem 2005 45 487-94

18() Senel S McClure SJ Potential applications of chitosan in veterinary medicine Adv Drug Deliv Rev 2004 56(10) 1467-8019

() Martino AD Sittinger M Risbud MV Chitosan a versatile biopolymer for orthopaedic tissue-engineering Biomaterials 2005 26 5983-90

20() Li Q Grandmaison EW Goosen MFA Dunn ET Applications and properties of chitosan J Bioact Compat Polym 1992 7(4) 370-97

21() Khor E Lim LY Implantable applications of chitin and chitosan Biomaterials 2003 24(13) 2339-49

22() Foda NH El-Laithy HM Tadros MI Implantable biodegradable sponges effect of interpolymer complex formation of chitosan with gelatin on the release behavior of tramadol hydrochloride Drug Dev Ind Pharm 2007 33(1) 7-1723

() Senel S McClure SJ Potential applications of chitosan in veterinary medicine Adv Drug Deliv Rev 2004 56(10) 1467-80

24() Wu ACM Determination of molecular weight distribution of chitosan by high-performance liquid chromatography J Chromatogr A 1976 128(1) 87-99

25() Beri G Walker J Reese ET Rollings JE Characterization of chitosans via coupled size-exclusion chromatography and multipleangle laser light-scattering technique Carbohydr Res 1993 238 11-26

26() Lee VF Solution and shear properties of chitin and chitosan PhD Dissertation University of Washington University MicrofilmsAnn Arbor USA 1974

27() Rathke TD Hodson SM Review of chitin and chitosan as fibre and film formers J Macromol Sci Rev Macromol Chem Phys 1994 C34(3) 375-43728

() Kumar MNVR Chitin and chitosan fibres a review Bull Mat Sci1999 22 905-15

29() Kurita K Sannan T Iwakura Y Studies on chitin 4 Evidence for formation of block and random copolymers of N-acetyl-Dglucosamine and D-glucosamine by hetero- and homogeneous hydrolyses Makromol Chem 1977 178 3197-202

30() Kurita K Koyama Y Nishimura S Kamiya M Facile preparation of water-soluble chitin from chitosan Chem Lett 1989 18(9) 1597-8

31() Kurita K Kamiya M Nishimura S Solubilization of a rigid polysaccharide controlled partial N-acetylation of chitosan todevelop solubility Carbohydr

Polym 1991 16 83-92 32

33() Kang DW Choi HR Kweon DK Selective adsorption capacity for metal ions of amidocimated chitosan bead-g-PAN copolymer Polymer(Korea) 1996 20 989-95

34() Kang DW Choi HR Kweon DK Stability constants of amidoximated chitosan-g-poly(acrylonitrile) copolymer for heavy metal ions J Appl Polym Sci 1999 73(4) 469-7635

() Yang JM Lin HT Wu TH Chen CC Wettability and antibacterial assessment of chitosan containing radiation-induced graft nonwoven fabric of polypropylene-g-acrylic acid J Appl Polym Sci 2003 90(15) 1331-636

() Song SH Yeom BY Shim WS Hudson SM Hwang TS Synthesis of biocompatible CS-g-CMS ion exchangers and their adsorption behavior for heavy metal ions J Ind Eng Chem 2007 13(6) 1009-1637

() Tojima T Katsura H Han SM Tanida F Nishi N Tokura S Preparation of an 1048577-cyclodextrin-linked chitosan derivative via reductive amination strategy J Polym Sci Pol Chem 1998 36(11) 1965-8

38() Sreenivasan K Synthesis and evaluation of a beta cyclodextrinbased molecularly imprinted copolymer J Appl Polym Sci 1988 69(6) 1051-5

39() Chen S Wang Y Study on 1048577-cyclodextrin grafting with chitosan and slow release of its inclusion complex with radioactive iodine J Appl Polym Sci 2001 82(10) 2414-21

40() Aoki N Nishikawa M Hattori K Synthesis of chitosan derivatives bearing cyclodextrin and adsorption of p-nonylphenol and bisphenol A Carbohydr Polym 2003 52(3) 219-2341

( ) Madihally SV Matthew HWT Porous chitosan scaffolds for tissue engineering Biomaterials 1999 20(12) 1133-42

42() Subramanian A Vu D Larsen GF Lin HY Preparation and evaluation of the electrospun chitosanPEO fibers for potential applications in cartilage tissue engineering J Biomater Sci ndashPolym Ed 2005 16(7) 861-7343

() Bhattarai N Edmondson D Veiseh O Matsen FA Zhang MQ Electrospun chitosan-based nanofibers and their cellular compatibility Biomaterials 2005 26(31) 6176-8444

() Ueno H Mori T Fujinaga T Topical Formulations and Wound Healing Applications of Chitosan Adv Drug Deliv Rev 2001 52(2) 105-1545

() Chen J Chang G Chen J Electrospun collagenchitosan nanofibrous membrane as wound dressing Colloid Surf APhysicochem Eng Asp 2008 313-314(1) 183-8

46() Jayakumar R Prabaharan M Reis RL Mano JF Graft copolymerized chitosan-present status and applications Carbohydr Polym 2005 62(2) 142-5847

() Prabaharan M Reis RL Mano JF Chitosan-based particles as controlled drug delivery systems Drug Deliv 2005 12(1) 41-5748

() Burns JW Skinner K Colt J Prevention of tissue injury and postsurgical adhesions by precoating tissues with hyaluronic acid solutions J Surg Res 1995 59 644-5249

() One K Saito Y Yura H et al Photocrosslinkable chitosan as a biological adhesive J Biomed Mater Res Part A 2000 49(2) 289-9550

() Hoemann CD Jun J Leacutegareacute A McKee MD Buschmann MD Osteoarthritis Cartilage 2005 13(4) 318-2951

() Journal of Emergency Medicine 74ndash81 January 200852

() Pusateri A E S J McCarthy K W Gregory R A Harris L Cardenas A T McManus amp C W Goodwin Jr (2003) Effect of a chitosan-based hemostatic dressing on blood loss and survival in a model of severe venous hemorrhage and hepatic injury in swine Journal of Trauma 4 (1) 177ndash182 doi10109700005373-200301000-00023 PMID 12544915

53() Kevin McCue (March 3 2003) New Bandage Uses Biopolymer (- Scholar search) Chemistryorg (American Chemical Society) Archived from the original on November 28 2005 Retrieved 2006-07-10

54() a b US patent 8106030 Craig Hardy Lee Johnson amp Paul LukschHemostatic Material issued 2012-01-3155

() a b Baldrick P (2009) The safety of chitosan as a pharmaceutical excipient Regul Toxicol Pharmacol 56 (3) 290ndash29956

() Stevens WF Rao MS Chandrakranchang S Eds Chitin and chitosan environmental friendly and versatile biomaterials Bangkok AIT 199657

() Hirano S Chitin and chitosan as novel biotechnological materials Polym Int 1999 48 732-458

() Teo WE Ramakrishna S A review on electrospinning design and nanofibre assemblies Nanotechnology 2006 17 R89-10659

() Sun T Xu P Liu Q Xue J Xie W Graft copolymerization of methacrylic acid onto carboxymethyl chitosan Eur Polym J 2003 39(1) 189-9260() Barber MS Bertram RE Ride JP Chitin oligosaccharides elicit lignification in wounded wheat leaves Physiol Mol Plant Pathol 1989343ndash12

61() Felix G Baureithel K Boller T Desensitization of the perception system for chitin fragments in tomato cells Plant Physiol 1998117643ndash650 [PMC free article] [PubMed]

62() Kaku H Shibuya N Xu P Aryan AP Fincher GB N acetylchitooligosaccharide elicitor expression of a single 13-β-glucanase gene in suspension-cultured cells from barley (Hordeum vulgare) Physiol Plant 1997100111ndash118

63( ) Yamada A Shibuya N Kodama O Akatsuka T Induction of phytoalexin formation in suspension cultured rice cells by N-acetylchitooligosaccharides Biosci Biotechnol Biochem 199357405ndash40964

() Nojiri H Sugimori M Yamane H Nishimura Y Yamada A Shibuya N Kodama O Murofushi N Ohmori T Involvement of jasmonic acid in elicitor induced phytoalexin production in suspension-cultured rice cells Plant Physiol 1996110387ndash392 [PMC free article] [PubMed]65

() Conrath U Domard A Kauss H Chitosan-elicited synthesis of callose and of coumarin derivatives in parsley cell suspension cultures Plant Cell Rep 19898152ndash15566

() Guan YJ Hu J Wang XJ Shao CX Seed priming with chitosan improves maize germination and seedling growth in relation to physiological changes under low temperature stress J Zhejiang Univ Sci B 200910427ndash433 [PMC free article] [PubMed]67

() Shao CX Hu J Song WJ Hu WM Effects of seed priming with chitosan solutions of different acidity on seed germination and physiological characteristics of maize seedling J Zhejiang Univ Agric Life Sci 20051705 70868

() Zhou YG Yang YD Qi YG Zhang ZM Wang XJ Hu XJ Effects of chitosan on some physiological activity in germinating seed of peanut J Peanut Sci 20023122ndash2569

() Ruan SL Xue QZ Effects of chitosan coating on seed germination and salt-tolerance of seedlings in hybrid rice (Oryza sativa L) Acta Agron Sinica 200228803ndash808

70() Rabea EI El Badawy MT Stevens CV Smagghe G Steurbaut W Chitosan as antimicrobial agent Applications and mode of action Biomacromolecules 200341457ndash1465 [PubMed]

71() Khan W Prithiviraj B Smith DL Effect of foliar application of chitin and chitosan oligosaccharides on photosynthesis of maize and soybean Photosynth Res 200240621ndash624

72() M Flury M Campbell GS Nichols EJ Reduction of transpiration through foliar application of chitosan Agric Forest Meteorol 2001107167ndash17573

() Iriti M Picchi V Rossoni M Gomarasca S Ludwig N Garganoand M Faoro F Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure Env Exp Bot 200966493ndash50074

() Faoro F Maffi D Cantu D Iriti M Chemical-induced resistance against powdery mildew in barley the effects of chitosan and benzothiadiazole BioControl 200853387ndash40175

() Murphy JG Rafferty SM Cassells AC Stimulation of wild strawberry (Fragaria vesca) arbuscular mycorrhizas by addition of shellfish waste to the growth substrate interaction between mycorrhization substrate amendment and susceptibility to red core (Phytophthora fragariae) Appl Soil Ecol 200015153ndash15876

() Uppal AK El Hadrami A Adam LR Tenuta M Daayf F Biological control of potato Verticillium wilt under controlled and field conditions using selected bacterial antagonists and plant extracts Biol Control 20084490ndash100

77() Al-Saadi A Yu CH Khutoryanskiy VV Shih S-J Crossley A Tsang SC JPhys Chem C 2009 113 15260-15265





Contents - PageNo

633 -Potential industrial use -


642- Applied as foliar treatment agents -

643 - Applied as soil amendment -

653 - Glycol chitosan-coated MRI(Magnetic-Resonance-Imaging) agent safer effective in detecting breast cancer -

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


Acknowledgement




2





























4


















5




17




18






22



6

Abbreviations -







(CD) Cyclodextrin



(ABA) Abscisic Acid



7

1- Introduction -




















8





applications














9

























2000)











11















al 2004 Qin et al 2006) (7)


12















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo




































4


















5




17




18






22



6

Abbreviations -







(CD) Cyclodextrin



(ABA) Abscisic Acid



7

1- Introduction -




















8





applications














9

























2000)











11















al 2004 Qin et al 2006) (7)


12















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo

























5




17




18






22



6

Abbreviations -







(CD) Cyclodextrin



(ABA) Abscisic Acid



7

1- Introduction -




















8





applications














9

























2000)











11















al 2004 Qin et al 2006) (7)


12















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo











17




18






22



6

Abbreviations -







(CD) Cyclodextrin



(ABA) Abscisic Acid



7

1- Introduction -




















8





applications














9

























2000)











11















al 2004 Qin et al 2006) (7)


12















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo








Abbreviations -







(CD) Cyclodextrin



(ABA) Abscisic Acid



7

1- Introduction -




















8





applications














9

























2000)











11















al 2004 Qin et al 2006) (7)


12















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo








1- Introduction -




















8





applications














9

























2000)











11















al 2004 Qin et al 2006) (7)


12















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo












applications














9

























2000)











11















al 2004 Qin et al 2006) (7)


12















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo
































2000)











11















al 2004 Qin et al 2006) (7)


12















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo






















al 2004 Qin et al 2006) (7)


12















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo






















13
























(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo































(Stevens 2001)

14
























15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo































15


(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo









(Muzzarelli 1973)





















16


17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo









17







of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo














of chitosan















18












19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo



















19






































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo













































21






chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo













chitinase








25 NaOH

22



23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo










23













24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo




















24








glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo















glucosamine









toxins






25












(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo



















(18) (19)











26
















27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo























27









al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo
















al 2001)













28








generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo















generated
















29



















30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo


























30











above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo


















above














31

















Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo
























Orozco et al 2004)





32















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo






















nature








33











Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo


















Rajapakse 2005)











34



















fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo


























fabrications(32)




35


2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo









2005)




not men











processing outlets






36














and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo





















and









38 39 40)

37











38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo


















38


























39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo

































39





















40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo




























40












41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo



















41










growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo

















growth











42
















delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo























delivery (47)






hernia repairs (48)



43























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo






























44


















acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo

























acts as an antacid





Weight loss


45









control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo
















control (56)










46


















remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo

























remains zero






47

























48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo
































48











capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo


















capsule













49



















50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo


























50



infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo










infections















genes





51

















species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo
























species (70)







52

























stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo
































stomatal closure

53
























54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo































54



resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo










resistance (76)














drugs


55






















56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo





























56

























57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo
































57











producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo


















producing bacteria












time

58



1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo










1999)







properties





References

59
































Polym 1991 16 83-92 32

















































Contents - PageNo







































Polym 1991 16 83-92 32

















































Contents - PageNo















































Contents - PageNo








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