review article chitooligosaccharide and its derivatives...

Review ArticleChitooligosaccharide and Its Derivatives Preparation andBiological Applications

Gaurav Lodhi12 Yon-Suk Kim12 Jin-Woo Hwang12

Se-Kwon Kim3 You-Jin Jeon4 Jae-Young Je5 Chang-Bum Ahn6

Sang-Ho Moon7 Byong-Tae Jeon7 and Pyo-Jam Park127

1 Department of Biotechnology Konkuk University Chungju 380-701 Republic of Korea2Department of Applied Life Science Konkuk University Chungju 380-701 Republic of Korea3 Specialized Graduate School of Convergence Science and Technology Department of Marine Bioconvergence ScienceBusan 608-737 Republic of Korea

4 School of Marine Biomedical Sciences Jeju National University Jeju 690-756 Republic of Korea5 Department of Marine Bio-Food Sciences Chonnam National University Yeosu 550-749 Republic of Korea6Division of Food and Nutrition Chonnam National University Gwangju 550-757 Republic of Korea7Nokyong Research Center Konkuk University Chungju 380-701 Republic of Korea

Correspondence should be addressed to Pyo-Jam Park parkpjkkuackr

Received 3 January 2014 Accepted 22 January 2014 Published 3 March 2014

Academic Editor Yoshihiko Hayashi

Copyright copy 2014 Gaurav Lodhi et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chitin is a natural polysaccharide ofmajor importanceThis biopolymer is synthesized by an enormous number of living organismsconsidering the amount of chitin produced annually in the world it is the most abundant polymer after cellulose The mostimportant derivative of chitin is chitosan obtained by partial deacetylation of chitin under alkaline conditions or by enzymatichydrolysis Chitin and chitosan are known to have important functional activities but poor solubility makes them difficult to usein food and biomedicinal applications Chitooligosaccharides (COS) are the degraded products of chitosan or chitin prepared byenzymatic or chemical hydrolysis of chitosan The greater solubility and low viscosity of COS have attracted the interest of manyresearchers to utilize COS and their derivatives for various biomedical applications In light of the recent interest in the biomedicalapplications of chitin chitosan and their derivatives this review focuses on the preparation and biological activities of chitinchitosan COS and their derivatives

1 Introduction

Synthetic polymers are gradually being replaced by biode-gradable materials especially those derived from replen-ishable natural resources [1] Natural biopolymers haveseveral advantages such as availability from replenishableagricultural or marine food resources biocompatibility andbiodegradability thereby leading to ecological safety and thepossibility of preparing a variety of chemically or enzymat-ically modified derivatives for specific end uses Polysaccha-rides as a class of natural macromolecules have the tendencyto be extremely bioactive and are generally derived fromagricultural feedstock or crustacean shell wastes [2]

Chitosan is a natural nontoxic biopolymer produced bythe deacetylation of chitin amajor component of the shells ofcrustaceans such as crab shrimp and crawfish chitooligosac-charides (COS) are the degraded products of chitosan or chit-in prepared by enzymatic or chemical hydrolysis of chitosan

Chitosan and its derivatives have shown various func-tional properties that have made them possible to be usedin many fields including food [3] cosmetics [4] biomedicine[5] agriculture [6] environmental protection [7] and waste-water management [8] Furthermore biodegradable non-toxic and nonallergenic nature of chitosan especially encour-ages its potential use as a bioactive material [9] Even thoughchitosan is known to have important functional activities

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 654913 13 pageshttpdxdoiorg1011552014654913

2 BioMed Research International

poor solubilitymakes themdifficult to use in food and biome-dicinal applications Unlike chitosan its hydrolyzed productsand COS are readily soluble in water due to their shorterchain lengths and free amino groups in D-glucosamine units[10]The low viscosity and greater solubility of COS at neutralpH have attracted the interest of many researchers to utilizechitosan in its oligosaccharide form Especially research onCOS in food and nutrition fields has emphasized their abilityto improve food quality and human health progression

In light of the recent interest in the biomedical appli-cations of chitin chitosan and its derivatives this reviewfocuses on the preparation and biological activities of chitinchitosan COS and their derivatives

2 Chitin

Chitin (Figure 1) a mucopolysaccharide and the supportingmaterial of crustaceans and insects is the second most abun-dant polymer after cellulose found in nature it is producedby many living organisms and is present usually in a complexwith other polysaccharides and proteins Chitin was found asa major component in arthropods (insects crustaceans ara-chnids and myriapods) nematodes algae and fungi [11ndash14]

Its immunogenicity is exceptionally low in spite of thepresence of nitrogen It is highly insoluble material resem-bling cellulose with its solubility and low chemical reactivityIt may be regarded as cellulose with hydroxyl at position C-2replaced by an acetamido group Like cellulose it functionsnaturally as a structural polysaccharide It is white hardinelastic nitrogenous polysaccharide [15 16]

Chitin is a linear polysaccharide composed of (1 rarr 4)linked 2-acetamido-2-deoxy-120573-d-glucopyranosyl units andoccurs naturally in three polymorphic forms with differentorientations of the microfibrils known as 120572- 120573- and 120574-chitin[1 17] The 120572-form has antiparallel chains and is a commonand the most stable polymorphic form of chitin in naturewhich is prevalent in crustaceans and in insect chitinouscuticles [18ndash20]The 120573-form of chitin is rare it occurs in pensof mollusks and is characterized by a loose-packing paral-lel chains fashion with weak intermolecular interactions andhigher solubility and swelling than 120572-form 120573-chitin was pre-pared from the pens of the squidOmmastrephes bartrami [2122] Loligo species and cuttlefish (Sepia officinalis) [18 23ndash25] The 120574-form is characterized by a mixture of antiparalleland parallel chains and was found in the cocoons of insects[26]

Besides its application as a starting material for thesynthesis of chitosan and chitooligosaccharides chitin itselfhas been a center of many therapeutic applications and isthought to be a promising biomaterial for tissue engineeringand stem cell technologies [27]

Bae et al [28] demonstrated that oral administrationof chitin (120572 and 120573 forms) is beneficial in preventing foodallergies the oral administration of chitin was accomplishedby milling it to particle size less than 20 120583m and mixing itwith feed Their results showed that 120572-form reduced serumlevels of peanut-specific IgE and both the forms decreasedthe levels of interleukins (IL) IL-5 and IL-10 and increased

the levels of IL-12 Dietary supplementation of chitin hasshown to exert positive immunomodulatory effects [29 30]antibacterial activity of chitin prepared from shrimp shellwaste was reported by Benhabiles et al [31]

3 Chitosan

Chitosan [poly-(120573-14)-2-amino-2-deoxy-D-glucopyranose]is a natural nontoxic biopolymer produced by the deacetyla-tion of chitin Chitosan (Figure 1) has three types of reactivefunctional groups an amino group as well as both primaryand secondary hydroxyl groups at the C-2 C-3 and C-6 posi-tions respectively Chemical modifications of these groupshave provided numerous useful materials in different fieldsof application Currently chitosan has received considerableattention for its commercial applications in the biomedicalfood and chemical industries [9 32ndash36]

Chitosan solubility biodegradability reactivity and ad-sorption of many substrates depend on the amount of pro-tonated amino groups in the polymeric chain and thereby onthe proportion of acetylated and nonacetylated glucosamineunits The amino groups (pKa from 62 to 70) are completelyprotonated in acids with pKa smaller than 62 making chitos-an soluble Chitosan is insoluble in water organic solventsand aqueous bases and it is soluble after stirring in acids suchas acetic nitric hydrochloric perchloric and phosphoric[37ndash39]

Applications of chitin are limited compared to chitosanbecause chitin is chemically inert and is insoluble in bothwater and acid while chitosan is relatively reactive and can beproduced in various forms Chitosan is normally insoluble inneutral or basic pH conditions while being soluble in acidicpH The solubility of chitosan depends upon the distributionof free amino and N-acetyl groups In dilute acids (pH lt6) the free amino groups are protonated and the moleculebecomes soluble [40]

Due to its unique biological characteristics including bi-odegradability and nontoxicity many applications have beenfound either alone or blended with other natural polymers(starch gelatin and alginates) in the food pharmaceuticaltextile agriculture water treatment and cosmetics industries[41ndash46]

Chitosan lacks irritant or allergic effects and is biocom-patible with both healthy and infected human skin [47]When chitosan was administered orally in mice the LD

50

was found to be in excess of 16 gkg which is higher thanthat of sucrose [48] The intriguing properties of chitosanhave been known previously and the polymer has been usedin the fields of agriculture industry and medicine In agri-culture chitosan has been described as a plant antivirusan additive in liquid multicomponent fertilizers [49] andit has also been investigated as a metal-recovering agent inagriculture and industry [50] Chitosan has been noted for itsapplication as a film-forming agent in cosmetics [51] a dyebinder for textiles a strengthening additive in paper [52] anda hypolipidic material in diets [53] It has been used exten-sively as a biomaterial [54] owing to its immunostimulatoryactivities [55] anticoagulant properties [56] antibacterial

BioMed Research International 3

Chitin

Chitosan

Chitooligosaccharide

OO

NH

O

OO

OH

OH

NH

O

HO

HO

O

OH

O

HO

OO

HO

HOOH

OHOH

HO

O

NHR

OH

O

HO

OO

HO

HO

NHRNHR

OH

OHOH

HO

n

R = H or Ac n = 0 to 8

NH2NH2

NH2

CH3

CH3

n

n

Figure 1

and antifungal action [57] and its action as a promoter ofwound healing in the field of surgery [58]

Chitosan and its derivatives possess some special prop-erties for use in regenerative medicine Several studies haveexamined the host tissue response to chitosan based implantsIn general these materials are nontoxic and biodegradablewith living tissues and evoke aminimal foreign body reactionwith little or no fibrous encapsulation [59]

Antimicrobial activity of chitosan has been demonstratedagainst many bacteria filamentous fungi and yeasts [60ndash63]Chitosan has wide spectrum of activity and high killing rateagainst Gram-positive and Gram-negative bacteria but lowertoxicity toward mammalian cells [64 65]

Owing to the presence of hydroxyl amine and acetylatedamine groups chitosan low molecular weight chitosan andCOS interact readily with various cell receptors that trigger a


cascade of interconnected reactions in living organisms re-sulting in anti-inflammatory [66] anticancerogenic [67]antidiabetic [68] antimicrobial [69] anti-HIV-1 [70] antiox-idant [71] antiangiogenic [72] neuroprotective [73] and im-munostimulative [74] effects

4 Chitooligosaccharides

Chitosans with degrees of polymerization (DPs) lt20 andan average molecular weight less than 3900Da are calledchitosan oligomers chitooligomers or chitooligosaccharides[75] COS (Figure 1) are generated by depolymerizationof chitin or chitosan using acid hydrolysis hydrolysis byphysical methods and enzymatic degradation [76]

Hydrolysis of chitosan can progress by use of acidas hydrochloric acid acid with electrolytes nitrous acidphosphoric acid hydrofluoric acid or oxidative reductivemethods with hydrogen peroxide or persulfate Other acidsas lactic acid [77] trichloroacetic acid [78] formic acid [79]and acetic acid have also been studied for their degradativeeffect on chitin or chitosan However due to the complexityof controlling the progress of the reaction these treatmentsalso result in the formation of secondary compounds that aredifficult to remove [75] With hydrolysis by physical methodssuch as irradiation with low-frequency ultrasound (20 kHz)partial depolymerization is obtained reducing the averageMW from 2000 kDa down to 450 kDa or from 300 kDa to50 kDa but the reduction of molecular weight is limited[75]

Enzymatic methods for the hydrolysis of chitosan involvethe use chitosanases and other nonspecific enzymes thismethod is performed in gentle conditions and theMWdistri-bution of the product can be controlled [80 81] Pantaleoneet al [82] reported the hydrolytic susceptibility of chitosan toa wide range of enzymes including glycanases proteases andlipases derived from bacterial fungal mammalian and plantsources

Recently COS have been the subject of increased atten-tion in terms of their pharmaceutical and medicinal applica-tions due to their nontoxic and high solubility properties aswell as their positive physiological effects

Recent advances have insight into the health benefits ofCOS possessing several beneficial biological effects includinglowering blood cholesterol lowering high blood pressureprotective effects against infections controlling arthritisimprovement of calcium uptake and enhancing antitumorproperties

The radical scavenging effects of COS were reportedby Mendis et al [83] They reported that COS successivelyparticipated in scavenging intracellular radicals and alsosuppressed NF-120581B activation

The antitumor activity of COS was first reported in early1970s [84] This activity was suggested mainly due to itscationic property exerted by amino groups and later it wasaccepted that the molecular weight also plays a major rolefor the antitumor activity [85] Some researchers found thatantitumor effects of COS were due to increased activity

of natural killer lymphocytes as observed in sarcoma 180-bearing mice [86] There have also been studies showing theapoptotic effect of COS on human hepatocellular carcinomacells by the upregulation of Bax (Bcl-2-associated X protein)[87] Pangestuti et al [88] suggested that COS with molecu-lar weight lt1 kDa exhibit potent anti-inflammatory activityand also that treatment with COS results in attenuationof proinflammatory mediators in LPS (lipopolysaccharides)stimulated BV2 microglia by MAPK (mitogen activatedprotein kinases) signaling pathway Administration of COShas been found to be effective against cyclophosphamideinduced immunosuppression in mice [89] The study of Meiet al [89] also suggested that COS may also be effective inenhancing systemic immune responses and in modulatingthe functions of immunocompetent cells

Wei et al [90] showed that a high concentration of COSsignificantly proliferated the mice marrow cells and inducedCD34+ cells into megakaryocyte progenitor cells Howeverin their study COS could not enhance the proliferation ofCD19+ and CD4+ and promote CD34+ cells to differentiateinto lymphoid progenitor cells suggesting that COS canpromote hematopoietic stemprogenitor cells hyperplasia inmice

Senevirathne et al [91] studies on the effect of COSon tert-butyl hydroperoxide (t-BHP) induced damage inChang liver cells showed that COS protected Chang liver cellsagainst oxidative damage induced by t-BHP via inhibitingproduction of ROS and lipid peroxidation and the elevationof the levels of antioxidant enzymes

The inhibitory effects of COS on degranulation andcytokine generation in rat basophilic leukemia RBL-2H3 cellswere investigated by Vo et al [92] Their results indicatedthat COS contribute to attenuation of allergic reactionsTheir study suggested that COS (MW 1 to 3 kDa) possessthe highest inhibitory effects on degranulation and cytokinegeneration of mast cells They also suggested that COS withdifferent molecular weight ranges might lead to variableabsorption which corresponds to the different inhibitoryeffects in mast cells

Efficacy of COS in the management of diabetes in alloxaninduced mice was evaluated by Katiyar et al [93]Their studysuggested that COS have significant effect of antidiabeticactivity hypolipidemic activity and antioxidative propertiesin alloxan induced diabetic mice

5 Chitooligosaccharide Derivatives

51 Amino Derived COS COS hydrolytic products of chi-tosan have amino groups or acetamide groups atC-2 positiondepending on their degree of acetylation Partly based onthese functional groups they have exhibited a number ofbiological activities

Ngo et al [94] synthesized aminoethyl COS (AE-COSFigure 2) by grafting aminoethyl functionality to improveits angiotensin I converting enzyme inhibitor potentialACE plays an important physiological role in regulatingblood pressure by converting an inactive form of decapep-tide angiotensin I to a potent vasoconstrictor octapeptide


O

OR

O

HOn

R1 = COCH3

R = (CH2)2NH2

NHR1

Figure 2 Aminoethyl chitooligosaccharide

angiotensin II and by inactivating catalytic function ofbradykinin which has depressor actionTherefore inhibitionofACE is considered to be an important therapeutic approachfor controlling hypertension [95]

Ngo et al [94] replaced the hydroxyl group at C-6 posi-tion by aminoethyl group because the C-6 hydroxyl groupshad the highest reactivity for aminoethylation while thestructure of COS was maintained the AE-COS thus formedwas completely soluble in water In their study AE-COSwith molecular mass ranging between 80079 and 4765Dawas prepared from COS with molecular weight 800ndash3000Daby grafting aminoethyl groups at C-6 position of pyranosering and its effects on ACE inhibition were investigatedTheir study showed that AE-COS exhibited an 893 ACEinhibition at 25mgmL and its IC50 valuewas 08017mgmL

Ngo et al [96] investigated the inhibitory effects of AE-COS on oxidative stress in mouse macrophages (RAW 2647cells) They prepared AE-COS by adding 2-chlorethylaminohydrochloride to COS while stirring at 40∘C NaOH wasadded to the reaction mixture dropwise and continuouslystirred for 48 h The solution was then filtered and thereaction mixture was acidified with 01 N HCl and dialyzedagainst water for 2 days The product was freeze-dried togive AE-COS (Figure 3) The results of Ngo et al [96]indicated that in HL-60 cells AE-COS exhibited a greaterinhibitory effect (a 20 increase) onmyeloperoxidase activityas compared to COS at 100 120583gmL They also reported thatAE-COS can prevent oxidative stress to membrane protein oflive cells and exhibits inhibitory effect against DNA oxidationin live cells Ngo et al [96] investigated the direct scavengingeffects of AE-COS and COS in live cell system For that RAW2647 cells were labeled with fluorescence probe DCFH-DA(dichlorodihydrofluorescein diacetate) which is the specificprobe for reactive oxygen species (ROS) that was used Theirstudy confirmed that AE-COS has antioxidant activity indose- and time-dependent manner and has potent direct freeradicals scavenging ability in a cellular environment

The antioxidant and anti-inflammatory activities of AE-COS in murine microglial cells (BV-2) were investigated byNgo et al [97] Neuroinflammation or inflammation of thebrain is closely involved in pathogenesis of several neurode-generative diseases such as Parkinsonrsquos disease Alzheimerrsquosdisease human immunodeficiency virus-associated demen-tia and multiple sclerosis It was found that the treatment

COS

Reaction mixture

2-Chlorethylamino HCl

Constant stirring

NaOH dropwise

Filtered

Acidified with 01 N HCl

Freeze-dried

40∘C

Stirred continuously for 48hours

Dialyzed against water for 2days

Figure 3 Synthesis of AE-COS

of AE-COS in BV-2 cells at 100120583gmL inhibited ROS DNAprotein and lipid oxidation AE-COS was also studied forits inhibitory activity against lipopolysaccharide inducedinflammatory responses in BV-2 cells It was found that AE-COS reduced the level of nitric oxide (NO) and prostaglandinE2 production by diminishing the expression of inducibleNOsynthase and cyclooxygenase-2 without significant cytotoxic-ityThey also observed that in LPS treatedBV-2 cells AE-COSlowered the levels of TNF-120572 and IL-1120573 in a dose-dependentmanner

Karagozlu et al [98] synthesized water-soluble aminoderivatized COS derivatives aminoethyl COS (AE-COS)dimethyl aminoethyl COS (DMAE-COS) and diethylaminoethyl COS (DEAE-COS) and evaluated their apoptoticactivity on human stomach adenocarcinoma (AGS cells)Their study showed that exposure of AGS cells to theaminoethylated COS derivatives resulted in the inhibitionof cell proliferation in a dose-dependent manner at 50 and500120583gmL AE-COS inhibited the cell proliferation by 22and 84 DMAE-COS by 45 and 85 and DEAE-COS by68 and 86 Karagozlu et al [98] also suggested that COSand amino derivatized COS derivatives induced apoptosisin AGS cancer cells by mitochondrial pathways via theupregulation of Bax expression and activation caspases

Antiproliferative effects of amino derivatized COS onAGS human gastric cancer cells were investigated by Karago-zlu et al [99]They were able to deduce that exposure of AGScells to increasing concentrations of amino derivatized COSresulted in a dose- and time-dependent decrease in cell via-bility relative to control cells and that amino derivatized COS


O O

OH

NRO

O

O

CH2OH

R = H or COCH3

Figure 4 Carboxylated chitooligosaccharide

induce cell death in AGS cells through a typical apoptoticpathway Karagozlu et al [99] reported that AE-COS inducedapoptosis via induction of p53 and p21 and inhibition of Bcl-2and Bax and DEAE-COS induced apoptosis via induction ofp53 and p21 activation of Bax and inhibition of Bcl-2

52 Carboxylated COS The functional properties of COSderivatives are mainly dependent upon their functionalgroups and molecular weight Low molecular weight COSderivatives not only are easily solubilized in aqueous mediabut also are preferred to be used for numerous applicationsRajapakse et al [100] synthesized carboxylated chitooligosac-charides (CCOS Figure 4) by introducing carboxyl group(COCH

2CH2COOminus) to the amino position of pyranose unit

They accomplished this by adding methanol to a solutionof COS in 10 acetic acid followed by addition of differentamounts of succinic anhydride dissolved in acetone drop-wise at room temperature for 1 h to obtain CCOS withdifferent substitution degrees (Figure 5) Rajapakse et al[100] evaluated the effect of these synthesized CCOS onMMP-9 (matrix metalloproteinases-9) expression in humanfibrosarcoma cells

MMPs are a structurally related class of zinc-bindingproteases (metzincin) which selectively cleave polypeptidebonds in extracellular matrix (ECM) and remodel structuralproteins However in several disease states pathologicaltissue degradation and remodeling take place due to overexpression and imbalanced activation of MMPs In cancerMMP-9 (gelatinase B 92 kDa) is thought to play a majorrole in tumor growth angiogenesis and metastasis [101]Rajapakse et al [100] reported in their study that CCOSexerted a dose- dependent inhibitory effect on MMP-9 inhuman fibrosarcoma cell line (HT1080) they observed thatreduction in MMP-9 expression was due to downregulationof MMP-9 transcription that was mediated via inhibitionof AP-1 (activator protein-1) and this inhibition of MMP-9expression led to inhibition of tumor invasiveness

Effect of CCOS onACE inhibitionwas assessed byHuanget al [102] They observed that attachment of carboxyl grouptoCOSwas beneficial for ACE inhibition because it enhancedthe binding ability of COS to the obligatory active site of theenzyme

Succinic anhydride

(different amounts)

acetone

DropwiseRoom temperature

COS in 10 acetic acid

Reaction mixture

COS with different degrees of substitution

Methanol

+

Figure 5 Synthesis of CCOS

Rajapakse et al [103] tested the cellular antioxidant effectsof CCOS by assessing the oxidation inhibition potentialon cellular biomolecules such as lipids proteins and directscavenging of reactive oxygen species (ROS) Their resultsindicated that CCOS inhibited membrane lipid peroxidationin a dose-dependent manner and at low concentrations italso showed a significantly higher inhibition of cellular lipidperoxidation as compared to COS The study of Rajapakse etal [103] also demonstrated that CCOS inhibited membraneprotein oxidation and myeloperoxidase and their free radicalscavenging studies using 2101584071015840-dichlorofluorescein diacetate(DCFH-DA) on RAW2647 cells showed that oxidation pro-tection effects exerted by CCOS are due to direct scavengingof cellular radicals

53 Gallyl COS Phenolic compounds are some of the mostwidespread molecules among plant secondary metabolites[104] These phenolics are rich in some foods and medicinalplants and considered as natural antioxidants [105] Amongthem gallic acid is particularly abundant in processed bever-ages such as redwine and green tea [106] It has been reportedthat gallic acid possesses a wide range of biological activitiesincluding antioxidant anti-inflammatory antimicrobial andanticancer activities [107]

Ngo et al [108] synthesized gallic acid conjugated COS(G-COS Figure 6) to evaluate its inhibitory effects onintracellular free radical generation G-COS was synthesizedby mixing two solutions solution A which was preparedby dissolving COS in distilled water (DW) and methanoland the pH of this solution was adjusted to 68 withtriethylamine and solution B which was prepared by dis-solving gallic acid in methanol and mixing it with DCC-(dicyclohexylcarbodiimide-) methanol mixture Solution Bwas gradually added to solution Awith constant stirring for 5hours the mixture was filtered and kept overnight at 2∘C anddiethyl ether was added the precipitate formed was filtereddissolved in DW and dialyzed (dialysis membranes molecu-lar weight cutoff below 1 kDa) against DW the solution wasthen freeze- dried to obtain G-COS (Figure 7)

Ngo et al [108] findings suggest that G-COS is a potentscavenger of free radicals and is able to inhibit and pre-vent oxidative damage to DNA proteins and lipids inSW1353 cells it increased the levels of intracellular antioxi-dant enzymes (superoxide dismutase (SOD) and glutathione(GSH)) and suppressed the NF-120581B activation and expression


O OO

OO

OHOHOH

OH

NH

HOHO

HOHO

OC

OH

OH

HO

n

NH2

NH2

Figure 6 Gallyl chitooligosaccharide

COS dissolved in distilledwater and methanol Gallic acid dissolved methanol

pH adjusted to 68 withtriethylamine Mixing DCC-methanol mixture

Solution A

Solution BGradually with constant

Filtered

Diethyl ether added

Precipitate formed filtered

Dissolved in distilled water

Dialyzed against distilled water

Freeze-dried

Kept overnight at 2∘C

stirring for 5h

Figure 7 Synthesis of Gallyl COS


inH2O2induced SW1353 cells thereby reducing and prevent-

ing the oxidative damage to cellular biomolecules in livingcells by both indirect and direct ways

Ngo et al [109] evaluated the antioxidant effect of gallatechitooligosaccharides inmousemacrophage RAW2647 cellsThey reported that G-COS was able to scavenge cellularradicals in RAW2647 cells and was able to inhibit oxidativedamage to lipids proteins and DNA they also deduced thatG-COS could decrease the activation and expression of NF-120581B and increase the level of intracellular antioxidant enzymes(SOD and GSH) in oxidative stress induced RAW2647 cells

Allergy is considered as a disorder of the immune systemin which an exaggerated response occurs when a personis exposed to normally harmless environmental substancessuch as animal dander house dust mites foods polleninsects and chemical agents [110 111] The effect of G-COSon antigen induced allergic reactions in RBL-2H3 mast cellswas studied by Vo et al [112] they observed the effect ofG-COS on the degranulation of mast cells by measuringthe amount of histamine released and found that G-COSinhibited the release of histamine in a dose- dependent man-ner They concluded that diminished histamine productionon treatment with G-COS may be due to the decreased inexpression of HDC (L-histidine decarboxylase) The studyof Vo et al [112] also showed that G-COS suppressedintracellular Ca2+ elevation and IL-4 and TNF-120572 in antigenstimulated RBL-2H3mast cellsThe suppression in cytokinesIL-4 and TNF-120572 might be due to regulation of the MAPKsand NF-120581B activation [113] The overall conclusion of Voet al [112] was that the inhibitory effects of G-COS onantigen induced allergic reactions in RBL-2H3 mast cellswere shown due to suppression of Fc120576RI expression whichmight cause the inhibition of intracellular signaling activationand subsequent inhibition of intracellular Ca2+ elevationcytokine generation and histamine release and productionFc120576RI has been well known to play a central role in theinduction and maintenance of allergic reactions [114]

54 Sulfated COS Glucose sensing is a very importantfunction of pancreatic beta cells and serves in maintainingan appropriate blood glucose level Impairment of glucosesensing by beta cells results in an alteration in the glucoseconcentration dependence of insulin secretion in pancreaticbeta cells which may lead to type 2 diabetes [113 115]Ishihara et al [116] characterized the properties of transportphosphorylation and utilization of glucose inMIN6 cells anddemonstrated that these characteristics are very similar tothose in normal pancreatic islets and beta cells

Protective effect of different substituted sulfated COS(COS-S Figure 8) on MIN6 cells was evaluated by Lu etal [117] They synthesized COS-S with different degreesof substitution by using two kinds of sulfating reagentchlorosulfonic acid and anhydrous formamide in differentratios (1 8 and 1 12) [117] Chitosan was suspended inanhydrous formamide by stirring for 30min and the sul-fating reagents were added drop by drop The mixture wasmaintained at 50∘C for 3 h with continuous stirring At theend of the reaction the mixture was cooled neutralized with

O

O

NHO

OO

R1

R1

R2

R1 = SO3 H

R2 = SO3 H OAc

Figure 8 Sulfated chitooligosaccharide

NaOH and treated by adding ethanol The precipitate wasdissolved in DW dialyzed exhaustively against DW and thenlyophilized (Figure 9) [117]The study of Lu et al [117] showedthat COS-S with different degrees of substitution protectedMIN6 from H

2O2induced apoptosis their study suggested

that COS-S prevented apoptosis probably by downregulatingH2O2induced Bax mRNA expression Caspase-3 mRNA

expression andNF-120581Bp65 activation and upregulating Bcl-2mRNA expression

Lu et al [118] evaluated the protective effects of COS-Sagainst hydrogen peroxide-induced damage in MIN6 cellsThey tested the effect of COS-S on cell viability morphologyinsulin contents malondialdehyde (MDA) inhibition lactatedehydrogenase (LDH) release and levels of antioxidantenzymes including catalase (CAT) SOD and glutathioneperoxidase (GPx) under oxidative damage by H

2O2 The

study of Lu et al [118] demonstrated that COS-S exhibitedantioxidant effect and enhanced the cell viability it attenu-ated the production of ROS MDA and LDH Lu et al [118]suggested that the protective effects of COS-S can be partlyattributed to increase of antioxidant enzyme activities andreduction of intracellular ROS along with the capacity ofsuppressing MIN6 cell apoptosis

The antioxidant effect of COS-S on H2O2induced oxida-

tive damage in Chang cells was reported by Lee et al [119]COS-S was prepared by deacetylating chitin using sodiumhydroxide to get chitosan the chitosan was then hydrolyzedtoCOS by using lactic acid andUFmembrane reactor systemThe chitosan and COS were lyophilized and dispersed inDWand anhydrous sodiumcarbonate trimethylamine sulfurtrioxide was added to it and the mixture was heated at 65∘Cuntil a viscous solution or gel was formed The mixture wascooled and dialyzed exhaustively against DW and freeze-dried to get sulfated chitosan and COS-S Lee et al [119]tested these synthesized sulfated chitosan andCOS-S for theirprotective effects against H

2O2induced oxidative damage in

Chang cells and concluded that sulfated chitosan and COS-S possess potent protective effect on liver and DNA againstoxidative stress


Chitosan

Sulphating agents

Reaction mixture

Cooled

Neutralized with NaOH

Treated with ethanol

Precipitate dissolved in distilled water


Freeze-dried

Dropwise

Stirred at 50∘C for 3h

Suspended in anhydrous formamidewith stirring for 30minutes

Figure 9 Synthesis of sulfated chitooligosaccharides

The inhibitory effects of COS-S with different molecularweights on angiotensin I-converting enzyme were reportedby Qian et al [120] Their study concluded that molecularweights of COS and COS derivatives are important factorsof ACE inhibition and mediummolecular weight COS-S andlow molecular weight COS-S inhibited ACE by specificallybinding to the active site of ACE and by competing with itsnatural substrate

The effect of COS-S as inhibitor of prolyl endopeptidase(PEP) was reported by Je et al [121] PEP is a proline-specificendopeptidase with a serine-type mechanism and hydrolyzespeptide bonds at the carboxyl terminus of prolyl residuesit has been reported to be involved in neurodegenerativedisorders Je et al [121] synthesized COS with high mediumand low molecular weight and with different degrees ofdeacetylation (90 75 and 50) which were then convertedinto their corresponding sulphates Je et al [121] reported thatmedium molecular weight 50 deacetylated COS-S showedthe maximum inhibitory action on PEP

The anti-HIV-1 activity of low molecular weight COS-Swas reported by Artan et al [122] They reported that COS-Swith molecular weight range between 3 and 5 kDa was mosteffective in inhibiting HIV-1 replication At nontoxic con-centrations COS-S exhibited remarkable inhibitory activitieson HIV-1 induced syncytia formation lytic effect and p24antigen production In contrast unsulfated chitooligosaccha-rides showed no activity against HIV-1 Furthermore Artan

et al [122] concluded that COS-S blocked viral entry andvirus-cell fusion probably by disrupting the binding of HIV-1gp120 to CD4 cell surface receptor

Karadeniz et al [123] reported the antiadipogenic effectof COS-S in 3T3-L1 adipocytes Their study suggested thatCOS-S inhibited the mRNA expressions and protein levelsof key adipogenic markers such as peroxisome proliferatoractivated receptor (PPAR)-120574 and CCAAT enhancer bindingprotein (CEBP)-120572 thereby highlighting its efficacy in themanagement of obesity

Ryu et al [124] reported the effects of COS-S withdifferent molecular weights on the degradation of articularcartilage through unregulated collagenase expression Theirstudy suggested that COS-S withmolecular weight between 3and 5 kDa effectively inhibited the expressions of collagenases1 and 3 and thereby prevented TNF-120572 induced degrada-tion of collagen in human chondrosarcoma cells (SW-1353)They concluded that COS-S prevented collagen degradationby inhibiting collagenases 1 and 3 via suppressing TNF-120572induced NF-120581B signaling

55 Phenolic Acid Conjugated COS Phenolic acids espe-cially hydroxycinnamic acids such as p-coumaric caffeicferulic and sinapinic acid and benzoic acids derivatives suchas p-hydroxybenzoic protocatechuic vanillic and syringicacids are natural plant hydrophilic antioxidants [125]


Eom et al [126] synthesized various derivatives of COSconjugating them with protocatechuic 4-hydroxybenzoicvanillic syringic p-coumaric caffeic ferulic and sinapinicacids and tested their antioxidant activity Their studiesshowed that among all the PA-c-COSs synthesized proto-catechuic acid conjugated COSs and caffeic acid conjugatedCOS showed the strongest antioxidant activities

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was carried out with the support of the ldquoCoop-erative Research Program for Agriculture Science and Tech-nologyDevelopment (Project no PJ907038)rdquo Rural Develop-ment Administration Republic of Korea

References

[1] R N Tharanathan ldquoBiodegradable films and composite coat-ings past present and futurerdquo Trends in Food Science andTechnology vol 14 no 3 pp 71ndash78 2003

[2] H P Ramesh and R N Tharanathan ldquoCarbohydrates therenewable rawmaterials of high biotechnological valuerdquoCriticalReviews in Biotechnology vol 23 no 2 pp 149ndash173 2003

[3] F Shahidi J K V Arachchi and Y-J Jeon ldquoFood applicationsof chitin and chitosansrdquo Trends in Food Science and Technologyvol 10 no 2 pp 37ndash51 1999

[4] M N V Ravi Kumar ldquoA review of chitin and chitosan applica-tionsrdquo Reactive and Functional Polymers vol 46 no 1 pp 1ndash272000

[5] O Felt P Buri and R Gurny ldquoChitosan a unique polysac-charide for drug deliveryrdquo Drug Development and IndustrialPharmacy vol 24 no 11 pp 979ndash993 1998

[6] A Yamada N Shibbuya O Komada et al ldquoInduction ofphytoalexin formation in suspension-cultured rice cells byN-acetylchitooligosaccharidesrdquo Bioscience Biotechnology andBiochemistry vol 57 no 3 pp 405ndash409 1993

[7] C Peniche-Covas L W Alvarez and W Arguelles-MonalldquoAdsorption of mercuric ions by chitosanrdquo Journal of AppliedPolymer Science vol 46 no 7 pp 1147ndash1150 1992

[8] C Jeuniaux ldquoChitosan as a tool for the purification of watersrdquoin Chitin in Nature and Technology pp 551ndash570 Plenum PressNew York NY USA 1986

[9] K Kurita ldquoChemistry and application of chitin and chitosanrdquoPolymer Degradation and Stability vol 59 no 1ndash3 pp 117ndash1201998

[10] Y-J Jeon F Shahidi and S-K Kim ldquoPreparation of chitinand chitosan oligomers and their applications in physiologicalfunctional foodsrdquo Food Reviews International vol 16 no 2 pp159ndash176 2000

[11] K M Rudall and W Kenchington ldquoThe chitin systemrdquo Biolog-ical Reviews of the Cambridge Philosophical Society vol 48 no4 pp 597ndash636 1973

[12] R A A Muzzarelli Chitin Pergamon Press New York NYUSA 1977

[13] J H Willis ldquoCuticular proteins in insects and crustaceansrdquoAmerican Zoologist vol 39 no 3 pp 600ndash609 1999

[14] J Synowiecki and N A Al-Khateeb ldquoProduction propertiesand some new applications of chitin and its derivativesrdquoCriticalReviews in Food Science andNutrition vol 43 no 2 pp 145ndash1712003

[15] R A A Muzzarelli Natural Chelating Polymers PergamonPress New York NY USA 1973

[16] J P Zikakis Chitin Chitosan and Related Enzymes AcademicPress Orlando Fla USA 1984

[17] K M Rudall ldquoThe chitinprotein complexes of insect cuticlesrdquoAdvances in Insect Physiology vol 1 pp 257ndash313 1963

[18] B Focher A Naggi G Torri A Cosani and M TerbojevichldquoStructural differences between chitin polymorphs and theirprecipitates from solutions-evidence from CP-MAS 13C-NMRFT-IR and FT-Raman spectroscopyrdquo Carbohydrate Polymersvol 17 no 2 pp 97ndash102 1992

[19] HMerzendorfer and L Zimoch ldquoChitinmetabolism in insectsstructure function and regulation of chitin synthases andchitinasesrdquo Journal of Experimental Biology vol 206 no 24 pp4393ndash4412 2003

[20] T Kameda M Miyazawa H Ono and M Yoshida ldquoHydrogenbonding structure and stability of 120572-chitin studied by 13C solid-state NMRrdquo Macromolecular Bioscience vol 5 no 2 pp 103ndash106 2005

[21] K Kurita K Tomita T Tada S Ishii S-I Nishimura and KShimoda ldquoSquid chitin as a potential alternative chitin sourceDeacetylation behavior and characteristic propertiesrdquo Journalof Polymer Science A vol 31 no 2 pp 485ndash491 1993

[22] S S Kim S H Kim and Y M Lee ldquoPreparation character-ization and properties of 120573-chitin and N-acetylated 120573-chitinrdquoJournal of Polymer Science B vol 34 no 14 pp 2367ndash2374 1996

[23] A Chandumpai N Singhpibulporn D Faroongsarng andP Sornprasit ldquoPreparation and physico-chemical character-ization of chitin and chitosan from the pens of the squidspecies Loligo lessoniana and Loligo formosanardquo CarbohydratePolymers vol 58 no 4 pp 467ndash474 2004

[24] K van de Velde and P Kiekens ldquoStructure analysis and degreeof substitution of chitin chitosan and dibutyrylchitin by FT-IRspectroscopy and solid state13C NMRrdquo Carbohydrate Polymersvol 58 no 4 pp 409ndash416 2004

[25] M Rhazi J Desbrieres A Tolaimate A Alagui and P VotteroldquoInvestigation of different natural sources of chitin Influenceof the source and deacetylation process on the physicochemicalcharacteristics of chitosanrdquo Polymer International vol 49 no 4pp 337ndash344 2000

[26] W KenchingtonThe Insect Integument Elsevier Science Ams-terdam The Netherlands 1976

[27] A C A Wan and B C U Tai ldquoCHITINmdasha promisingbiomaterial for tissue engineering and stem cell technologiesrdquoBiotechnology Advances vol 31 no 8 pp 1776ndash1785 2013

[28] M J Bae H S Shin En-Kyoung Kim et al ldquoOral administra-tion of chitin and chitosan prevents peanut-induced anaphy-laxis in a murine food allergy modelrdquo International Journal ofBiological Macromolecules vol 61 pp 164ndash168 2013

[29] R Harikrishnan J-S Kim C Balasundaram and M-S HeoldquoDietary supplementation with chitin and chitosan on haema-tology and innate immune response in Epinephelus bruneusagainst Philasterides dicentrarchirdquo Experimental Parasitologyvol 131 no 1 pp 116ndash124 2012


[30] R Harikrishnan J-S Kim C Balasundaram and M-S HeoldquoImmunomodulatory effects of chitin and chitosan enricheddiets in Epinephelus bruneus against Vibrio alginolyticus infec-tionrdquo Aquaculture vol 326-329 pp 46ndash52 2012

[31] M S Benhabiles R Salah H Lounici N Drouiche M FA Goosen and N Mameri ldquoAntibacterial activity of chitinchitosan and its oligomers prepared from shrimp shell wasterdquoFood Hydrocolloids vol 29 no 1 pp 48ndash56 2012

[32] D Knorr ldquoUse of chitinous polymers in food a challenge forfood research and developmentrdquo Food Technology vol 38 no 1pp 85ndash97 1984

[33] E Furusaki Y Ueno N Sakairi N Nishi and S Tokura ldquoFacilepreparation and inclusion ability of a chitosan derivative bear-ing carboxymethyl-120573-cyclodextrinrdquo Carbohydrate Polymersvol 29 no 1 pp 29ndash34 1996

[34] K Kurita ldquoChemical modifications of chitin and chitosanrdquo inChitin in Nature and Technology pp 287ndash293 Plenum PressNew York NY USA 1986

[35] R R A Muzzarelli Chitin Enzymology European ChitinSociety Ancona Italy 1996

[36] A Razdan and D Pettersson ldquoEffect of chitin and chitosan onnutrient digestibility and plasma lipid concentrations in broilerchickensrdquoBritish Journal of Nutrition vol 72 no 2 pp 277ndash2881994

[37] M W Anthonsen and O Smidsroslashd ldquoHydrogen ion titration ofchitosans with varying degrees of N-acetylation by monitoringinduced 1H-NMR chemical shiftsrdquo Carbohydrate Polymers vol26 no 4 pp 303ndash305 1995

[38] M Rinaudo ldquoChitin and chitosan properties and applicationsrdquoProgress in Polymer Science vol 31 no 7 pp 603ndash632 2006

[39] N Sankararamakrishnan and R Sanghi ldquoPreparation andcharacterization of a novel xanthated chitosanrdquo CarbohydratePolymers vol 66 no 2 pp 160ndash167 2006

[40] S V Madihally and H W T Matthew ldquoPorous chitosanscaffolds for tissue engineeringrdquoBiomaterials vol 20 no 12 pp1133ndash1142 1999

[41] I S Arvanitoyannis A Nakayama and S-I Aiba ldquoChitosanand gelatin based edible films state diagrams mechanical andpermeation propertiesrdquo Carbohydrate Polymers vol 37 no 4pp 371ndash382 1998

[42] I S Arvanitoyannis ldquoTotally and partially biodegradable poly-mer blends based on natural and synthetic macromoleculespreparation physical properties and potential as food packag-ing materialsrdquo Journal of Macromolecular Science C vol 39 no2 pp 205ndash271 1999

[43] T Haque H ChenW Ouyang et al ldquoSuperior cell delivery fea-tures of poly(ethylene glycol) incorporated alginate chitosanand poly-L-lysine microcapsulesrdquo Molecular Pharmaceuticsvol 2 no 1 pp 29ndash36 2005

[44] H-J Kim F Chen X Wang and N C Rajapakse ldquoEffect ofchitosan on the biological properties of sweet basil (Ocimumbasilicum L)rdquo Journal of Agricultural and Food Chemistry vol53 no 9 pp 3696ndash3701 2005

[45] G A F Roberts Chitin Chemistry MacMillan Press LondonUK 1992

[46] K Yamada YAkiba T Shibuya AKashiwada KMatsuda andMHirata ldquoWater purification through bioconversion of phenolcompounds by tyrosinase and chemical adsorption by chitosanbeadsrdquo Biotechnology Progress vol 21 no 3 pp 823ndash829 2005

[47] W Malette H Quigley and E Adickes Chitin in Nature andTechnology Plenum Press 1986

[48] K Arai T Kinumaki and T Fujita Bulletin of Tokai RegionalFisheries Research Laboratory 1968

[49] H Struszczyk H Pospieszny and S Kotlinski ldquoSome newapplication of chitosanrdquo in Chitin and Chitosan G Skjoak-Brok T Anthonsen and P Sandford Eds pp 733ndash742 ElseiverScience London UK 1989

[50] E Onsoyen and O Skaugrud ldquoMetal recovery using chitosanrdquoJournal of Chemical Technology and Biotechnology vol 49 no4 pp 395ndash404 1990

[51] G Lang and T Clausen ldquoThe use of chit os an in cosmeticsrdquoin Chitin and Chitosan G Skjoak-Brok T Anthonsen and PSandford Eds pp 139ndash147 Elseiver Science London UK 1989

[52] N A Ashford D B Hattis and A E Murray MIT Sea GrantProgram Massachusetts Institute of Technology 1977

[53] Y Fukada K Kimura and Y Ayaki ldquoEffect of chitosan feedingon intestinal bile acid metabolism in ratsrdquo Lipids vol 26 no 5pp 395ndash399 1991

[54] Y Shigemasa and SMinami Biotechnology ampGenetic Engineer-ing Reviews Intercept 1995

[55] K Nishimura S-I Nishimura and H Seo ldquoEffect of multi-porous microspheres derived from chitin and partially deacety-lated chitin on the activation ofmouse peritonealmacrophagesrdquoVaccine vol 5 no 2 pp 136ndash140 1987

[56] J Dutkiewicz and M Kucharska Advances in Chitin andChitosan Elsevier Science 1992

[57] H Seo K Mitsuhashi and H Tanibe Advances in Chitin andChitosan Elsevier Science 1992

[58] R Muzzarelli G Biagini A Pugnaloni et al ldquoReconstructionof parodontal tissue with chitosanrdquo Biomaterials vol 10 no 9pp 598ndash603 1989

[59] C Shi Y Zhu X Ran et al ldquoTherapeutic potential of chitosanand its derivatives in regenerative medicinerdquo Journal of SurgicalResearch vol 133 no 2 pp 185ndash192 2006

[60] S Hirano and N Nagao ldquoEffects of chitosan pectic acidlysozyme andchitinase on the growth of several phytopatho-gensrdquo Agricultural and Biological Chemistry vol 53 no 11 pp3065ndash3066 1989

[61] D F Kendra and L A Hadwiger ldquoCharacterization of thesmallest chitosan oligomer that is maximally antifungal toFusarium solani and elicits pisatin formation in Pisum sativumrdquoExperimental Mycology vol 8 no 3 pp 276ndash281 1984

[62] Y Uchida M Izume and A Ohtakara Chitin and ChitosanElsevier London UK 1989

[63] K Ueno T Yamaguchi N Sakairi et al Advances in ChitinScience Jacques Andre Lyon France 1997

[64] T J Franklin and G A Snow Biochemistry of AntimicrobialAction Chapman and Hall London UK 3rd edition 1981

[65] K Takemono J Sunamoto and M Askasi Polymers andMedical Care Mita Tokyo 1989

[66] J C Fernandes H Spindola V De Sousa et al ldquoAnti-inflammatory activity of chitooligosaccharides in vivordquoMarineDrugs vol 8 no 6 pp 1763ndash1768 2010

[67] R Huang E Mendis N Rajapakse and S-K Kim ldquoStrongelectronic charge as an important factor for anticancer activityof chitooligosaccharides (COS)rdquo Life Sciences vol 78 no 20 pp2399ndash2408 2006

[68] C Ju W Yue Z Yang et al ldquoAntidiabetic effect and mecha-nism of chitooligosaccharidesrdquo Biological and PharmaceuticalBulletin vol 33 no 9 pp 1511ndash1516 2010


[69] E J Jung D K Youn S H Lee H K No J G Ha andW Prinyawiwatkul ldquoAntibacterial activity of chitosans withdifferent degrees of deacetylation and viscositiesrdquo InternationalJournal of Food Science and Technology vol 45 no 4 pp 676ndash682 2010

[70] M Artan F Karadeniz M Z Karagozlu M-M Kim and S-K Kim ldquoAnti-HIV-1 activity of low molecular weight sulfatedchitooligosaccharidesrdquo Carbohydrate Research vol 345 no 5pp 656ndash662 2010

[71] D-N Ngo S-H Lee M-M Kim and S-K Kim ldquoProductionof chitin oligosaccharides with different molecular weightsand their antioxidant effect in RAW 2647 cellsrdquo Journal ofFunctional Foods vol 1 no 2 pp 188ndash198 2009

[72] H Quan F Zhu X Han Z Xu Y Zhao and Z Miao ldquoMecha-nism of anti-angiogenic activities of chitooligosaccharides maybe through inhibiting heparanase activityrdquoMedical Hypothesesvol 73 no 2 pp 205ndash206 2009

[73] Y Gong L Gong X Gu and F Ding ldquoChitooligosaccharidespromote peripheral nerve regeneration in a rabbit commonperoneal nerve crush injury modelrdquo Microsurgery vol 29 no8 pp 650ndash656 2009

[74] J-S Moon H-K Kim H C Koo et al ldquoThe antibacterial andimmunostimulative effect of chitosan-oligosaccharides againstinfection by Staphylococcus aureus isolated from bovine masti-tisrdquo Applied Microbiology and Biotechnology vol 75 no 5 pp989ndash998 2007

[75] V K Mourya N N Inamdar and Y M Choudhari ldquoChi-tooligosaccharides synthesis characterization and applica-tionsrdquo Polymer Science A vol 53 no 7 pp 583ndash612 2011

[76] B B Aam E BHeggset A L NorbergM Soslashrlie KMVarumand V G H Eijsink ldquoProduction of chitooligosaccharides andtheir potential applications in medicinerdquo Marine Drugs vol 8no 5 pp 1482ndash1517 2010

[77] A V Ilrsquoina and V P Varlamov ldquoHydrolysis of chitosan in lacticacidrdquo Applied Biochemistry and Microbiology vol 40 no 3 pp300ndash303 2004

[78] T Ando and S Kataoka ldquoAcylation of chtin in trichloroaceticacid based solventrdquo Kobunshi Ronbunshu vol 37 no 1 pp 1ndash71980

[79] R Yamaguchi Y Arai and T Itoh ldquoA microfibril formationfrom depolymerized chitosan by N-acetylationrdquo Agriculturaland Biological Chemistry vol 46 no 9 pp 2379ndash2381 1982

[80] Y-J Jeon P-J Park and S-K Kim ldquoAntimicrobial effect ofchitooligosaccharides produced by bioreactorrdquo CarbohydratePolymers vol 44 no 1 pp 71ndash76 2001

[81] C Qin B Zhou L Zeng et al ldquoThe physicochemical propertiesand antitumor activity of cellulase-treated chitosanrdquo FoodChemistry vol 84 no 1 pp 107ndash115 2004

[82] D PantaleoneM Yalpani andM Scollar ldquoUnusual susceptibil-ity of chitosan to enzymic hydrolysisrdquo Carbohydrate Researchvol 237 pp 325ndash332 1992

[83] E Mendis M-M Kim N Rajapakse and S-K Kim ldquoAnin vitro cellular analysis of the radical scavenging efficacy ofchitooligosaccharidesrdquo Life Sciences vol 80 no 23 pp 2118ndash2127 2007

[84] R A A Muzzarelli Chitin Oxford Pergamon Press LondonUK 1977

[85] C Qin Y Du L Xiao Z Li and X Gao ldquoEnzymic preparationof water-soluble chitosan and their antitumor activityrdquo Interna-tional Journal of Biological Macromolecules vol 31 no 1ndash3 pp111ndash117 2002

[86] Y Maeda and Y Kimura ldquoAntitumor effects of various low-molecular-weight chitosans are due to increased natural killeractivity of intestinal intraepithelial lymphocytes in sarcoma 180-bearing micerdquo Journal of Nutrition vol 134 no 4 pp 945ndash9502004

[87] Q Xu J Dou P Wei et al ldquoChitooligosaccharides induceapoptosis of human hepatocellular carcinoma cells via up-regulation of Baxrdquo Carbohydrate Polymers vol 71 no 4 pp509ndash514 2008

[88] R Pangestuti S-S Bak and S-K Kim ldquoAttenuation of pro-inflammatory mediators in LPS-stimulated BV2 microglia bychitooligosaccharides via the MAPK signaling pathwayrdquo Inter-national Journal of Biological Macromolecules vol 49 no 4 pp599ndash606 2011

[89] Y X Mei H X Chen J Zhang et al ldquoProtective effectof chitooligosaccharides against cyclophosphamide-inducedimmunosuppression inmicerdquo International Journal of BiologicalMacromolecules vol 62 pp 330ndash335 2013

[90] X Wei W Chen F Mao et al ldquoEffect of chitooligosaccharideson mice hematopoietic stemprogenitor cellsrdquo InternationalJournal of Biological Macromolecules vol 54 pp 71ndash75 2013

[91] M Senevirathne C-B Ahn and J-Y Je ldquoHepatoprotectiveeffect of chitooligosaccharides against tert-butylhydroperoxide-induced damage in Chang liver cellsrdquo Carbohydrate Polymersvol 83 no 2 pp 995ndash1000 2011

[92] T-S Vo C-S Kong and S-K Kim ldquoInhibitory effects of chi-tooligosaccharides on degranulation and cytokine generation inrat basophilic leukemia RBL-2H3 cellsrdquoCarbohydrate Polymersvol 84 no 1 pp 649ndash655 2011

[93] D Katiyar B Singh A M Lall and C Haldar ldquoEfficacy of chi-tooligosaccharides for the management of diabetes in alloxaninduced mice A correlative study with antihyperlipidemicand antioxidative activityrdquo European Journal of PharmaceuticalSciences vol 44 no 4 pp 534ndash543 2011

[94] D-N Ngo Z-J Qian J-Y Je M-M Kim and S-KKim ldquoAminoethyl chitooligosaccharides inhibit the activity ofangiotensin converting enzymerdquo Process Biochemistry vol 43no 1 pp 119ndash123 2008

[95] P-J Park J-Y Je and S-K Kim ldquoAngiotensin I convertingenzyme (ACE) inhibitory activity of hetero-chitooligosaccha-rides prepared from partially different deacetylated chitosansrdquoJournal of Agricultural and Food Chemistry vol 51 no 17 pp4930ndash4934 2003

[96] D-N Ngo M-M Kim and S-K Kim ldquoProtective effectsof aminoethyl-chitooligosaccharides against oxidative stress inmouse macrophage RAW 2647 cellsrdquo International Journal ofBiological Macromolecules vol 50 no 3 pp 624ndash631 2012

[97] D-H Ngo D-N Ngo T-S Vo B Ryu Q Van Ta and S-K Kim ldquoProtective effects of aminoethyl-chitooligosaccharidesagainst oxidative stress and inflammation in murine microglialBV-2 cellsrdquo Carbohydrate Polymers vol 88 no 2 pp 743ndash7472012

[98] M Z Karagozlu F Karadeniz C-S Kong and S-K KimldquoAminoethylated chitooligomers and their apoptotic activity onAGS human cancer cellsrdquo Carbohydrate Polymers vol 87 no 2pp 1383ndash1389 2012

[99] M Z Karagozlu J-A Kim F Karadeniz C-S Kong andS-K Kim ldquoAnti-proliferative effect of aminoderivatized chi-tooligosaccharides on AGS human gastric cancer cellsrdquo ProcessBiochemistry vol 45 no 9 pp 1523ndash1528 2010

[100] N Rajapakse M-M Kim E Mendis R Huang and S-K KimldquoCarboxylated chitooligosaccharides (CCOS) inhibit MMP-9


expression in human fibrosarcoma cells via down-regulation ofAP-1rdquo Biochimica et Biophysica Acta vol 1760 no 12 pp 1780ndash1788 2006

[101] Y Jiang and R J Muschel ldquoRegulation of matrix metalloprote-inase-9 (MMP-9) by translational efficiency in murine prostatecarcinoma cellsrdquo Cancer Research vol 62 no 6 pp 1910ndash19142002

[102] R Huang E Mendis and S-K Kim ldquoImprovement of ACEinhibitory activity of chitooligosaccharides (COS) by carboxylmodificationrdquo Bioorganic and Medicinal Chemistry vol 13 no11 pp 3649ndash3655 2005

[103] N Rajapakse M-M Kim E Mendis and S-K Kim ldquoInhibi-tion of free radical-mediated oxidation of cellular biomoleculesby carboxylated chitooligosaccharidesrdquo Bioorganic and Medici-nal Chemistry vol 15 no 2 pp 997ndash1003 2007

[104] J H J R Makoi and P A Ndakidemi ldquoBiological ecologicaland agronomic significance of plant phenolic compounds inrhizosphere of the symbiotic legumesrdquo African Journal ofBiotechnology vol 6 no 12 pp 1358ndash1368 2007

[105] T Li X Zhang and X Zhao ldquoPowerful protective effects ofgallic acid and tea polyphenols on human hepatocytes injuryinduced by hydrogen peroxide or carbon tetrachloride in vitrordquoJournal of Medicinal Plant Research vol 4 no 3 pp 247ndash2542010

[106] H N Graham ldquoGreen tea composition consumption andpolyphenol chemistryrdquo Preventive Medicine vol 21 no 3 pp334ndash350 1992

[107] S-H Kim C-D Jun K Suk et al ldquoGallic acid inhibitshistamine release and pro-inflammatory cytokine productionin mast cellsrdquo Toxicological Sciences vol 91 no 1 pp 123ndash1312006

[108] D-H Ngo Z-J Qian D-N Ngo T-S Vo IWijesekara and S-K Kim ldquoGallyl chitooligosaccharides inhibit intracellular freeradical-mediated oxidationrdquo Food Chemistry vol 128 no 4 pp974ndash981 2011

[109] D-H Ngo Z-J Qian T-S Vo B Ryu D-N Ngo and S-KKim ldquoAntioxidant activity of gallate-chitooligosaccharides inmouse macrophage RAW2647 cellsrdquo Carbohydrate Polymersvol 84 no 4 pp 1282ndash1288 2011

[110] S H Arshad ldquoDoes exposure to indoor allergens contribute tothe development of asthma and allergyrdquo Current Allergy andAsthma Reports vol 10 no 1 pp 49ndash55 2010

[111] E Milian and A M Dıaz ldquoAllergy to house dust mites andasthmardquo Puerto Rico health sciences journal vol 23 no 1 pp47ndash57 2004

[112] T S Vo D H Ngo and S K Kim ldquoGallic acid-grafted chi-tooligosaccharides suppress antigen-induced allergic reactionsin RBL-2H3 mast cellsrdquo European Journal of PharmaceuticalSciences vol 47 no 2 pp 527ndash533 2012

[113] N Vionnet M Stoffel J Takeda et al ldquoNonsense mutation inthe glucokinase gene causes early-onset non-insulin-dependentdiabetes mellitusrdquo Nature vol 356 no 6371 pp 721ndash722 1992

[114] D Von Bubnoff N Novak S Kraft and T Bieber ldquoThe centralrole of Fc120576Ri in allergyrdquoClinical and Experimental Dermatologyvol 28 no 2 pp 184ndash187 2003

[115] H Katagiri T Asano H Ishihra et al ldquoNonsense mutation ofglucokinase gene in late-onset non-insulin-dependent diabetesmellitusrdquo Lancet vol 340 no 8831 pp 1316ndash1317 1992

[116] H Ishihara T Asano K Tsukuda et al ldquoPancreatic beta cellline MIN6 exhibits characteristics of glucose metabolism andglucose-stimulated insulin secretion similar to those of normalisletsrdquo Diabetologia vol 36 no 11 pp 1139ndash1145 1993

[117] X Lu H Guo L Sun et al ldquoProtective effects of sulfatedchitooligosaccharides with different degrees of substitution inMIN6 cellsrdquo International Journal of Biological Macromoleculesvol 52 pp 92ndash98 2013

[118] X Lu H Guo and Y Zhang ldquoProtective effects of sul-fated chitooligosaccharides against hydrogen peroxide-induceddamage in MIN6 cellsrdquo International Journal of BiologicalMacromolecules vol 50 no 1 pp 50ndash58 2012

[119] S J Lee E K Kim J W Hwang et al ldquoAntioxidative effectsof sulfated chitooligosaccharides on oxidative injuryrdquo Journal ofChitin and Chitosan Science vol 14 no 4 pp 192ndash196 2009

[120] Z J Qian T K Eom B Ryu et al ldquoAngiotensin I-convertingenzyme inhibitory activity of sulfated chitooligosaccharideswith different molecular weightsrdquo Journal of Chitin and Chi-tosan Science vol 15 no 2 pp 75ndash79 2010

[121] J-Y Je E-K Kim C-B Ahn et al ldquoSulfated chitooligosaccha-rides as prolyl endopeptidase inhibitorrdquo International Journal ofBiological Macromolecules vol 41 no 5 pp 529ndash533 2007


[123] F Karadeniz M Z Karagozlu S-Y Pyun and S-K KimldquoSulfation of chitosan oligomers enhances their anti-adipogeniceffect in 3T3-L1 adipocytesrdquoCarbohydrate Polymers vol 86 no2 pp 666ndash671 2011

[124] B Ryu S W A Himaya R J Napitupulu T-K Eom and S-K Kim ldquoSulfated chitooligosaccharide II (SCOS II) suppresscollagen degradation in TNF-induced chondrosarcoma cells viaNF-120581B pathwayrdquo Carbohydrate Research vol 350 pp 55ndash612012

[125] M-C Figueroa-Espinoza and P Villeneuve ldquoPhenolic enzy-matic lipophilizationrdquo Journal of Agricultural and Food Chem-istry vol 53 no 8 pp 2779ndash2787 2005

[126] T K Eom M Senevirathne and S K Kim ldquoSynthesis ofphenolic acid conjugated chitooligosaccharides and evaluationof their antioxidant activityrdquo Environmental Toxicology andPharmacology vol 34 no 2 pp 519ndash527 2012

Submit your manuscripts athttpwwwhindawicom

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poor solubilitymakes themdifficult to use in food and biome-dicinal applications Unlike chitosan its hydrolyzed productsand COS are readily soluble in water due to their shorterchain lengths and free amino groups in D-glucosamine units[10]The low viscosity and greater solubility of COS at neutralpH have attracted the interest of many researchers to utilizechitosan in its oligosaccharide form Especially research onCOS in food and nutrition fields has emphasized their abilityto improve food quality and human health progression

In light of the recent interest in the biomedical appli-cations of chitin chitosan and its derivatives this reviewfocuses on the preparation and biological activities of chitinchitosan COS and their derivatives

2 Chitin

Chitin (Figure 1) a mucopolysaccharide and the supportingmaterial of crustaceans and insects is the second most abun-dant polymer after cellulose found in nature it is producedby many living organisms and is present usually in a complexwith other polysaccharides and proteins Chitin was found asa major component in arthropods (insects crustaceans ara-chnids and myriapods) nematodes algae and fungi [11ndash14]

Its immunogenicity is exceptionally low in spite of thepresence of nitrogen It is highly insoluble material resem-bling cellulose with its solubility and low chemical reactivityIt may be regarded as cellulose with hydroxyl at position C-2replaced by an acetamido group Like cellulose it functionsnaturally as a structural polysaccharide It is white hardinelastic nitrogenous polysaccharide [15 16]

Chitin is a linear polysaccharide composed of (1 rarr 4)linked 2-acetamido-2-deoxy-120573-d-glucopyranosyl units andoccurs naturally in three polymorphic forms with differentorientations of the microfibrils known as 120572- 120573- and 120574-chitin[1 17] The 120572-form has antiparallel chains and is a commonand the most stable polymorphic form of chitin in naturewhich is prevalent in crustaceans and in insect chitinouscuticles [18ndash20]The 120573-form of chitin is rare it occurs in pensof mollusks and is characterized by a loose-packing paral-lel chains fashion with weak intermolecular interactions andhigher solubility and swelling than 120572-form 120573-chitin was pre-pared from the pens of the squidOmmastrephes bartrami [2122] Loligo species and cuttlefish (Sepia officinalis) [18 23ndash25] The 120574-form is characterized by a mixture of antiparalleland parallel chains and was found in the cocoons of insects[26]

Besides its application as a starting material for thesynthesis of chitosan and chitooligosaccharides chitin itselfhas been a center of many therapeutic applications and isthought to be a promising biomaterial for tissue engineeringand stem cell technologies [27]

Bae et al [28] demonstrated that oral administrationof chitin (120572 and 120573 forms) is beneficial in preventing foodallergies the oral administration of chitin was accomplishedby milling it to particle size less than 20 120583m and mixing itwith feed Their results showed that 120572-form reduced serumlevels of peanut-specific IgE and both the forms decreasedthe levels of interleukins (IL) IL-5 and IL-10 and increased

the levels of IL-12 Dietary supplementation of chitin hasshown to exert positive immunomodulatory effects [29 30]antibacterial activity of chitin prepared from shrimp shellwaste was reported by Benhabiles et al [31]

3 Chitosan

Chitosan [poly-(120573-14)-2-amino-2-deoxy-D-glucopyranose]is a natural nontoxic biopolymer produced by the deacetyla-tion of chitin Chitosan (Figure 1) has three types of reactivefunctional groups an amino group as well as both primaryand secondary hydroxyl groups at the C-2 C-3 and C-6 posi-tions respectively Chemical modifications of these groupshave provided numerous useful materials in different fieldsof application Currently chitosan has received considerableattention for its commercial applications in the biomedicalfood and chemical industries [9 32ndash36]

Chitosan solubility biodegradability reactivity and ad-sorption of many substrates depend on the amount of pro-tonated amino groups in the polymeric chain and thereby onthe proportion of acetylated and nonacetylated glucosamineunits The amino groups (pKa from 62 to 70) are completelyprotonated in acids with pKa smaller than 62 making chitos-an soluble Chitosan is insoluble in water organic solventsand aqueous bases and it is soluble after stirring in acids suchas acetic nitric hydrochloric perchloric and phosphoric[37ndash39]

Applications of chitin are limited compared to chitosanbecause chitin is chemically inert and is insoluble in bothwater and acid while chitosan is relatively reactive and can beproduced in various forms Chitosan is normally insoluble inneutral or basic pH conditions while being soluble in acidicpH The solubility of chitosan depends upon the distributionof free amino and N-acetyl groups In dilute acids (pH lt6) the free amino groups are protonated and the moleculebecomes soluble [40]

Due to its unique biological characteristics including bi-odegradability and nontoxicity many applications have beenfound either alone or blended with other natural polymers(starch gelatin and alginates) in the food pharmaceuticaltextile agriculture water treatment and cosmetics industries[41ndash46]

Chitosan lacks irritant or allergic effects and is biocom-patible with both healthy and infected human skin [47]When chitosan was administered orally in mice the LD

50

was found to be in excess of 16 gkg which is higher thanthat of sucrose [48] The intriguing properties of chitosanhave been known previously and the polymer has been usedin the fields of agriculture industry and medicine In agri-culture chitosan has been described as a plant antivirusan additive in liquid multicomponent fertilizers [49] andit has also been investigated as a metal-recovering agent inagriculture and industry [50] Chitosan has been noted for itsapplication as a film-forming agent in cosmetics [51] a dyebinder for textiles a strengthening additive in paper [52] anda hypolipidic material in diets [53] It has been used exten-sively as a biomaterial [54] owing to its immunostimulatoryactivities [55] anticoagulant properties [56] antibacterial


Chitin

Chitosan


OO

NH

O

OO

OH

OH

NH

O

HO

HO

O

OH

O

HO

OO

HO

HOOH

OHOH

HO

O

NHR

OH

O

HO

OO

HO

HO

NHRNHR

OH

OHOH

HO

n


NH2NH2

NH2

CH3

CH3

n

n

Figure 1
























O

OR

O

HOn

R1 = COCH3

R = (CH2)2NH2

NHR1






COS

Reaction mixture


Constant stirring

NaOH dropwise

Filtered


Freeze-dried

40∘C








O O

OH

NRO

O

O

CH2OH

R = H or COCH3








Succinic anhydride

(different amounts)

acetone



Reaction mixture


Methanol

+







O OO

OO

OHOHOH

OH

NH

HOHO

HOHO

OC

OH

OH

HO

n

NH2

NH2




Solution A


Filtered

Diethyl ether added




Freeze-dried


stirring for 5h









O

O

NHO

OO

R1

R1

R2

R1 = SO3 H

R2 = SO3 H OAc







2O2 The







Chitosan

Sulphating agents

Reaction mixture

Cooled





Freeze-dried

Dropwise















Acknowledgment


References










































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Chitin

Chitosan


OO

NH

O

OO

OH

OH

NH

O

HO

HO

O

OH

O

HO

OO

HO

HOOH

OHOH

HO

O

NHR

OH

O

HO

OO

HO

HO

NHRNHR

OH

OHOH

HO

n


NH2NH2

NH2

CH3

CH3

n

n

Figure 1
























O

OR

O

HOn

R1 = COCH3

R = (CH2)2NH2

NHR1






COS

Reaction mixture


Constant stirring

NaOH dropwise

Filtered


Freeze-dried

40∘C








O O

OH

NRO

O

O

CH2OH

R = H or COCH3








Succinic anhydride

(different amounts)

acetone



Reaction mixture


Methanol

+







O OO

OO

OHOHOH

OH

NH

HOHO

HOHO

OC

OH

OH

HO

n

NH2

NH2




Solution A


Filtered

Diethyl ether added




Freeze-dried


stirring for 5h









O

O

NHO

OO

R1

R1

R2

R1 = SO3 H

R2 = SO3 H OAc







2O2 The







Chitosan

Sulphating agents

Reaction mixture

Cooled





Freeze-dried

Dropwise















Acknowledgment


References










































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






















O

OR

O

HOn

R1 = COCH3

R = (CH2)2NH2

NHR1






COS

Reaction mixture


Constant stirring

NaOH dropwise

Filtered


Freeze-dried

40∘C








O O

OH

NRO

O

O

CH2OH

R = H or COCH3








Succinic anhydride

(different amounts)

acetone



Reaction mixture


Methanol

+







O OO

OO

OHOHOH

OH

NH

HOHO

HOHO

OC

OH

OH

HO

n

NH2

NH2




Solution A


Filtered

Diethyl ether added




Freeze-dried


stirring for 5h









O

O

NHO

OO

R1

R1

R2

R1 = SO3 H

R2 = SO3 H OAc







2O2 The







Chitosan

Sulphating agents

Reaction mixture

Cooled





Freeze-dried

Dropwise















Acknowledgment


References










































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




O O

OH

NRO

O

O

CH2OH

R = H or COCH3








Succinic anhydride

(different amounts)

acetone



Reaction mixture


Methanol

+







O OO

OO

OHOHOH

OH

NH

HOHO

HOHO

OC

OH

OH

HO

n

NH2

NH2




Solution A


Filtered

Diethyl ether added




Freeze-dried


stirring for 5h









O

O

NHO

OO

R1

R1

R2

R1 = SO3 H

R2 = SO3 H OAc







2O2 The







Chitosan

Sulphating agents

Reaction mixture

Cooled





Freeze-dried

Dropwise















Acknowledgment


References










































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




O OO

OO

OHOHOH

OH

NH

HOHO

HOHO

OC

OH

OH

HO

n

NH2

NH2




Solution A


Filtered

Diethyl ether added




Freeze-dried


stirring for 5h









O

O

NHO

OO

R1

R1

R2

R1 = SO3 H

R2 = SO3 H OAc







2O2 The







Chitosan

Sulphating agents

Reaction mixture

Cooled





Freeze-dried

Dropwise















Acknowledgment


References










































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










O

O

NHO

OO

R1

R1

R2

R1 = SO3 H

R2 = SO3 H OAc







2O2 The







Chitosan

Sulphating agents

Reaction mixture

Cooled





Freeze-dried

Dropwise















Acknowledgment


References










































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Chitosan

Sulphating agents

Reaction mixture

Cooled





Freeze-dried

Dropwise















Acknowledgment


References










































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







Acknowledgment


References










































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials















































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



review article chitooligosaccharide and its derivatives...

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