Synthesis of Quaternary Ammonium Salts of Chitosan Bearing Halogenated Acetate for Antifungal

14 Pages·2017·1.67 MB·English

polymers
Article
Synthesis of Quaternary Ammonium Salts of
Chitosan Bearing Halogenated Acetate for Antifungal
and Antibacterial Activities
JingjingZhang1,2,WenqiangTan1,2,FangLuan1,2,XiuliYin1,*,FangDong1,QingLi1
andZhanyongGuo1,2,* ID
1 KeyLaboratoryofCoastalBiologyandBioresourceUtilization,YantaiInstituteofCoastalZoneResearch,
ChineseAcademyofSciences,Yantai264003,China;[email protected](J.Z.);[email protected](W.T.);
fl[email protected](F.L.);[email protected](F.D.);[email protected](Q.L.)
2 UniversityofChineseAcademyofSciences,Beijing100049,China
* Correspondence:[email protected](X.Y.);[email protected](Z.G.);Tel.:[phone](Z.G.)
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:19April2018;Accepted:10May2018;Published:15May2018
Abstract: In this paper, quaternary ammonium salts of chitosan bearing halogenated acetate,
includingN,N,N-trimethylchitosanchloroacetate(TMCSC),N,N,N-trimethylchitosandichloroacetate
(TMCSDC), N,N,N-trimethyl chitosan trichloroacetate (TMCSTC), and N,N,N-trimethyl chitosan
trifluoroacetate(TMCSTF),werepreparedviaN,N,N-trimethylchitosaniodide(TMCSI).Theobtained
chitosanderivativeswerecharacterizedbyFT-IR,1HNMRspectra,13CNMRspectra,andelemental
analysis. Their antifungal property against Fusarium oxysporum f. sp. cucumebrium Owen
(F.oxysporumf.sp.cucumebriumOwen),Botrytiscinerea(B.cinerea),andPhomopsisasparagi(P.asparagi)
were evaluated by hyphal measurement method at concentrations ranging from 0.08 mg/mL to
0.8 mg/mL. Meanwhile, two common bacteria, Escherichia coli (E. coli) and Staphylococcus aureus
(S.aureus),wereselectedasthemodelGram-negativeandGram-positivebacteriatoevaluatethe
antibacterialpropertyofthechitosanderivativesbyagarwelldiffusionmethod. Theresultsshowed
thatTMCSC,TMCSDC,TMCSTC,andTMCSTFhadbetterantifungalandantibacterialactivities
thanchitosanandTMCSI.Inparticular,aruleshowedthattheinhibitoryactivitydecreasedinthe
order: TMCSTF>TMCSTC>TMCSDC>TMCSC>TMCSI>chitosan,whichwasconsistentwith
the electron-withdrawing property of different halogenated acetate. Apparently, the quaternary
ammoniumsaltsofchitosanwithstrongerelectronwithdrawingabilitypossessedrelativelygreater
antifungalandantibacterialactivities. Thisexperimentprovidesapotentiallyfeasiblemethodforthe
furtherutilizationofchitosaninfieldsofantifungalandantibacterialbiomaterials.
Keywords:quaternaryammoniumsaltsofchitosan;halogenatedacetate;characterization;antifungal
activity;antibacterialactivity
1. Introduction
Phytopathogenicfungihavebeensevereconstraintsofcropproductionandfoodsecurityglobally,
whichmayleadtogreatlossestohumans[1]. Inaddition,E.coliandS.aureushavehistoricallybeen
themajorhumanpathogenandcontinuetobethemostcommonlyimplicatedbacteriacausinghuman
disease[2]. Inthepastfewdecades,thelarge-scaleuseofchemicalfungicidesandantibioticswas
thedominanttooltoaddressthesedisasters[3]. However,theextensiveapplicationofsuchchemical
fungicides and antibiotics often causes drug resistance in the fungi and bacteria. Drug-resistant
bacterialandfungalstrainscanposeaseriousthreattohumanhealthgloballyanditobligesresearchers
toseeknewalternatives[4,5].Therefore,developingsecureandecofriendlyalternativesthatcancontrol
microorganismsaswellasdecreaseenvironmentalriskhasbecomeaneffectivetactic[6].
Polymers2018,10,530;doi:10.3390/polym10050530 www.mdpi.com/journal/polymers
Polymers2018,10,530 2of14
As one of the most abundant natural polysaccharides, chitosan has unique physiochemical
characteristics and bioactivities including biocompatibility, low toxicity, hemostatic activity,
wound-healingproperties,andantimicrobialactivitythatcanbeappliedinthefieldsofbiotechnology,
pharmaceutics,wastewater,cosmetics,agriculture,foodscience,textiles,andsoon[7–10]. Meanwhile,
duetotheseexcellentproperties,chitosancanbeconsideredasagreatalternativeofantimicrobial
medicineorfungicide.However,theapplicationofpristinechitosanasafungicidehasbeenlimiteddue
toitslowersolubilityandweakeractivitycomparedtocurrentantimicrobialsonthemarket[11–13].
In order to meet the requirements of commercial medicine or fungicides, modifying the structure
of chitosan is a common means to improve its solubility and other properties. Exactly, various
derivativesofchitosanwithbetterwatersolubilityandbioactivitieshavebeensynthesizedthrough
chemicalmodificationsuchasSchiffbasesofchitosan,N-substitutedchitosan,carboxymethylchitosan,
quaternizedchitosan,andsoon[14,15]. Amongvariousderivativesofchitosan,quaternizedchitosan
has drawn people’s attention for its excellent bioactivity, especially antimicrobial and antioxidant
activities. Furthermore, we found that quaternized chitosan had better antioxidant activity than
chitosan, Schiff bases of chitosan, and N-substituted chitosan, which was caused by the cation of
quaternizedchitosan[14]. Andtheantifungalandantioxidantactivitiesofquaternizedchitosanare
affectedbytheactivegroups,whichgraftedtochitosanaccordingtoearlierreports[12,16].
Except quaternized chitosan, chitosan ammonium salt is another kind of chitosan derivative
whichhasanobviouscation. Meanwhile,thebetterantifungalactivityofchitosanammoniumsalt
thanchitosanagainstC.cucumerinum(Ell.) etArthur,M.fructicola(Wint.) Honey,andF.oxysporumf.
sp. cucumissativusLwerealsoreportedinapastpaper[17]. Nowthatbothquaternizedchitosan
andchitosanammoniumsalthavemorepositivecharge,whichistheprincipalfactorthatcausesthe
enhancementofchitosan’sbioactivities,quaternaryammoniumsaltofchitosanmaybeanexcellent
antifungalorantibacterialagent. Inaddition,theelectron-withdrawingsubstitution–halogenscanplay
keyrolesintheantifungalandantibacterialpropertiesofcompounds,whichcandisruptcellwalls
andmembranestoleadtothedeathofmicroorganisms[18–20]. Therefore,theintroductionofthese
groupsbearingvarioustypesandquantitiesofhalogensintoquaternaryammoniumsaltofchitosan
couldhelpenhancethebioactivitiesofquaternaryammoniumsaltofchitosan.
In order to study the effect of halogen atoms on the bioactivities of chitosan, the synthesis
of TMCSC, TMCSDC, TMCSTC, and TMCSTF were reported. Firstly, the C –NH of chitosan
2 2
wasmodifiedasquaternizedchitosan,whichactedasanintermediatethatcouldformquaternary
ammonium salts of chitosan bearing different halogenated acetate. Then, the final products were
synthesizedthroughionexchangebetweenN,N,N-trimethylchitosaniodideandsodiumhaloacetic.
ThechemicalstructuresofthederivativeswerecharacterizedbyFT-IR,1HNMRspectra,13CNMR
spectra, and elemental analysis. Meanwhile, their antifungal activity against Fusarium oxysporum
f. sp. cucumerium Owen (F. oxysporum f. sp. cucumerium Owen: ATCC42357), Botrytis cinerea
(B. cinerea: ATCC48340), and Phomopsis asparagi (P. asparagi: ATCC24625) were measured in this
paper. Furthermore,wetestedtheinhibitingabilityofsynthesizedderivativeswithbacteria,including
Escherichiacoli(E.coli: ATCC27325)andStaphylococcusaureus(S.aurous: ATCC25923). Accordingly,we
havediscussedtherelationshipbetweenthestructuresandbioactivitiesofchitosanderivativesbriefly.
2. MaterialsandMethods
2.1. Materials
Chitosan was purchased from Qingdao Yunzhou Biochemistry Co., Ltd. (Qingdao, China).
The degree of deacetylation was 81% and the viscosity average molecular weight was 7.8 × 103.
Iodomethane, sodium iodide, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, and
trifluoroaceticacidwerepurchasedfromSigma-AldrichChemicalCorp. (Shanghai,China). N-methyl-
2-pyrrolidone,ethanol,sodiumhydroxide,andsodiumchloridewereallpurchasedfromSinopharm
ChemicalReagentCo.,Ltd. (Shanghai,China). Thereagentswereanalyticalgradeandusedwithout
Polymers2018,10,530 3of14
further purification. Fungi Medium was purchased from Qingdao Hop Bio-Technogy Co., Ltd.
(Qingdao,China). AgarPowderwaspurchasedfromBeijingChembaseTechnologyCo.,Ltd. (Beijing,
China). TryptoneandYeastExtractPowderwerepurchasedfromBeijingAoboxingBio-TechCo.,Ltd.
(Beijing,China).
2.2. AnalyticalMethods
2.2.1. FourierTransformInfrared(FT-IR)Spectroscopy
FT-IRspectrometers,intherangeof4000–400cm−1withresolutionof4.0cm−1,wererecorded
on a Jasco-4100 (Tokyo, Japan, provided by JASCO China (Shanghai) Co. Ltd., Shanghai, China).
AllsamplesweregroundandmixedwithKBrdisksfortesting.
2.2.2. NuclearMagneticResonance(NMR)Spectroscopy
1H Nuclear Magnetic Resonance (1H NMR) spectra and 13C Nuclear Magnetic Resonance
(13CNMR)spectrawereallmeasuredusingaBrukerAVIII-500Spectrometer(500MHz,Switzerland,
providedbyBrukerTech. andServ. Co.,Ltd.,Beijing,China)at25◦Candusing99.9%Deuterium
Oxide(D O)assolvents.
2
2.2.3. ElementalAnalysis
The elemental analyses by combustion were used to evaluate the degrees of substitution in
chitosanderivatives. Theanalysesofelementalcarbon,hydrogen,andnitrogeninchitosanderivatives
were performed on a Vario Micro Elemental Analyzer (Elementar, Germany). The degrees of
substitution(DS)ofchitosanderivativeswerecalculatedonthebasisofthepercentagesofcarbonand
nitrogenaccordingtothefollowingequations[21]:
n ×M −M × W
DS = 1 C N C/N
1 n ×M
2 C
M × W +n ×M ×DS −n ×M
DS = N C/N 2 C 1 1 C
2 n ×M
3 C
M × W +n ×M ×DS −n ×M −n ×M ×DS
DS = N C/N 2 C 1 1 C 3 C 2
3 n ×M
4 C
whereDS ,DS ,andDS representthedeacetylationdegreeofchitosan,thedegreesofsubstitution
1 2 3
ofN,N,N-trimethylchitosaniodide, anddegreesofsubstitutionofquaternaryammoniumsaltsof
chitosanbearinghalogenatedacetate,respectively;M andM arethemolarmassesofcarbonand
C N
nitrogen, M = 12, M = 14, respectively; n , n , n , and n are the number of carbons of chitin,
C N 1 2 3 4
acetamidogroup,trimethyl,andhaloaceticacidgroup,n =8,n =2,n =3,n =2,respectively;W
1 2 3 4 C/N
representsthemassratiobetweencarbonandnitrogeninchitosanderivatives.
2.3. SynthesisofChitosanDerivatives
2.3.1. SynthesisofN,N,N-TrimethylChitosanIodide(TMCSI)
TMCSIwaspreparedaccordingtothefollowingmethod[1]: Chitosan(10mmol)wasdispersed
in80mLofN-methyl-2-pyrrolidone(NMP)andstirredatroomtemperaturefor1h. Then,NaI(4.5g),
15%NaOHaqueoussolution(15mL),andCH I(15mL)wereadded,subsequently. Themixturewas
3
refluxedforanadditional2hat60◦C.Afterrefluxreaction,thesolutionwaspouredintoethanolto
affordsomeflavescentprecipitateandTMCSIcollectedbyfiltrationwasobtainedbyfreeze-drying
overnightinvacuum.
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2.3.2. SynthesisofQuaternaryAmmoniumSaltofChitosanBearingHalogenatedAcetate
2.3.2. Synthesis of Quaternary Ammonium Salt of Chitosan Bearing Halogenated Acetate
TMCSI was then dissolved in 15% of sodium haloacetic in order to replace the iodide ions
TMCSI was then dissolved in 15% of sodium haloacetic in order to replace the iodide ions with
with haloacetic ions. The solution was dialyzed with distilled water for 3 days, and quaternary
haloacetic ions. The solution was dialyzed with distilled water for 3 days, and quaternary
ammonium salts of chitosan bearing halogenated acetate including N,N,N-trimethyl chitosan
ammonium salts of chitosan bearing halogenated acetate including N,N,N-trimethyl chitosan
chloroacetate (TMCSC), N,N,N-trimethyl chitosan dichloroacetate (TMCSDC), N,N,N-trimethyl
chloroacetate (TMCSC), N,N,N-trimethyl chitosan dichloroacetate (TMCSDC), N,N,N-trimethyl
chitosantrichloroacetate(TMCSTC),andN,N,N-trimethylchitosantrifluoroacetate(TMCSTF)were
chitosan trichloroacetate (TMCSTC), and N,N,N-trimethyl chitosan trifluoroacetate (TMCSTF) were
obtainedafterbeingfreeze-dried(Scheme1).
obtained after being freeze-dried (Scheme 1).

Scheme 1. Synthesis pathway of chitosan derivatives.
Scheme1.Synthesispathwayofchitosanderivatives.
2.4. Antifungal Assays
2.4. AntifungalAssays
The antifungal ability was assessed by the model of the earlier method [22]. Firstly, the samples
Theantifungalabilitywasassessedbythemodeloftheearliermethod[22]. Firstly,thesamples
(chitosan and chitosan derivatives) were dispersed in distilled water at a concentration of 6 mg/mL
(chitosanandchitosanderivatives)weredispersedindistilledwaterataconcentrationof6mg/mLas
as stock solutions. Then, each sample solution was added to fungi media, which was prepared from
stocksolutions. Then,eachsamplesolutionwasaddedtofungimedia,whichwaspreparedfroma
a mixture with a mass ratio of fungi medium, agar powder, and sterile water of 2.85:1.8:100,
mixturewithamassratiooffungimedium,agarpowder,andsterilewaterof2.85:1.8:100,respectively,
respectively, to give final concentrations of 0.08, 0.4, and 0.8 mg/mL. The final solutions were poured
togivefinalconcentrationsof0.08,0.4,and0.8mg/mL.Thefinalsolutionswerepouredintosterilized
into sterilized Petri dishes (9 cm). After the mixture was cooled, the fungi mycelium of 5.0 mm
Petri dishes (9 cm). After the mixture was cooled, the fungi mycelium of 5.0 mm diameter was
tdraianmsfeetrerre dwtaos tthraentsefsetrpreladt etoa nthdei tnecsut bpalatetde aantd27 in◦cCufboarte2d– 3atd 2a7y s°.CW fohre 2n–t3h deamyys.c Welihuemn othfefu mnygcierleiaucmhe odf
fungi reached the edges of the control plate (without added samples), the antifungal index was
theedgesofthecontrolplate(withoutaddedsamples),theantifungalindexwascalculatedasfollows:
calculated as follows:
AAnnttiiffuunnggaal lininddexe x(%(%))==(1(1−−D(cid:2911)D(cid:3415)a))× ×110000
DDb(cid:2912)
wwhheerree DDa iiss tthhee ddiiaammeetteerr ooff tthhee ggrroowwtthh zzoonnee iinn tthhee tteesstt ppllaatteess aanndd DDb iiss tthhee ddiiaammeetteerr ooff tthhee ggrroowwtthh
a b
zone in the control plate.
zoneinthecontrolplate.
22..55.. AAnnttiibbaacctteerriiaall AAssssaayyss
TThhee aannttiibbaacctteerriaialla abbiliiltiytyo focf hcihtoitsoasnaann adncdh icthositaonsadne rdivearitvivaetsivwesa swdaest edrmeteinrmedinuesdin gustihnega tghaer wagealrl
dwifeflul sidoinffumseiothno dm[e2t3h,2o4d] .[F2i3rs,2tl4y],. auFtiorsctllayv, edau(tsotecrlialve)edn u(tsriteenritlea)g anrumtreideniat (aagmarix tmureedoiaf T(ray pmtoinxetu,yree aostf
eTxrtyrpactotnpeo,w ydeaesrt, aegxtarrapcto pwodwedr,esro, daiguamr pcohwlodriedr,e ,soadnidusmte crhilleowridatee, rainndt hsteermilaes wsaratetiro ino fth10e :m5:1a8s:s1 r0a:1ti0o0 o0)f
w10a:s5:p18re:1p0a:1re0d00.)T wheans, pitrwepaasrepdo.u Trehdenin, tiot swtearsi lepopularteeds ainntdo csuteltruilree dplmateeds iaanwde rceulatlulorewde dmteodsiao liwdeifrye.
0a.l1lomwLedE .tcoo lsioalniddifSy.. a0u.r1e umsL(1 E05. cCoFliU a/nmd LS). wauerreeuisn (o1c0u5 lCatFeUd/omnLth) ewneuretr iiennotcauglaatremd eodni athaen dnuthtreinenspt raegaadr
omnetdhiea eanntdir ethseunrf aspcereoafdt hoenm thede ieunmtirbey ssuterfrailcees opfa ttuhlea .mNeedxiut,mfo ubry wsteelrlsileo fs5pamtumlad. iNamexett,e frowure rweeblolsr eodf
u5s minmg adsiatemrieletebr owreerrein beoarcehd pulsaitneg. Ian setearcihlew beolrle,r2 5inµ eLaochf spalmatpe.l eIsn (eaatcthh ewceolnl,c 2e5n tμraLt ioofn saomf2p0lems g(a/tm thLe)
wcoenrecelonatrdaetdio.nB aocft e2r0i amlpgl/amteLs)w wereerein lcouabdaetded. Batac3t7e◦riCalo pvelartneisg hwtearned iznocnuebsaotefdin ahti b3i7ti o°nC woevreernmigeahstu arnedd
azfotenres2 4ofh i.nGhribeaittieorni nwheibreit mioneazsounreeds ianfdteicr a2t4e dh.h Gigrheeartearn itnibhaibcitteiroinal zaocntievsi tiyn.dicated higher antibacterial
activity.
Polymers 2018, 10, x FOR PEER REVIEW 5 of 14
Polymers2018,10,530 5of14
2.6. Statistical Analysis
2.6. StaAtisltl icthaleA enxapleyrsiims ents were performed in triplicate and the data were expressed as means ± the
standard deviation (SD, n = 3). Significant difference analysis was determined using Scheffe’s multiple
Alltheexperimentswereperformedintriplicateandthedatawereexpressedasmeans±the
range test. A level of p < 0.05 was considered statistically significant.
standarddeviation(SD,n=3). SignificantdifferenceanalysiswasdeterminedusingScheffe’smultiple
rangetest. Alevelofp<0.05wasconsideredstatisticallysignificant.
3. Results and Discussion
3. ResultsandDiscussion
3.1. Structure of Quaternary Ammonium Salts of Chitosan Bearing Halogenated Acetate
3.1. StructureofQuaternaryAmmoniumSaltsofChitosanBearingHalogenatedAcetate
Each step of the synthesis was followed by FT-IR spectra (Figure 1), 1H NMR spectra (Figure 2),
andE a1c3hC sNteMpoRf sthpeecstyrna th(Feisgiusrwe a3s)f. oMlloewanewdhbiyleF, Tt-hIRe sepleemcternat(aFli gaunraely1s)e,s1,H yNieMldsR, sapnedc ttrhae( Fdigeugrreee2s) ,of
ansdub13sCtituNtMionR osfp eqcutarater(Fniagruyr aem3)m. oMnieuamnw sahlitlse ,otfh ceheitloesmaenn btaelarainnagl yhsaelso,gyeinealdtesd, aancdetathtee adree gsrheoews onf in
suTbastbilteu t1i.o n of quaternary ammonium salts of chitosan bearing halogenated acetate are shown in
Table1.
CS
3432.67 2877.27 TMCSI 1654.631600.63 1079.84
1469.49
775.24
TMCSC
TMCSDC 767.53
TMCSTC
833.10
TMCSTF
1195.951126.23
4000 3500 3000 2500 2000 1500 1000 500
Wavenum ber (cm-1)

FigFuigruer1e. 1F.T F-ITR-IsRp sepctercatroaf ocfh cithoistaonsa,nT,M TCMSCI,STI,M TCMSCCS,CT,M TCMSCDSCD,CT,M TCMSCTSCT,Can, danTdM TCMSCTSF.TF.
The FT-IR spectra of chitosan, TMCSI, TMCSC, TMCSDC, TMCSTC, and TMCSTF are shown in
TheFT-IRspectraofchitosan,TMCSI,TMCSC,TMCSDC,TMCSTC,andTMCSTFareshown
Figure 1. The spectrum of chitosan shows characteristic absorption bands at approximately 3432.67 cm−1
in Figure 1. The spectrum of chitosan shows characteristic absorption bands at approximately
34(3a2n.6g7uclamr −d1e(faonrmgualtaiornd eofof rOm–aHtio annodf ON––HH)a, n2d87N7–.2H7) ,c2m87−17 .(2–7CcHm −st1re(–tcChHingst)r, e1tc6h0i0n.6g3), 1cm60−01 .6(3vicbmra−ti1on
(vimbroadtieosn ofm aomdienso ogfroaumpi)n, oangdr o1u07p9).,84a ncdm−11 0(7th9e.8 a4bcsmor−ba1n(cteh eofa βb-s(o1r,4b)a gnlcyecoosfidβic-( 1in,4 c)hgitloyscaons)i d[2ic5–i2n8].
chFitoors aTnM) [C25S–I,2 8a] .nFeowr TpMeaCk SIa,papenaerws pate aakboauptp e1a4r6s9a tcmab−o1, uwt 1h4ic6h9 cims −as1s,iwgnheidch tios athssei gcnheadratcotetrhisetic
chaabrascotrebraisntcice aobfs Nor–bCaHnc3e [1o,f1N6]–. CFoHr T[M1,1C6S]C. F, oTrMTCMSCDSCC, ,aTnMd CTSMDCCS,TaCn,d thTeMreC aSrTeC n,etwhe preeaakres napewpepaeriankgs at
3
ap7p7e5a.r2i4n,g 7a6t77.5735,. 2a4n,d7 6873.35.31,0a cnmd−81,3 r3e.1sp0eccmti−v1e,lyre, swphecictihv sehlyo,uwldh ibche ashssoiuglndedbe toa stshige naebdsotorbtahnecaeb osfo trhbea nCc–eCl
ofbthoendC –[C12l]b. oMndea[1n2w].hMilee,a nnwewhi lpe,enaekws appeapkesara papt e1a1r9a5t.91519 5a.n9d5 a1n1d261.12236 .c2m3−c1m f−or1 fToMrTCMSTCFS,T Fw,hwichhic hare
assigned to the characteristic absorbance of C–F [12]. At the same time, there are characteristic peaks
areassignedtothecharacteristicabsorbanceofC–F[12]. Atthesametime,therearecharacteristic
peoafk Nso–fCNH–3 CaHt abaotuatb 1o4u7t01 c4m70−1,c mwh−i1c,hw phriocvhep trhoavte ththea qtuthaeteqrnuaartyer anmarmyoanmiummo nsiaultms gsraoltuspgsr sotuilpl sexsitsiltl in
3
the molecules of TMCSC, TMCSDC, TMCSTC, and TMCSTF. The above-mentioned results
existinthemoleculesofTMCSC,TMCSDC,TMCSTC,andTMCSTF.Theabove-mentionedresults
preliminarily demonstrated that quaternary ammonium salts of chitosan derivatives were obtained.
preliminarilydemonstratedthatquaternaryammoniumsaltsofchitosanderivativeswereobtained.
Polymers2018,10,530 6of14
Polymers 2018, 10, x FOR PEER REVIEW 6 of 14
CH OH
2
O
O
OH
n
A
N+(CH ) OOCR
3 3
CH2OH D O
2
O
O
OH
A
A n pyranosering
N+(CH3)3I
B
A
B
R= Cl
Cl A
B
R= Cl
B
A
Cl
Cl
R=
Cl
A
F
F
R=
F

FFiigguurree 22.. 11HH NNMMRR ssppeeccttrraa ooff TTMMCCSSII, ,TTMMCCSSCC, ,TTMMCCSSDDCC, ,TTMMCCSSTTCC,, aanndd TTMMCCSSTTFF..
The structures of the synthesized products were further demonstrated by 1H NMR spectra
The structures of the synthesized products were further demonstrated by 1H NMR spectra
(Figure 2) and 13C NMR spectra (Figure 3). As shown in the figures, all spectra show signals at
(Figure 2) and 13C NMR spectra (Figure 3). As shown in the figures, all spectra show signals at
2.0–5.5 ppm (1H NMR spectra) and 50–100 ppm (13C NMR spectra), which are assigned to the
2.0–5.5 ppm (1H NMR spectra) and 50–100 ppm (13C NMR spectra), which are assigned to the
diagnostic chemical shifts of chitosan [29,30]. In 1H NMR spectrum of TMCSI, the chemical shift at
diagnostic chemical shifts of chitosan [29,30]. In 1H NMR spectrum of TMCSI, the chemical shift
3.1 ppm is assigned to the protons of –N+(CH3)3 groups [1,31]. Similarly, the trimethyl groups show
at 3.1 ppm is assigned to the protons of –N+(CH ) groups [1,31]. Similarly, the trimethyl groups
the prominent peak at 53 ppm in 13C NMR spect3ru3m. As compared with TMCSI, new peaks are
showtheprominentpeakat53ppmin13CNMRspectrum. AscomparedwithTMCSI,newpeaks
observed at 4.0 ppm (CH2ClCOO– in TMCSC) and 6.0 ppm (CHCl2COO– in TMCSDC) in 1H NMR
are observed at 4.0 ppm (CH ClCOO– in TMCSC) and 6.0 ppm (CHCl COO– in TMCSDC) in 1H
spectra of products, which a2re assigned to protons of halogenated ac2etic anions [32]. As far as
NMRspectraofproducts, whichareassignedtoprotonsofhalogenatedaceticanions[32]. Asfar
TMCSTC and TMCSTF, the spectra are similar to TMCSI and no new peaks appear because of
as TMCSTC and TMCSTF, the spectra are similar to TMCSI and no new peaks appear because of
absence of protons of halogenated acetic anions. As to the 13C NMR spectra of quaternary
absenceofprotonsofhalogenatedaceticanions. Astothe13CNMRspectraofquaternaryammonium
ammonium salts of chitosan bearing halogenated acetate, new signals at about 175, 170, 166, and 161
ppm are related to carbons of COO– groups in TMCSC, TMCSDC, TMCSTC, and TMCSTF [33].
Polymers2018,10,530 7of14
saltsofchitosanbearinghalogenatedacetate, newsignalsatabout175, 170, 166, and161ppmare
Polymers 2018, 10, x FOR PEER REVIEW 7 of 14
relatedtocarbonsofCOO–groupsinTMCSC,TMCSDC,TMCSTC,andTMCSTF[33]. Furthermore,
Fnuerwthreersmoonraen, cenepwea kressaopnpaenacre apte4a4kps pampp(CeaHr 2aCtl 4in4 TpMpmCS C(C),H629Cpl pinm T(CMHCCSlC2)i,n 6T9M pCpSmD C(C),H95Clp2 pimn
T(CMCCl3SDinCT)M, 9C5 SpTpCm), (aCnCdl31 1in7 TpMpmCS(CTFC3),i nanTdM 1C17T Fp)p,mco n(CfiFrm3 iinn gTMthCeTpFre),s econncefiromftihneg hthaleo pgerensaetnedcem oef tthhyel
hcaalrobgoennsa[t1e2d, 3m4]e.thInyla cdadrbitoionns, [t1h2e,3c4h].e mIni caadldsihtiioftns, othfe– Nch+e(mCHic3a)l3 sahtifatsb oouf t–3N.1+(CppHm3)3( a1Ht aNboMutR 3s.1p epcptrma)
(a1Hnd N5M3pRp smpe(c1t3rCa) NanMdR 53s ppepcmtra ()13sCti lNleMxRis tsipnecthtrea)s psteicllt reaxiosft qinu athteer snpaercytraam omf qounaiutemrnsaarlyts aomfmchointoiusamn
sbaeltasr inogf hcahloitgoesanna tebdeaarcientgat eh.aTlohgeerenfaotreed, thaecesetadtea. taTahdeerqefuoartee, lythpersoev edtahtea suacdceeqsusfautelllyy sypnrothvees isthoef
scuhcictoessasfnudlleyr sivyanttihveessi.s of chitosan derivatives.
CH OH
2
O
O
OH
A n B
N+(CH ) OOCR
CH2OH 3 3
O
O
OH
A n pyranosering
N+(CH3)3I A
B C
C
A
R= Cl
C
Cl
B C
A
R= Cl
Cl
Cl
R= A
B C Cl C
F
F
A
R=
B C F
C

FFiigguurree 33.. 113C3C NNMMRR spspeecctrtara oof fTTMMCCSSI,I ,TTMMCCSSCC, ,TTMMCCSSDDCC, ,TTMMCCSSTTCC, ,aanndd TTMMCCSSTTFF..

The degrees of substitution (DS) for chitosan derivatives were calculated on the basis of the
percentagesofcarbonandnitrogenandtheresultsareshowninTable1. Asisshown,theintermediate
TMCSIpresentsthehighestdegreeofsubstitution. TheDSoffourfinalproducts(TMCSC,TMCSDC,
TMCSTC, and TMCSTF) are various. For example, compared with TMCSDC with the DS of 0.60,
Polymers 2018, 10, x FOR PEER REVIEW 8 of 14
Table 1. The elemental analyses, yields, and the degrees of substitution of chitosan derivative.
Polymers2018,10,530 Elemental Analyses (%) 8of14
Compounds Yields (%) Degrees of Substitution Deacetylation
C N H C/N
CS 40.851 7.474 6.879 5.47 - 0.81
theDSvalueofTMCSTFisonly0.32. Generally,thedegreesofsubstitutionofproductswoulddirectly
TMCSI 87.6 29.211 4.076 5.485 7.17 0.66 -
affect tThMeiCrSbCio activity7.3.T5 he low39e.r28d eg4r.e9e41o f s6u.4b61st itu7t.9io5 n as well as0.l4e6s s functional group- s mean a
weakeTrMbiCoSaDctCiv ity. Th6e9re.8f ore,e3v8e.n43i9f TM4.C68S9T F7h.2a1s7t he8s.t2r ongestelectro0n.6e gativity,itsbiologi-c alactivity
maynToMtbCeStThCe bestbe7c5a.3u seof4t3h.e05l4o w5D.3S61in s7o.8m37e ca8s.e0s3. 0.5 -
TMCSTF 74.6 42.88 5.556 6.998 7.72 0.32 -
Table1.Theelementalanalyses,yields,andthedegreesofsubstitutionofchitosanderivative.
The degrees of substitution (DS) for chitosan derivatives were calculated on the basis of the
percentages of carbon and nitrogeEnle manednt atlhAe narelyssuelsts( %a)re showDne girne esTaobfle 1. As is shown, the
Compounds Yields(%) Deacetylation
intermediate TMCSI presents the ChighestN degreeH of suCb/sNtitutioSnu.b sTthiteu tiDonS of four final products
(TMCSC, TCMSCSDC, TMCSTC, an40d. 8T51MC7S.4T7F4) ar6e. 8v79ariou5s.4. 7For examp-le, compared 0w.81ith TMCSDC
with the DTMS CoSf I0.60, the 8D7.S6 value2 9o.f2 1T1MC4.S0T76F is 5o.4n8ly5 0.372..1 7Generally0,. 6t6he degrees of s-ubstitution of
productsT wMoCuSlCd directly73 .a5ffect th3e9.i2r8 bio4a.c9t4i1vity.6 .T46h1e low7.9e5r degree 0o.4f6 substitution a-s well as less
TMCSDC 69.8 38.439 4.689 7.217 8.2 0.6 -
functional groups mean a weaker bioactivity. Therefore, even if TMCSTF has the strongest
TMCSTC 75.3 43.054 5.361 7.837 8.03 0.5 -
electronegativity, its biological activity may not be the best because of the low DS in some cases.
TMCSTF 74.6 42.88 5.556 6.998 7.72 0.32 -
3.2. Antifungal Activity
3.2. AntifungalActivity
The antifungal activity of chitosan and chitosan derivatives against three destructive
Theantifungalactivityofchitosanandchitosanderivativesagainstthreedestructivephytopathogenic
phytopathogenic fungi, including F. oxysporum f. sp. cucumebrium Owen, B. cinerea, and P. asparagi,
fungi, including F. oxysporum f. sp. cucumebrium Owen, B. cinerea, and P. asparagi, are shown in
are shown in Figures 4–6. Their antifungal indices and the relationship between the structure and
Figures4–6. Theirantifungalindicesandtherelationshipbetweenthestructureandantifungalactivity
antifungal activity of chitosan derivatives are discussed as follows.
ofchitosanderivativesarediscussedasfollows.
0.08 mg/mL
80
0.4 mg/mL
0.8 mg/mL
70
60
)
%
(
x 50
e
d
n
al I 40
g
n
u 30
ntif
A
20
10
0
CS TMCSI TMCSC TMCSDC TMCSTC TMCSTF

FFiigguurree 44.. TThhee aannttiiffuunnggaall aaccttiivviittyy ooff cchhiittoossaann aanndd cchhiittoossaann ddeerriivvaattiivveess aaggaaiinnsstt FF.. ooxxyyssppoorruumm ff.. sspp..
ccuuccuummeebbrriiuumm OOwweenn..
FF.. ooxxyyssppoorruumm iiss oonnee ooff tthhee mmoosstt imimppoorrttaanntt ssooiill--bboorrnnee ppaatthhooggeennss tthhaatt ccaauussee rroooott--rroott aanndd wwiilltt
ddiisseeaasseess iinn aa wwiiddee vvaarriieettyy ooff ccrrooppss.. IItt hhaass tthhee aabbiilliittyy ttoo ppeerrssiisstt ffoorr vveerryy lloonngg ppeerriiooddss iinn ssooiill wwiitthhoouutt aa
hhoosstt [[3355]].. FFiigguurree 44 sshhoowwss tthhee aannttiiffuunnggaall aaccttiivviittyy ooff cchhiittoossaann aanndd aallll ddeerriivvaattiivveess aaggaaiinnsstt FF.. ooxxyyssppoorruumm ff..
sspp.. ccuuccuummeebbrriuiummO Owwene.nA. Asiss issh oshwonw,sne, vseervaelrcaoln ccolunscilounssiocnasn cbaeng abien egdaiansefdo lalosw fos:llfiorwstsl:y ,fifrosrtlayl,l tfeosrt eadll
staemstepdle ssa,tmhepliensh, itbhiteo irnyhriabtietsorinyc rraetaesse iwncitrheathsee wrisiethi nthceo nricseen itnra ctioonnc.eTnhtrisattiroenn.d Tihsiosb tvreionuds iasn odbivsisouuista abnled
fiosr sauliltasabmle pfolers a.lFl osarminpstleasn.c Feo,rth inesitnahnicbei,t othrye rinahteisbiotforTyM raCtSeCs oafr eTM1.0C1S%C, a3r0e.4 17.%01,%an, 3d06.477.3%8,% anwdh 6e7n.3t8h%e
cworhreens ptohne dcionrgrecsopnocnednitnragt icoonnscaernetr0a.t0i8onms ga/rme 0L.,008. 4mmg/gm/Lm, L0,.4a nmdg0/.m8Lm, ga/nmd L0,.8r emspge/cmtiLv,e lrye.sSpeeccotinvdellyy,.
bSeeccaounsdeloyf, tbheecainutsreo doufc ttihoen ionfteroledcutrcotnio-nd raowf ienlegcgtrroonu-pdsr–ahwaliongge ngaroteudpas–cehtaaltoeg,ethneatfienda lapcerotadtue,c ttshhea fvienaal
b etterabilitytoinhibitF.oxysporumf. sp. cucumebriumOwencomparedwithchitosanandTMCSI,
andthisconclusionisapparentlyreinforcedat0.8mg/mLand0.4mg/mL.Thirdly,ofallthetested
Polymers 2018, 10, x FOR PEER REVIEW 9 of 14
products have a better ability to inhibit F. oxysporum f. sp. cucumebrium Owen compared with
Polymers2018,10,530 9of14
chitosan and TMCSI, and this conclusion is apparently reinforced at 0.8 mg/mL and 0.4 mg/mL.
Thirdly, of all the tested samples, the antifungal activity against F. oxysporum f. sp. cucumebrium
Owen decreases in the order: TMCSTF > TMCSTC > TMCSDC > TMCSC > TMCSI > chitosan. This
samples,theantifungalactivityagainstF.oxysporumf. sp. cucumebriumOwendecreasesintheorder:
TreMguClSaTriFty> iTs MidCenStTicCal> wTiMthC tShDe Cso>rtT oMrdCeSrC of> eTleMctCroSnIe>gcahtiivtoitsya n(–.CTFh3i s>r e–gCuClal3r i>t y–CisHidCeln2 t>ic –aClwHi2tChl)t hoef
substituent groups with halogens in chitosan quaternary ammonium salts. For example, the
sortorderofelectronegativity(–CF >–CCl >–CHCl >–CH Cl)ofsubstituentgroupswithhalogens
3 3 2 2
antifungal indices of TMCSTF, TMCSTC, TMCSDC, TMCSC, TMCSI, and chitosan are 77.15%,
inchitosanquaternaryammoniumsalts. Forexample,theantifungalindicesofTMCSTF,TMCSTC,
76.30%, 70.01%, 67.38%, 50.72%, and 20.25% at 0.8 mg/mL, respectively. The results above
TMCSDC,TMCSC,TMCSI,andchitosanare77.15%,76.30%,70.01%,67.38%,50.72%,and20.25%at
demonstrate that the difference of active groups grafting onto the synthesized chitosan derivatives
0.8mg/mL,respectively. Theresultsabovedemonstratethatthedifferenceofactivegroupsgrafting
contributed much to the antifungal action, but their effect on promoting antifungal activity varied
ontothesynthesizedchitosanderivativescontributedmuchtotheantifungalaction,buttheireffecton
according to the difference in electronegativity.
promotingantifungalactivityvariedaccordingtothedifferenceinelectronegativity.
0.08 mg/mL
80
0.4 mg/mL
0.8 mg/mL
70
60
)
%
(
x 50
e
d
n
Polymers 2018, 10, x Fal IOR P4E0ER REVIEW 10 of 14
g
n
fungi of F. oxysfuporum30 f. sp. cucumerium Owen and B. cinerea. The order, which is TMCSTC >
ti
n
TMCSDC > TMCASC, is in accord with earlier descriptions. The inhibitory property of TMCSTF is
20
slightly lower than TMCSTC. Although the electronegativity of the fluorine atom is stronger than
that of the chlorine atom, it is possible that the antifungal activity of TMCSTF is weaker than that of
10
TMCSTC because of its lower substitution degree (Table 1) and less active ingredient. In brief, these
results mentioned abo0ve indicate that the halogen atoms have a positive effect on antifungal activity
CS TMCSI TMCSC TMCSDC TMCSTC TMCSTF
when they are introduced into chitosan and the mechanism between the structure and antifungal

activity of chitosan derivatives will be simply discussed later.
Figure5.TheantifungalactivityofchitosanandchitosanderivativesagainstB.cinerea.
Figure 5. The antifungal activity of chitosan and chitosan derivatives against B. cinerea.
80
B. cinerea is an airborne p0.l0a8n tm pga/mthLogen with a necrotrophic lifestyle attacking over 200 crop
hosts (including field and row 0 c.4ro mpgs,/ mfrLuit, vegetables, and flowers) and it can cause destructive and
70
economically important plant 0d.8is meags/emsL worldwide. The antifungal activity of chitosan and chitosan
derivatives against B. cinerea was tested and the results are shown in Figure 5. Overall, most rules
60
against F. oxysp)orum f. sp. cucumerium Owen discussed above still can be appropriate for the
%
antifungal activx (ity o5f0 samples against B. cinerea. For example, all samples exhibit antifungal
e
properties againdst B. cinerea and the inhibitory indices are concentration-dependent. Besides, the
n
sample that posal Isesse4s0 the weakest inhibitory activity is still chitosan. The quaternary ammonium
salts of chitosang bearing halogenated acetate (TMCSC, TMCSDC, TMCSTC, and TMCSTF) also have
n
better antifungaful abi3li0ty than TMCSI and chitosan. Furthermore, the rule of inhibitory property
ti
against B. cinerean, which is TMCSTF > TMCSTC > TMCSDC > TMCSC > TMCSI > chitosan, is distinct
A
20
according to Figure 5. The inhibitory indices of TMCSTF, TMCSTC, TMCSDC, TMCSC, TMCSI, and
chitosan at 0.4 mg/mL against B. cinerea are 59.43%, 59.02%, 57.2%, 55.66%, 30.10%, and 9.25%,
10
respectively.
As a destructive pathogenic fungus, P. asparagi can cause severe stem blight of asparagus.
0
Figure 6 shows the antifungCaSl activiTtyM CoSfI chitoTsMaCnS Cand TcMhCitSoDsCan dTeMrCivSaTCtivesT MagCaSiTnFst P. asparagi. The

inhibitory indices of all the samples mounted up with increasing concentration. All chitosan
derivativesFF iibggeuuarrerei 6n6..g TT hhheea laaonngttieiffnuunangtgeaadll aaacccttiievvtiiattyyte oo ffs hcchhoiiwttoo ssbaanent aatennrdd accnhhiitttioofussaannng ddaeel rraiivvcaatittviivvieetyss aatgghaaaiinnnss ttT PPM.. aaCssppSaaIrr aaaggnii..d chitosan,
and the inhibitory indices of TMCSTF, TMCSTC, TMCSDC, and TMCSC are 73.54%, 74.69%, 72.36%,
3a.n3d. A6Bn9..t3cibi5na%cert eearati a0ils. 8Aa mcntigav/iimrtybL o,r nreesppelacntitveplayt,h wogheilne twhiet hinahinbeitcorroyt rroaptehsi cofl iTfeMstCylSeI aatntadc ckhinitgosoavne arr2e0 505.c4r7o%p
hanosdt s1T(0hi.ne8c %alun. dtHiibnoagwctfieeverliedarl, a atnhcdtei vrroiutwyle a cogrfoa piinnssh,tfi rbEui.t iocto,rlyvi eaagncetdtia vSbi.tl yea usa,rgeauansidn osflft ocPwh. ieatorssps)aaarnan gadin iidts ccaah nliittoctasleau nsde idfdfeeerrisvetnrautti cvftreiovsme wa tenhrdee
economicallyimportantplantdiseasesworldwide. Theantifungalactivityofchitosanandchitosan
evaluated through the agar well diffusion method. The results depicted in Table 2 reveal that the
d erivativesagainstB.cinereawastestedandtheresultsareshowninFigure5. Overall,mostrules
tested samples display a moderate inhibitory effect on the growth of bacterial pathogens. In general,
the results drawn from antibacterial activity are similar to antifungal activity. For instance, all of the
chitosan derivatives exhibit better antibacterial activity comparable to chitosan. Moreover, the
antibacterial activity decreases in the order of TMCSTF > TMCSTC > TMCSDC > TMCSC > TMCSI >
chitosan, which is identified with antifungal activity. The above results therefore suggest that
different substituents can markedly influence the antibacterial efficacy of the compounds and the
stronger electron-withdrawing capacity indicates better antibacterial activity.
Table 2. The measurements of antibacterial activity for chitosan and chitosan derivatives by agar
well diffusion method.
Zone of Inhibition in mm (Mean ± SD) n = 3
Microorganism
CS TMCSI TMCSC TMCSDC TMCSTC TMCSTF
E. coli 5.11 ± 0.25 5.72 ± 0.35 7.14 ± 0.46 7.66 ± 0.52 7.77 ± 0.47 7.86 ± 0.39
S. aureus 5.19 ± 0.21 6.42 ± 0.53 6.62 ± 0.48 7.36 ± 0.60 8.23 ± 0.49 8.77 ± 0.41
Values are expressed as mean ± SD. Different letters within a row mark significantly different values
(p < 0.05).
According to earlier reports, chitosan and its derivatives possess broad spectrum antimicrobial
activity and this activity can be affected by several factors such as pH, concentration, molecular
weight, degree of deacetylation, temperature, and so on with different mechanisms of action [36–38].
In this paper, the halogenated acetate that was selected to be grafted on chitosan is another
Polymers2018,10,530 10of14
against F. oxysporum f. sp. cucumerium Owen discussed above still can be appropriate for the
antifungalactivityofsamplesagainstB.cinerea. Forexample,allsamplesexhibitantifungalproperties
againstB.cinereaandtheinhibitoryindicesareconcentration-dependent. Besides,thesamplethat
possessestheweakestinhibitoryactivityisstillchitosan. Thequaternaryammoniumsaltsofchitosan
bearinghalogenatedacetate(TMCSC,TMCSDC,TMCSTC,andTMCSTF)alsohavebetterantifungal
abilitythanTMCSIandchitosan. Furthermore,theruleofinhibitorypropertyagainstB.cinerea,which
isTMCSTF>TMCSTC>TMCSDC>TMCSC>TMCSI>chitosan,isdistinctaccordingtoFigure5.
TheinhibitoryindicesofTMCSTF,TMCSTC,TMCSDC,TMCSC,TMCSI,andchitosanat0.4mg/mL
againstB.cinereaare59.43%,59.02%,57.2%,55.66%,30.10%,and9.25%,respectively.
Asadestructivepathogenicfungus,P.asparagicancauseseverestemblightofasparagus.Figure6
showstheantifungalactivityofchitosanandchitosanderivativesagainstP.asparagi. Theinhibitory
indicesofallthesamplesmountedupwithincreasingconcentration. Allchitosanderivativesbearing
halogenatedacetateshowbetterantifungalactivitythanTMCSIandchitosan,andtheinhibitoryindices
ofTMCSTF,TMCSTC,TMCSDC,andTMCSCare73.54%,74.69%,72.36%,and69.35%at0.8mg/mL,
respectively,whiletheinhibitoryratesofTMCSIandchitosanare55.47%and10.8%. However,the
ruleofinhibitoryactivityagainstP.asparagiisalittledifferentfromthefungiofF.oxysporumf. sp.
cucumeriumOwenandB.cinerea.Theorder,whichisTMCSTC>TMCSDC>TMCSC,isinaccordwith
earlierdescriptions. TheinhibitorypropertyofTMCSTFisslightlylowerthanTMCSTC.Although
theelectronegativityofthefluorineatomisstrongerthanthatofthechlorineatom,itispossiblethat
theantifungalactivityofTMCSTFisweakerthanthatofTMCSTCbecauseofitslowersubstitution
degree(Table1)andlessactiveingredient. Inbrief,theseresultsmentionedaboveindicatethatthe
halogenatomshaveapositiveeffectonantifungalactivitywhentheyareintroducedintochitosanand
themechanismbetweenthestructureandantifungalactivityofchitosanderivativeswillbesimply
discussedlater.
3.3. AntibacterialActivity
TheantibacterialactivityagainstE.coliandS.aureusofchitosanandchitosanderivativeswere
evaluated through the agar well diffusion method. The results depicted in Table 2 reveal that the
testedsamplesdisplayamoderateinhibitoryeffectonthegrowthofbacterialpathogens. Ingeneral,
the results drawn from antibacterial activity are similar to antifungal activity. For instance, all of
thechitosanderivativesexhibitbetterantibacterialactivitycomparabletochitosan. Moreover,the
antibacterialactivitydecreasesintheorderofTMCSTF>TMCSTC>TMCSDC>TMCSC>TMCSI>
chitosan,whichisidentifiedwithantifungalactivity. Theaboveresultsthereforesuggestthatdifferent
substituents can markedly influence the antibacterial efficacy of the compounds and the stronger
electron-withdrawingcapacityindicatesbetterantibacterialactivity.
Table2.Themeasurementsofantibacterialactivityforchitosanandchitosanderivativesbyagarwell
diffusionmethod.
ZoneofInhibitioninmm(Mean±SD)n=3
Microorganism
CS TMCSI TMCSC TMCSDC TMCSTC TMCSTF
E.coli 5.11±0.25 5.72±0.35 7.14±0.46 7.66±0.52 7.77±0.47 7.86±0.39
S.aureus 5.19±0.21 6.42±0.53 6.62±0.48 7.36±0.60 8.23±0.49 8.77±0.41
Valuesareexpressedasmean±SD.Differentletterswithinarowmarksignificantlydifferentvalues(p<0.05).
Accordingtoearlierreports,chitosananditsderivativespossessbroadspectrumantimicrobial
activityandthisactivitycanbeaffectedbyseveralfactorssuchaspH,concentration,molecularweight,
degree of deacetylation, temperature, and so on with different mechanisms of action [36–38]. In
thispaper,thehalogenatedacetatethatwasselectedtobegraftedonchitosanisanotherimportant
factor that could influence the bioactivity of chitosan. Up to now, many researches have proven