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S334

Document heading

Extraction, characterization and in vitro antioxidative potential of

chitosan and sulfated chitosan from Cuttlebone of Sepia aculeata

Orbigny, 1848

Aruldhason Barwin Vino*, Pasiyappazham Ramasamy, Vairamani Shanmugam, Annaian Shanmugam

Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai - 608 502, India

Asian Pacific Journal of Tropical Biomedicine (2012)S334-S341

Asian Pacific Journal of Tropical Biomedicine

journal homepage:www.elsevier.com/locate/apjtb

*Corresponding author: *Aruldhason Barwin Vino, Centre of Advanced Study inMarine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai - 608502, India. Tel: +91-9445453166 E-mail: [email protected]

1. Introduction

Naturally occurring sulfated biopolymers, such as heparinand chondroitin sulfate, are widely spread in nature anddemonstrate important functions in the regulation of cellularproliferation and differentiation[1,2]. Even polysaccharideswithout sulfate groups exhibit biological activities aftersulfation, e.g. cellulose sulfate shows anticoagulant andantiviral functions[3-5]. Among others, the application ofsulfated polysaccharides or polyanions to influence thebiological activity of growth factors has attracted broadattention in tissue engineering. Besides being used as partof scaffolds or for the encapsulation of proteins, the sulfatedpolysaccharides were also directly applied to control thebinding and activity of growth factors in cells[6-9].

Chitosan is a deacetylated derivative of chitin representingnaturally occurring polysaccharides. Chitosan has somebeneficial properties, such as antimicrobial activity,excellent biocompatibility and low toxicity that promoteits applications in many fields including food industry andpharmaceutics[10-12]. Moreover, chitosan is readily soluble insome inorganic and organic acids, including hydrochloric acidand acetic acid, while cellulose is only very poorly soluble insuch acids[12,13]. Chemical modification of chitosan and chitin has beenfrequently carried out, in order to prepare their derivativeswith advantageous properties, such as carboxy methylated,oxidized and sulfated chitosan/chitin[10,14,15]. These derivativeshave wide applications, e.g. as additives in food and cosmeticproducts, as drug and gene delivery system or as chiralstationary phases for HPLC[10,14-18]. Among many derivativesof chitosan, chitosan sulfate have been proven to beanticoagulant, antiviral, antimicrobial, and antioxidant[19-22]. Cortizo[23] investigated the characterization of chitin fromIllex argentinus squid pen. -Chitin was isolated by using

ARTICLE INFO ABSTRACT

Article history:

Received 25January 2012Received in revised form 5February 2012Accepted 23 March 2012Available online 28 April 2012

Keywords:

AntioxidantChitinChitosanSulfated chitosanDPPHDMF/HClSO3

Objective:To investigate antioxidant potency of chitosan and sulfated chitosan in variousestablishedin vitrosystems, such as superoxide (O2-)/hydroxyl (-OH)radicals scavenging,reducing power, metal ion chelating. Methods:Chitosan was prepared from deacetylation ofchitin from cuttlefish. FTIR, degree of acetylation and mineral studies was carried out from thechitosan. Chitosan was converted into sulfated chitosan using DMF/HClSO3and their antioxidantactivity was tested. Results:Mineral content of chitosan was Ca - 30ppm, Na- 0.092ppm, Cu-0.172ppm, Mg- 3.601, Mn- 0.264and Zn- 0.924ppm, DAwas 49.9%(IRspectroscopy)and 40.56%(UVspectrophotometer)recorded. Cuttlefish chitosan showed high level of superoxide radicalscavenging activity (88.6%)and hydroxyl radical scavenging activity (72.1%), moderate activity wasnoted in chelating activity (62.6%at 100gm/mL). At the same time low level of reducing powerwas observed (0.300Absorbance at 0.75mg/mL)when compared to standard BHAand Ascorbicacid (2.305and 2.05Absorbance). Conclusions:Finally, the scavenging rate, reducing power,chelating and reducing power of sulfated chitosan increased with their increasing concentration.

Contents lists available at ScienceDirect

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chemical methods and the pens composition determined.The structural characteristics of-chitin were identifiedwith infrared (IR). Sagheer[24]reported the extraction andcharacterization of chitin and chitosan from marine sourcesin Arabian Gulf. Two different forms of chitinand thehave been extracted from different marine crustaceanfrom the Arabian Gulf. Kucukgulmez[25] investigated thephysicochemical characterization of chitosan extracted fromMetapenaeus stebbingi shells. Recently, the physicochemicalcharacterization of biopolymers chitin and chitosan extractedfrom squid pen Doryteuthis sibogae[26]. The antioxidant capacity of sulfated polysaccharides hasbeen studied by different in vitro methods, including hydrogenperoxide, superoxide anion, and hydroxyl radical scavengingassays. DPPHradical scavenging assay is frequently used forthe analysis of food and substances obtained from naturalsources[27]. The aim of this work was preparation of chitosan from thecuttlebone of cephalopod molluscs Sepia acculeata. FT-IRspectrum of chitin, chitosan and sulfated chitosan was takenand degree of acetylation was calculated using IR stretchingbands and UV spectrophotometer. Furthermore sulfatedchitosan was prepared from cuttlefish chitosan and evaluate invitro antioxidant capacity of sulfated chitosan on superoxideradical scavenging assay, hydroxyl radical scavenging assay,metal ion chelating assay and reducing power.

2. Materials and methods

2.1. Chemicals and reagents

The animal (Sepia aculeata) was purchased from thelanding centre of Cuddalore (Lat.11042N; Long. 79046E)Tamil Nadu, India. Cuttlebone were removed from theanimal, and then washed and air dried. Standards (Chitinand Chitosan), DMF, HClSO3, PMS, NADH, NBT, DRand allthe other chemicals and reagents are purchased from sigmaaldrich and dialysis membrane also was bought from SigmaChemicals, molecular weight cut-off was 8000Da.

2.2. Extraction of chitin

Chitin was prepared by following the method of

Takiguchi[28]after removing the minerals and protein using2NHCIand 1NNaOH.

2.3. Conversion of chitin into chitosan

Chitin was deacetylated following the method ofTakiguchi[29]using 40%NaOHto obtain chitosan.

2.4. Mineral analysis of chitosan

Mineral study was carried out following the methodof Topping [30] using Inductively Coupled P lasma

spectrophotometer (ICP).

2.5. Fourier Transform - Infra Red spectroscopy (FT-IR)

The chitin, chitosan, standard chitin and chitosan were

determined using FT-IRspectrometer (Bio-Rad FTIS-40model, USA). Sample (10g)was mixed with 100g of driedPotassium Bromide (KBr)and compressed to prepare a saltdisc (10mm diameter).

2.6. Measurements of degree of N- acetylation

The DA of chitosan was calculated using two methods

i.e. FT-IRspectrometer and UVspectrophotometer. Theabsorbance of IRspectrum at 1637and 3430cm-1were usedto calculate the DAaccording to the following equation:

DA(%)= 100- (A1637/ A3430)115[31]. DAwas calculated using UV- spectrophotometer (Hitachi,220S)following the method of Liu et al[32]. DAwas calculatedaccording to the following equation:

DA= (161.1.A.V. - 0.0218m / 3.3615m - 42.1. A. V)

2.7. Preparation of sulfating reagent

Five milliliter of HClSO3was added drop wise with stirring

to 30mLof N, N-dimethyl formamide (DMF)which waspreviously cooled at 0-4. The reaction mixture was stirredwithout cooling until the solution (DMF.SO3)reached roomtemperature.

2.8. Preparation of sulfated chitosan

Chitosan sulfate was prepared by following the method

ofX

ing et al.[33]

.B

rieflyDMF

.SO

3was added a500

mL

offlask containing chitosan solution in a mixture of DMF-formic acid with swirling to get gelatinous chitosan. Thereaction was run at adequate temperature for 1-2.5hrs and95%of EtOHwas added to the precipitate the product. Theprecipitate was washed with EtOH, and then redissolved indistilled water. The solution was dialyzed against distilledwater. The product was then concentrated and lyophilized togive chitosan sulfates.

2.9. Determination of antioxidant activity

2.9.1. Super oxide radical scavenging assay

The superoxide scavenging ability of sulfated chitosan wasassessed by the method of Nishikimi et al.[34]. The reactionmixture, containing sulfated chitosan (0.05- 0.5mg/mL),PMS(30M), NADH(338M)and NBT(72M)in phosphatebuffer (0.1MpH7.4)was incubated at room temperature for 5min and the absorbance was read at 560nm against a blank.The capability of scavenging the superoxide radical wascalculated using the following equationScavenging effect (%)= (1-Asample 560nm)/Acontrol560nm

2.9.2. Hydroxyl radical scavenging assay

The reaction mixture containing sulfated chitosan (0.1- 3.2

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mg/mL), was incubated with deoxyribose (3.75mM), H2O2(1mM), FeCl3)(100M), EDTA(100M)and ascorbic acid (100M)in potassium phosphate buffer (20mM, pH7.4)for 60min at 37[35]. The reaction was terminated by adding 1ml ofTBA(1%, w/v)and 1ml of TCA(2%, w/v)and then heating thetubes in a boiling water bath for 15min. The contents werecooled, and the absorbance of the mixture was measuredat 535nm against reagent blank. Decreased absorbanceof the reaction mixture indicated decreased oxidation ofdeoxyribose.

2.9.3. Measurement of reducing power

The reducing power of sulfated chitosan was quantifiedby the method described by Xing et al.[36]. Briefly, reactionmixture 1mL, containing different concentration of sulfatedchitosan in phosphate buffer (0.2M, pH6.6), was incubatedwith potassium ferricyanide (1%, w/v)at 50 for 20min.The reaction was terminated by adding TCAsolution (10%.w/v) and the mixture was centrifuged at 3000 rpm for 10min. The supernatant was mixed with distilled water and

ferric chloride (0.1%, w/v)solution and the absorbance of thereaction measured at 700nm. Increased absorbance of thereaction mixture indicated increased reducing power.

2.9.4. Metal ion chelating assay

The ferrous ion-chelating potential of sulfated chitosan

was investigated according to the method of Decker andWelch[37], wherein the Fe2+ -chelating ability of sulfatedchitosan was monitored by measuring the ferrous iron-ferrozine complex at 562nm. Briefly, the reaction mixture,containing sulfated chitosan of different concentrations,FeCl2 (2mM)and ferrozine (5mM), was adjusted to a totalvolume of 0.8mLwith water, shaken well and incubated for10min at room temperature. The absorbance of the mixturewas measured at 562nm against blank. EDTAwas used as apositive control. The ability of sulfated chitosan to chelateferrous ion was calculated using the following equation:Chelating effect (%)= (1-Asample 562nm)/Acontrol 562nm

2.10. Statistical analysis

Statistical analysis was performed using one-way

analysis of variance (ANOVA)using SPSSSoftware followedby Duncuns multiple range test (DMRT). Results wereexpressed as meanS.D. from triplicate estimates in each

assays.Pvalues

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acculeata was 21% and 49.71%, respectively. Chitin wasprepared from cuttlebone ofS. aculeataby using acid andalkaline treatments; the yield of chitin was 21%in the totalweight of the dried cuttlebone, after N- acetylation, theyield of chitosans were in the range of 49.71%. Whereas inthe case of sulfated chitosan obtained from the chitosan of S.acculeata was 90.3%

The higher amount of protein content was present incuttlebone (30- 32%). At the same time no protein contentwas found in chitin as indicated by no absorbance at 280nm.The mineral content of chitin from cuttlefish was found to beCa 30ppm, Na 0.092ppm, Mg 3.601ppm, Mn 0.264ppm andZn 0.924ppm. But in the case of Kand Fe are absent in theabove chitin. IRspectrum of the cuttlefish chitin, chitosan, standardchitin and chitosan was given in Table 1and 2and Figure1-5. In the present investigation, the DAof chitosan showed49.9%(IRspectrum)and 40.56%(UVspectrophotometer).

4000 3000 2000 1000

Wavenumbers (cm-1)

1009896949290888684828078767472706866646260585654

5250

NT-1

%Transmittance

3766.2

7

3436.3

5

3268.6

33108.3

7

2961.3

2

2930.1

1

2886.8

1

2359.7

0

2334.0

62149.9

8

1661.5

0

1563.4

2 1418.7

41377.3

8

1315.4

0

1259.9

4

1203.4

0

1156.9

2

1116.1

2

1073.9

3

1026.3

3

952.2

9

752.3

3

690.9

9

606.6

8

530.1

1896.2

8

Figure 1.Showing the FT- IRspectrum of standard chitin.

4000 3000 2000 1500 1000 500 400

cm-1

100

95

90

85

80

75

70

65

60

55

5045

40

35

30

25

20

15

10

5

0

%T

3929

3404

29212853

2674

2546

25222499

2355

2326

1788

1477

116010821033

970948

712

699

637618

520425

407

Figure 2.Showing the FT- IRspectrum of S. aculeata chitin.

NT-A-1

4000 3000 2000 1000

Wavenumbers (cm-1)

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

5

0

%Transmittance 3

914.8

1

3785.8

8

3659.5

2

3377.9

5

2922.8

5 2361.4

1

1660.5

5

1587.9

4

1422.7

3

1380.7

81324.2

41259.5

4

1154.6

4

1077.9

3

1026.6

3

897.4

1

666.1

7

570.6

0

459.1

3

431.0

6

408.0

4

Figure 3. Showing the FT- IRspectrum of standard chitosan.

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

4000 3500 3000 2500 2000 1500 1000 500Wavenumbers (cm-1)

%T

ransmitance

3428.7

0

2936.9

9

2849.3

2

2816.4

4

2734.2

5

2182.7

6

1595.7

7

1416.4

5

1386.3

0

1353.7

6

1121.2

0

1024.6

6

873.6

2

772.6

0

471.2

3

645.5

8

516.18

Figure 4.Showing the FT- IRspectrum of S. aculeata chitosan.

101.0

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

5

04000.0 3000 2000 1500 1000 500400.0

cm-1

%T

3423

2922

2877

2519

23612344

1794

1774

1155

1081 1029

873

712

669617

411

1443

Figure 5.Showing the FT- IRspectrum of S. aculeata sulfatedchitosan.

3.1. Antioxidant activity

The inhibitory effect of sulfated chitosan scavengingactivity of superoxide radicals was marked and concentrationrelated. Asignificant scavenging effect (20.4- 88.6%)of superradicals was evident at all tested concentrations of sulfated

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chitosan (Figure 6). The effect of sulfated chitosan on oxidative damaged,induced by Fe3+/ H2O2on deoxyribose, is plotted in Figure7. Nearly 72.1% inhibition was observed at the highestconcentration (3.2mg/mL), 75.9and 88%was observed inAscorbic acid and BHArespectively. The ferrous ion-chelating effect of sulfated chitosan wasconcentrated related as shown in Figure 8. At 1mg/mL, chelating

ability of chitosan on ferrous ions was 62.6%. However, EDTAshowed a 68%chelating ability.

The reducing power of chitosan and sulfated chitosan wasincreased with the increasing concentration. Figure 9showsthe reducing power increased with increasing chitosan andsulfated chitosan concentration.

0.05 0.1 0.2 0.3 0.4 0.5

Concentration (mg/mL)

BHA

Ascorbic acid

ChitosanS. chitosan

120

100

80

60

40

20

0

Scaven

gingactivity(%)

Figure 6.Scavenging effect of sulfated chitosan of cuttlebone ofS.aculeata on superoxide radical.Values are meansSDof three determinations.

0.1 0.2 0.4 0.8 1.2 3.2


BHA

Ascorbic acid

Chitosan

S. chitosan

100

90

80

70

60

5040

30

20

10

0

Scavenging

activity(%)

Figure 7.Inhibitory effect of sulfated chitosan of cuttlebone andstandards of S. aculeata on deoxyribose oxidative damage.Values are meansSDof three determinations.

Chelatingability(%)

80

70

60

50

40

30

20

10

050 100 2500 500 1000

EDTA

Chitosan

S. chitosan


Figure 8.Chelating effect of sulfated chitosan and standards ofcuttlebone of S. aculeata on ferrous ions. Values are meansSDof

three determinations.

Absorbance(A)

BHA

Ascorbic acid

3

2.5

2

1.5

1

0.5

00.15 0.3 0.45 0.6 0.75


S. chitosan

Chitosan

Figure 9.Reducing power of sulfated chitosan and standards onsuperoxide radical. Values are meansSDof three determinations.

4. Discussion

The cuttlebone of Sanguisorba officinalis was found to be20%of chitin[38,39], whereas in general, the squid/ cuttlefish

reported 3-20% of chitin[40]. One of the major problemsrelated to the preparation of pure chitins is keeping astructure as close as possible than the native form is tominimize the partial deacetylation and chain degradationcaused by demineralization and deproteinization appliedduring process of the raw materials. Squid chitin showed nocolor and odor. Chitin occurs naturally partially deacetylated(with a low content of glucosamine units), depending onthe source[41]; nevertheless, both - and - forms areinsoluble in all the usual solvent, despite natural variationin crystallinity. The insolubility is a major problem thatconfronts the development mechanisms and uses of chitin.

The - chitin is more reactive than the - form, animportant property with regard to enzymatic and chemicaltransformations of chitin[42]. Cuttlefish chitin which is associated to organic andinorganic matter. Doryteuthis sibogae Squid pen found themineral content, Ca 21ppm, Mg 11.01ppm, Mn 0.260ppm, Cu0.905ppm and this was absent in Na, K, Fe and Zn BarwinVino[43]. D. sibogaepen chitosan contain only very smallless amount of mineral content (1.50and 1.37%). The similarless amount of mineral content were recorded in S. aculeatachitin. Generally the mineral salts content in squid pen andcuttlebone of cuttlefish (1%w/w)is much lower than shrimpshell (14%w/w). Consequently, in the preparation of chitinfrom squid pen, it is not need to be demineralized by acidtreatment as preparation from shrimp and crab shells[44]. The properties of chitosan depend on various intrinsicparameters such as percentage of degree of deacetylation(DDA)and molecular weight and thereby on the uses ofchitosan[45,46]. But in the case of quality parameters ofchitosan for chitin and chitosan is lacking [46,47]. Rinaudoand Domard[45]analysed the above parameters for differentcommercially available chitins and chitosans and foundthat they differed with the products. Besides this, numerousmethods are being used for the estimation of the same

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parameters leading to discrepancies. Hence there exists aneed for standard analytical parameters and methodologiesthat are recognized by all. The yield of high molecular weight sulfated chitosan wasrecorded as 173%at 50[33]and Vikhoreva et al.[48]reportedthe low molecular weight chitosan yield was 0.95to 1.20g/g.But in the present study only 90.3%of sulfated chitosan was

obtained.In the FT- IR spectrum of sulfated chitosan obtainedfrom Ulva pertusa[49]the peaks at 847cm-1, 1052cm-1, 1056cm-1, 1641cm-1and 3446cm-1were reported to be causedby the bending vibration of C-O-S of sulfate in axialposition, stretching vibration of C-O, S-Oof sulfate, C-Oof uronic acids and O-Hrespectively. In the present studyalso the observed peaks (873.62cm-1, 1024.66cm-1, 1353.78cm-1, 1595.77cm-1and 3428.70cm-1)represented the samestructural features. The inhibitory effect of sulfated chitosan on superoxideradical scavenging effect (20.4- 88.6%)of superoxide radicals

was evident at all concentration of sulfated chitosan (0.05-0.5mg/mL). Compared with low molecular weight chitosanand parent chitosan, their scavenging activity for superoxideradical was 80.3% and 13%at 0.5mg/mL, respectively[50].High molecular weight chitosan sulfate (HCTS) showsthe superoxide scavenging effect was 27.93- 97.21 in theconcentration of 0.005- 0.4mg/ml[33]. Although superoxideis a relatively weak oxidant, it decomposes to from strongerreactive species, such as single oxygen and hydroxyl radicals,which initiate peroxidation of lipids[51]. In the presentstudy, chitosan sulfate effectively scavenged superoxide ina concentration - dependent manner. Further, superoxides

are also known to indirectly initiate lipid peroxidation asa result of H2O2 formation, creating precursors of hydroxylradicals[52]. These results showed that the chitosan sulfatehad strong scavenging activity of superoxide radical andclearly suggested that the antioxidant activity of sulfatedchitosan was also related to its ability scavenge superoxideradical. Hydroxyl radical scavenging activity of sulfated chitosanwas obtained in the deoxyribose system. In this system,sulfated chitosan exhibited a concentration dependentinhibition of deoxyribose oxidation. Earlier Halliwell et al.[35]have employed this system to assess the biological activityof various natural plant - derived biomolecules. Smithetal.[52] reported that molecules that can inhibit deoxyribosedegradation are those that can chelate iron and render theminactive or poorly active in a fenton reaction. In the presentstudy, in another assay system, we found that the sulfatedchitosan has considerably soft ferrous ion chelating power,so it is impossible that the chelating effect of sulfatedchitosan on metal ions may be responsible that the inhibitionof deoxyribose oxidation. Therefore, the mechanism ofsulfated chitosan on scavenging hydroxyl radical needs to befurther researched.

Xing et al.[33]found that the ferrous ion - chelating effect

of all kinds of sulfated chitosan was concentration related withsulfatedTSCTSbeing the most of effective (~72%at 0.25mg/mL). Linand Chou[52]reported the chelating abilities of N- alkylateddisaccharide chitosan derivatives were less than theobserved with EDTA. However, at 1mg/ml, EDTAchelated~ 60%of cupric ions (Liu and Chou, 2004). Whereas Xing etal.[53] reported HCTSwas ~30% in 1mg/mLand GHshow

~5.5%. But, at 1mg/mL, chelating ability of chitosan B60andC60on ferrous ions was 88.7%and 90.3%respectively[54]. Transition metal ions can initiate lipid peroxidationand start a chain reaction, which lead to the deteriorationof flavor and test in food[55]. It has also proposed thatthe catalysis of metal ions might correlate to cancer andarthritis[35]. Since ferrous ions are the most effective pre-oxidants in the food system. In the present show thechelating effect of chitosan and sulfated chitosan was low,especially compared to EDTA.

Mau et al.[56] reported reducing powers were 0.80, 0.89and 0.92at 1.0mg/mLfor ascorbic acid, a-tocopherol and

BHA, respectively. However, the reducing power of differentmolecular weight chitosans and sulfated chitosans was lowerthan that of ascorbic acid, a-tocopherol and BHA[33]. Pin-Der Duh et al.[54]observed a direct correlation betweenantioxidant activities and reducing power of certain plantextracts. The reducing properties are generally associatedwith the presence of reductions which have been shown toexert antioxidant action by breaking the free radical chainby donating a hydrogen atom[57]. Redactors are also reportedto react with certain precursors of peroxide formation. Ourdata on the reducing power of sulfated chitosan suggestedthat it was likely to contribute significantly towards the

observed antioxidant effect. Further research is needed toevaluate the in vivo antioxidative potential of chitosan.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

Authors are thankful to the Director & Dean, CAS in

Marine Biology, Faculty of Marine Sciences, AnnamalaiUniversity for providing with necessary facilities. Theauthors (PR ) thankful to the Centre for Marine LivingResources and Ecology (CMLRE), Ministry of Earth Sciences,Cochin for providing the financial assistance.

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