Applied Radiation and Isotopes 69 (2011) 126–129

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Applied Radiation and Isotopes

0969-80

doi:10.1

n Tel.:

E-m

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

Effects of molecular weight and ratio of guluronic acid to mannuronic acid onthe antioxidant properties of sodium alginate fractions prepared byradiation-induced degradation

Murat S-en n

Hacettepe University, Department of Chemistry, Polymer Chemistry Division, Beytepe, 06800 Ankara, Turkey

a r t i c l e i n f o

Article history:

Received 18 February 2010

Accepted 18 August 2010

Keywords:

Radiation-induced degradation

Sodium alginate

Antioxidant properties

43/$ - see front matter & 2010 Elsevier Ltd. A

016/j.apradiso.2010.08.017

+90 312 2977989; fax: +90 312 2997973.

ail address: msen@hacettepe.edu.tr

a b s t r a c t

In this study, the effects of the molecular weight and ratio of guluronic acid (G) to mannuronic acid (M),

G/M, of some sodium alginate (NaAlg) fractions on their antioxidative properties were investigated.

Low-molecular-weight-fractions with various G/M were prepared by gamma radiation-induced

degradation of NaAlg. Change in their molecular weight was monitored. Antioxidant properties of

the fractions with various molecular weight and G/M were evaluated by determining the scavenging

ability of 1,1-diphenyl-2-picrylhydrazyl free radical (DPPHd). 50% inhibition concentrations of the

50 kGy-irradiated NaAlgs having molecular weights of 20.5, 17.7, and 16.0 kDa were found to be 11.0,

18.0, and 24.0 mg/ml, respectively, whereas the fractions of the same molecular weight with a lower

G/M exhibited a better DPPHd scavenging activity. The results demonstrated that its molecular weight

and G/M were important factors in controlling the antioxidant properties of NaAlg.

& 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Considerable attention has recently been directed to themodification and preparation of low-molecular-weight-fractionsor oligosaccharites of natural polymers and identification of theirantioxidant properties (Sun et al., 2009; Xing et al., 2005a, 2005b).Kappa carrageenan (Yuan et al., 2005), sodium alginate (Trommerand Neubert, 2005), chitosan (Xie et al., 2001; Yen et al., 2008),carboxymethyl chitosans (Sun et al., 2007), fucoidan andlaminarin (Choi et al., 2009), guar and locust bean gum (Trommerand Neubert, 2005) are only a fraction of these polysaccharides.

Lately, Sun et al. (2007) synthesized low-molecular-weightcarboxymethyl chitosan (CMCTS) fractions by an oxidativedegradation method involving hydrogen peroxide (H2O2) withoutor with microwave radiation and examined their antioxidantproperties. They investigated the influence of the molecularweight and concentration of CMCTSs on antioxidant efficacy byevaluating the superoxide anion scavenging activity. It was foundthat the 50% inhibition concentrations (IC50s) of CMCTSs havingmolecular weights of 1130, 2430, and 4350 Da were 10.36, 17.57,and 23.38 mg/ml, respectively; CMCTSs with a lower molecularweight had a better superoxide anion scavenging activity.

Xie et al. (2001) synthesized water-soluble chitosan deriva-tives by graft copolymerization of maleic acid sodium onto

ll rights reserved.

hydroxypropyl chitosan and carboxymethyl chitosan sodium,and characterized their scavenging activities for hydroxyl radicalsOH by a chemiluminescence technique. It was found that thechitosan derivatives exhibited IC50 values ranging from 246 to498 mg/ml, which was attributed to their different contents ofhydroxyl and amino groups and different substituting groups.

Alginate is one of the natural polysaccharides that occur insubstantial amounts in brown seaweeds. Alginates are un-branched binary co-polymers of (1–4)-linked residues of b-D-mannuronic acid (M) and a-L-guluronic acids (G). Alginic acid andits water-soluble sodium salt have been manufactured fordecades for their great ability to give highly viscous solutionseven at moderate concentrations and mainly used in food,pharmaceutical, and chemical industries as thickening agents,drug carrier, stabilizers, and plant growth stimulator (Østberget al., 1994; Garcia and Ghaly, 1996; Hien et al., 2000). An aqueoussolution of sodium alginate forms stable gels in the presence ofmultivalent cations such as Ca2 + and Mg2 +. Gel formation occursdue to the ionic interaction between the G residues from two ormore alginate chains and cations, yielding a three-dimensionalnetwork of alginate molecules well described by the ‘‘egg-boxmodel’’ (Grant et al., 1973). Functional and physical properties,mechanical strength, porosity, gel uniformity, biocompatibility,influence on encapsulated cells properties of alginate gels andradiation-induced degradation yields vary widely depending onthe ratio of M to G, the frequency and size of G-blocks, and themolecular weight of the polymer (Smidsrod, 1974; Klock et al.,1994; S-en et al., 2010).

M. S- en / Applied Radiation and Isotopes 69 (2011) 126–129 127

In recent years, even more interest has focused on theradiation modification and degradation of natural polymers suchas kappa carrageenan, sodium alginate chitosan and galactoman-nans, and on the use of these low-molecular-weight-polysacchar-ides or oligosaccharites in plant growth promoters and plantprotectors (Relleve et al., 2005; Thama et al., 2001; Nagasawaet al., 2000; S-en et al., 2007). Also, radiation-induced degradationwas widely employed as a method of enhancing the antioxidantactivity of polysaccharides (Choi et al., 2009; Feng et al., 2008).

More recent studies of Feng et al. (2008) indicated thatchitosan degraded via radiation induction exhibited a highreductive capacity and a good inhibition of linoleic acidperoxidation. They employed the radical mediated lipid peroxida-tion inhibition, reducing power, superoxide anion radical, andhydroxyl radical quenching methods for the evaluation of theantioxidant activity of the irradiated chitosan, and observed thatthe superoxide and hydroxyl radical scavenging percentage ofchitosan could be increased up to 74.2% and 63.8%, respectively,by increasing the irradiation dose up to 20 kGy.

Given the literature mentioned above, the general consensus isthat the molecular weight of polysaccharides is an importantfactor in controlling their antioxidant properties. So far, none-theless, no relationship has been established between themolecular weight and structural properties (i.e. G/M) of NaAlg,and its antioxidant properties. In the present study, the antiox-idant properties of a NaAlgs fraction with various G/M ratios wereinvestigated for the first time. NaAlg samples were irradiated withgamma rays in air at ambient temperature in the solid state at lowdose rates. Change in their molecular weight was monitored bysize exclusion chromatography (SEC). Variation of their antiox-idant properties was characterized by their scavenging activity for1,1-diphenyl-2-picrylhydrazyl free radical (DPPHd).

20

25

30

35 LF120 LF200 LF240

n x10

4

2. Materials and methods

2.1. Chemicals

Sodium alginate samples were obtained from FCM Biopoly-mers Company, Norway, and used as received. The initialmolecular weights of NaAlgs, G, and M, the G/M, and otherabbreviations used throughout the text are given in Table 1.

2.2. Gamma irradiation

To prepare the NaAlg fractions, the polymer samples wereplaced in tightly closed containers, and irradiated at various doses(2.5, 5, 10, 15, 20, 25, and 50 kGy) using a Gammacell 220 type60Co-gamma irradiator at room temperature in air.

2.3. Determination of the molecular weight of NaAlgs

Molecular weight analysis of both the unirradiated andirradiated samples were performed using a Waters Breeze modelGel Permeation Chromatograph. Water 2000–1000–500 hydrogelcolumns were employed; NaNO3 (0.1 M) was used as the elutingsolvent. The universal calibration was constructed by using

Table 1Initial molecular weights and G/Ms of NaAlg samples used in the study.

Polymer Mn Mw G/M

LF120 340.0�103 670.0�103 70/30

LF200 310.0�103 690.0�103 50/50

LF240 275.0�103 500.0�103 45/55

poly(ethylene oxide) standards with narrow-molecular-weightdistribution. The Mark–Houwink parameters K and a used for PEOwere 6.4�10�5 dl/g and 0.82, respectively, and for NaAlg, theywere taken as 7.3�10�5 dl/g and 0.92, respectively (De Ramoset al., 1997).

2.4. DPPHd scavenging activity

Scavenging activity of the DPPHd was measured according tothe method described by Yuan et al. (2005) and Sun et al. (2009).A 0.1 mM solution of DPPHd in a mixture of ethanol and water(with a mass ratio of 1:1) was prepared, and 3 ml of this solutionwas added to 1 ml of a sample solution that containedpolysaccharides of various molecular weights. This solution wasincubated at room temperature for 30 min, and the absorbance(A1) was read at 525 nm. The concentration of the sample in theassay mixture was appropriately diluted to meet the calibrationrange of the spectrometer. The absorbance of a blind control (A2)containing DPPHd and deionized water instead of a polysacchar-ide solution was also read. The following equation was used tocalculate the DPPHd scavenging activity:

Scavenging activityð%Þ ¼A2�A1

A2� 100

3. Results and discussion

3.1. Preparation of NaAlg fractions

It is very well known that polysaccharides in dry form or insolution degrade when exposed to ionizing radiation; in thisstudy, raw NaAlgs were irradiated with gamma rays at variousdoses in order to prepare the NaAlg fractions. To investigate theeffect of gamma rays on the molecular weight of NaAlgs, their Mw

and Mn values were evaluated using SEC. Unimodal chromato-grams were obtained for all NaAlg samples. As the irradiationdose increased, the SEC chromatogram of the NaAlg sampleshifted to higher retention volumes, indicating that the molecularweight of the sample decreased with irradiation. By using theuniversal calibration curve, the average molecular weight of theNaAlg samples was evaluated. Changes in the weight and numberaverage molecular weight (Mw and Mn, respectively) withirradiation dose are given in Figs. 1 and 2, respectively. As canbe seen from these figures, both average molecular weightsdecreased rapidly up to 20 kGy. The efficiency of radiation-induced events is expressed by the so-called G-value. The G-value,which is equal to the number of events per 100 eV of energy

0 5 10 15 20 25 40 505

10

15

Dose (kGy)

M

Fig. 1. Change in the number average molecular weight (M n) of NaAlgs with dose.

0 5 10 15 20 25 40 50

20

30

40

50

60

70 LF120 LF200 LF240

Dose (kGy)

Mw

x10

4

Fig. 2. Change in the weight average molecular weight (M w) of NaAlgs with dose.

0 10 20 30 40 500

10

20

30

40

50

60

70

80

90

LF120 Unirradiated LF200 Unirradiated LF240 UnirradiatedD

PPH

rad

ical

sca

veng

ing

activ

ity (

%)

Concentration (mg/ml)

Fig. 3. Variation of the DPPH scavenging activity of the unirradiated raw NaAlgs as

a function of concentration.

0 10 20 30 40 500

10

20

30

40

50

60

70

80

90

LF120 LF200 LF240 D

PPH

rad

ical

sca

veng

ing

activ

ity (

%)

M. S- en / Applied Radiation and Isotopes 69 (2011) 126–129128

absorbed, is customarily used to measure the radiation chemicalyield. In a previous study of the author of this article’s, the effectof G/M on the radiation-induced degradation of sodium alginatewas investigated in detail (S-en et al., 2010). It was concluded inthat study that the chain scission yield (G(S)) values weredependent on the structure (G/M) of the sample and followedthe order LF2404LF2004LF120 or 70/30o50/50o45/55 G/M.Another conclusion was that G/M was an important factor incontrolling the degradation rate of sodium alginate.

Concentration (mg/ml)

Fig. 4. Variation of the DPPH scavenging activity of the 50 kGy-irradiated NaAlgs

as a function of concentration.

0 5 10 15 20 25 40 500

10

20

30

40

50

60 LF120 LF200 LF240

DPP

H r

adic

al s

cave

ngin

g ac

tivity

(%

)

Dose (kGy)

Concentration : 5 mg/ml

Fig. 5. Effect of irradiation dose on the DPPH scavenging activity of NaAlgs.

3.2. DPPH scavenging activity of NaAlg fractions

In order to evaluate the antioxidant properties of NaAlgfractions, the effect of concentration of NaAlg on the scavengingactivity of DPPHd was investigated firstly. Fig. 3 shows the DPPHd

inhibiting efficiency of NaAlg LF120, LF200 and LF240 (340, 310,275 kDa, respectively) at different concentrations. Scavengingability for DPPHd was observed in the case of the three differentG/M and NaAlgs with close initial molecular weights whereas thesame concentration exhibited a different inhibiting efficacy. Theconcentration of LF240 NaAlg was 25 mg/ml in the solution; aDPPHd inhibiting efficacy of approximately 50% was achieved.This indicated that the 50% inhibition concentration (IC50) was25 mg/ml. In the case of the concentration of 40 mg/ml, on theother hand, the DPPH inhibiting efficacies of LF120 and LF200NaAlg were approximately 38% and 41%, respectively. Thisdecrease was probably due to the decrease in the G content ofNaAlg. It is also well known that the diaxial linkage in G-blocksresults in a large, hindered rotation around the glycosidic linkage,

which may account for the stiff and extended nature of alginatechains (Smidsrod et al., 1973).

The DPPHd scavenging activity of the 50 kGy-irradiated NaAlgswith reduced molecular weight as a function of concentration isgiven in Fig. 4. As can be seen from the figure, the DPPHd

scavenging activity increased with concentration as expected, andthe increase in efficacy followed the order LF2404LF2004LF120.The IC50 values of the 50 kGy-irradiated LF120, LF200, and LF240NaAlgs (180, 150, and 140 kDa) were approximately 23, 16, and11 mg/ml, respectively. The decrease in the IC50 value of NaAlgwith irradiation was attributed to the decrease in molecularweight with irradiation and to different G and M contents in thechain structure. The effect of G/M on the DPPHd scavengingactivity of NaAlg is mentioned in the following sections. Due tothe low molecular weight of the 50 kGy-irradiated LF240, LF200,and LF120, and the molecular weight order Mn(LF240)oMnðLF200Þ

oMn(LF120), the following order was obtained for the IC50 value ofthis fraction: IC50 (LF240)o IC50 (LF200)o IC50 (LF120).

The effect of irradiation dose on the DPPHd scavenging efficacyof NaAlg is illustrated in Fig. 5. As clearly seen from this figure, theantioxidant activity of NaAlgs increased with irradiation dose, andthe scavenging efficacy increased rapidly up to 5.0 kGy, whichthen slowed down at higher doses. Higher scavenging activity wasobserved at all irradiation doses for LF240 NaAlg. This highefficacy was ascribed to the higher extent of degradation of LF240at higher irradiation doses. These results showed that NaAlg witha low molecular weight had a relatively strong scavenging abilityfor PDDHd.

In order to investigate the effects of the structural propertiesof NaAlg on its antioxidant properties, the number average

10 15 20 25 30 35

7

14

21

28

35

42 LF120 LF200 LF240

DPP

H r

adic

al s

cave

ngin

g ac

tivity

(%

)

Mn x104

Fig. 6. Effect of the molecular weight and G/M of NaAlg on the DPPH scavenging

activity.

M. S- en / Applied Radiation and Isotopes 69 (2011) 126–129 129

molecular weight of the fractions was plotted against the PDDHd

scavenging activity (Fig. 6). It was evident from this figure that theantioxidant activity of NaAlg was independent of its molecularweight whereas the G/M appeared to be an important factor inaffecting the DPPHd scavenging activity of NaAlg. NaAlg with ahigher G/M had a relatively strong scavenging ability for PDDHd.This increase was attributed again to the increase in NaAlg chainmobility with decreasing G/M. All in all, the highest radicalscavenging activity for the polymer concentration of 5 mg/ml wasobtained for the NaAlg fraction with the lowest molecular weightand G/M.

4. Conclusions

The radical scavenging activity of NaAlg was found to bedependent on its molecular weight and chemical structure. Theeffect of irradiation on the molecular weight and radicalscavenging properties of NaAlg became more pronounced atlower G/M. It could therefore be concluded that its molecularweight and G/M were factors that affected the antioxidantproperties of NaAlg. The results also indicated that the antiox-idative behavior of NaAlg was not completely independent of theinitial G/M.

Acknowledgements

The authors gratefully acknowledge the support provided bythe International Atomic Energy Agency through Research Con-tract no. 14475/R01 and TUBITAK bilateral cooperation projectTBAG-U/175(106T442) and Hacettepe University 08G702001projects.

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