Radiolysis studies of aqueous κ-carrageenan

Nuclear Instruments and Methods in Physics Research B 268 (2010) 1607–1612

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Radiolysis studies of aqueous j-carrageenan L.V. Abad a,*, H. Kudo b, S. Saiki b,c, N. Nagasawa c, M. Tamada c, H. Fu b, Y. Muroya b, M. Lin b,d, Y. Katsumura b,d, L.S. Relleve a, C.T. Aranilla a, A.M. DeLaRosa a a

Philippine Nuclear Research Institute, Diliman, Quezon City, Philippines Nuclear Professional School, Graduate School of Engineering, The University of Tokyo, 2-22 Shirakata-Shirane, Tokai, Naka, Ibaraki 319-1188, Japan c Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292, Japan d Advanced Science Research Center, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Naka, Ibaraki 319-1195, Japan b

a r t i c l e

i n f o

Article history: Received 23 November 2009 Received in revised form 2 February 2010 Available online 18 February 2010 Keywords: j-Carrageenan Radiation degradation yield Pulse radiolysis Conformation

a b s t r a c t The effects on N2O and N2 gas on the radiation degradation yield of aqueous kappa (j-) carrageenan were investigated. The Gd of solution saturated with N2O solution was expectedly much higher than in air (1.7 and 1.2  107 mol J1). On the other hand, a lower Gd of 1.1  107 mol J1 was obtained from j-carrageenan solution saturated with N2. The rate constant of reaction of OH radicals with sonicated and irradiated j-carrageenan were determined using e-beam pulse radiolysis. The rate constant of OH interaction with sonicated j-carrageenan decreased with decreasing molecular weight. On the other hand, the OH interaction with irradiated jcarrageenan decreased but did not vary significantly with decreasing molecular weight. Metal ion (Na+) induced conformational transition into helical form decreased the rate constant of OH reaction with j-carrageenan. Likewise, the Gd in aqueous form was affected by the conformational state of j-carrageenan. The helical conformation gave a lower Gd (7  108 mol J1) than the coiled conformation (Gd = 1.2  107 mol J1). Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction

j-Carrageenan is a class of red seaweed from the Eucheuma species found abundantly in the Philippines. It is made of galactose residues made up of a-(1,4)-D-galactose-4-sulfate and b-(1,3)-3,6anhydro-D-galactose units. Carrageenans are known to have valuable biological functions. Due to the superior gelling and high viscosity properties of the native carrageenans, their utilization for biological applications is in most cases in the form of their oligomers. Oligo-kappa carrageenans induce secretion of laminarinase from Rubus cells and protoplast [1]. Degraded k-carrageenan is reported to have tumor inhibiting activities [2,3]. Oligomers from carrageenans suggest promising antiherpetic and anti-HIV (human immunodeficiency virus) activities [4–7]. Oligomers of carrageenan can easily be prepared through depolymerisation either by chemical or enzymatic hydrolysis. Recently, degradation by radiation processing of polysaccharides has gained much attention due to its technological effectiveness in producing low molecular weight oligomers [8–10]. Previous works have studied the physical, chemical and rheological properties of irradiated carrageenan [11–15]. Study on the use of irradiated carrageenan as plant growth promoter has also been reported [16]. The optimum dose

for plant growth promoter effect of solid j-carrageenan is at 100 kGy. The radiation degradation yield (Gd) both in solid and aqueous (1%) has already been investigated [17]. This research aims to determine some factors affecting the degradation of aqueous j-carrageenan. The effect of N2 and N2O gas on the Gd of aqueous j-carrageenan will be studied. The rate constant of reaction of OH radicals with degraded or depolymerized j-carrageenan (sonicated and irradiated j-carrageenan) as measured by electron beam pulse radiolysis will also be discussed. The effect of conformational transitions from helix to coil by the addition of Na+ on the rate constant of OH reaction with j-carrageenan and its Gd will be investigated. 2. Materials and methods 2.1. Materials Refined j-carrageenan was obtained from Shemberg Corporation, Philippines and was purified according to the procedure done previously [11]. 2.2. Irradiation of aqueous j-carrageenan

* Corresponding author. Address: Philippine Nuclear Research Institute, Commonwealth Ave., Quezon City, Philippines. Tel./fax: +63 2 9201655. E-mail address: [email protected] (L.V. Abad). 0168-583X/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2010.02.006

Purified j-carrageenan was dissolved in water at 1% concentration (w/v) and was divided into three different sets while placing

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them in separates tubes (five tubes per set). The different sets of tubes were then treated in the following manner: (a) Tubes were sealed with parafilm. (b) Tubes were purged with N2O gas for 30 min. and heat sealed while still purging the gas at the mouth of the tubes. (c) Tubes were purged with N2 gas for 30 min. and heat sealed while still purging the gas at the mouth of the tubes. In another experiment, purified j-, i- and k-carrageenan were dissolved in water or in 0.08 M NaClO4 solution at 1% concentration (w/v). These samples were irradiated using the Co-60 facility of the Takasaki Advanced Radiation Research Institute, Japan Atomic Energy Agency at a dose rate of 1 kGy/h with absorbed doses ranging from 1 to 6 kGy. 2.3. Irradiation of solid j-carrageenan Samples of purified j-carrageenan in powder form was irradiated at 25, 50 and 100 kGy in air and at ambient temperature at a dose rate of 10 kGy/h.

j-carrageenan was determined by the competition kinetics using KSCN as competitor scavenger. Polymer concentration ranged from 10 to 35 mM which is below its critical concentration of 40 mM. 2.8. UV–vis analysis UV–vis spectroscopy of carrageenan solutions was performed using a Shimadzu spectrophotometer UV-265 FW (wavelength range = 200–600 nm) at ambient temperature and at 0.025% (w/ v) concentration. 3. Results and discussion 3.1. The effect of N2 and N2O gas on the radiation degradation yield of j-carrageenan The Gd of j-carrageenan in aqueous (1%) solution has already been determined to be 1.2  107 mol J1 [14]. The presence of N2O and N2 gases in these solutions will definitely have an effect on its Gd. N2O reacts with solvated electrons to produce more OH radicals (Eq. (2)).

eaq þ N2 O þ H2 O ! OH þ N2 þ OH 2.4. Viscosity measurement Viscosity measurement of carrageenan solutions (1%) was done using a Tokimec Viscometer TV-20L with a THM-11 spindle at 25 °C and a rotor speed of 6 rpm. 2.5. Determination of radiation degradation yield of carrageenan Radiation degradation yield (Gd) of aqueous j-carrageenan was calculated from the molecular weights obtained from GPC experiments in Eq. (1). This equation is based on the assumption that the yield of crosslinking (Gx) is zero as it is irradiated in dilute solution (1%).

Gd ¼

  2c 1 1  D Mw Mw0

ð1Þ

where Mw is the weight-average molecular weight at absorption dose; Mw0 is the initial weight-average molecular weight; D is the absorbed dose in kGy; c is the fractional weight of carrageenan solution [16]. GPC analyses of samples (1 mg/ml in 0.1 M NaNO3) were performed on a Tosoh chromatograph equipped with DP8020 pump, CO-8020 column oven, RI-8020 refractive index detector and four TSK gel PWXL columns in series (G6000 PWXL, G4000 PWXL, G3000 PWXL and G2500 PWXL. Elution was carried out using 0.1 M NaNO3 as the mobile phase at a flow rate of 0.5 ml/ min. The temperatures of the column and detector were both maintained at 40 °C. A calibration curve was constructed using polyethylene oxide as standards. All molecular masses reported in this work are based on PEO standards and are not absolute.

ð2Þ

Previous results indicate that OH radicals react at a very fast rate with j-carrageenan (k 1.2  109 M1 s1). Thus, it would be interesting to know to what extent is the Gd of j-carrageenan affected by the presence of N2O and by purging N2 gas to remove air in the solution. Fig. 1 shows the plot of 1/Mw  Mw0 against radiation dose in the presence of these gases. From the graph, it is quite obvious that in the presence of N2O, Gd was increased substantially. Expectedly, the Gd of j-carrageenan in the presence of N2 was lowest when compared to N2O and in air. There was only a slight difference in the slope of the curves between N2 and air. The computed Gd values were as follows: 1.7, 1.3 and 1.1  107 mol J1 for N2O, air and N2, respectively. The Gd in air was quite close to the value of 1.2  107 mol J1 previously obtained [17]. Studies on alginate do not show any difference in the Gd of 1% solution in air and in N2 (Gd = 5.5  108 mol J1) [19]. In some cases, it has been reported that the radiation-chemical yield of scission has been reduced in oxygen containing polymeric solution. The radiation-chemical yields of chain scission for a 0.01 M chitosan are Gd = 3.4  107 mol J1 in N2O-saturated and Gd = 2.1  107 mol J1 in N2O– O2-saturated solutions [18]. Similar trends have been observed in pectic substances [20]. Fig. 2 shows the UV absorbance at 260 nm

14 12

in Air in N2O

10

in N 2

8 2.6. Sonication of j-carrageenan Sonochemical degradation of j-carrageenan was performed using Cole Parmer 4710 Series, Ultrasonic Homogenizer, with a frequency of 80 kHz. Solutions with concentration of 5%, 2.5% and 1% were sonicated for 30 min. 2.7. Pulse radiolysis of j-carrageenan Pulse radiolysis experiments were performed using an electron beam (10 ns) of 35 MeV delivered from a linear accelerator. The rate constant of the reaction of OH with sonicated and irradiated

6 4 2 0 0

1

2

3

4

5

6

7

Fig. 1. Reciprocal of Mw of 1% aqueous j-carrageenan at varying doses purged with different gases.

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1.6 Mw==436,915 Mw 437 kDa (Non (Non-sonicated) -sonicated) Mw==376,139 Mw 376 kDa Mw==267,467 Mw 267 kDa Mw==202,244 Mw 202 kDa

1.4

λ

A 0 / A -1 / a.u.

1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.0

2.0e-10 4.0e-10 6.0e-10 8.0e-10 1.0e-9 1.2e-9 1.4e-9 1.6e-9

[KC] / [K OH.] [SCN- ] / M -1 s -1

for aqueous j-carrageenan in the presence of N2O, N2 and in air with increasing radiation doses. This 260 nm peak is known to be attributed to the formation of carbonyl bond in the pyranose ring. A slight increase in intensity of this peak was seen with increasing radiation doses. The UV absorbance trend follows the same order as that of their Gd values where N2O > air > N2. Thus, the greater is the radiation degradation yield, the greater are the number of carbonyl groups formed. Free carbonyl groups are formed in two ways, first, by radiolytically induced hydrolytical cleavage of glycosidic bonds, and second, as the result of oxidation of carbohydrate radicals generated inside the galacto-pyranose ring [20]. 3.2. Pulse radiolysis studies of sonicated and irradiated j-carrageenan The molecular weight of the sonicated j-carrageenan solutions was analyzed by GPC. Mw was determined to be as follows: 376, 267 and 202 kDa (Table 1). The rate constant of OH reaction with j-carrageenan of these samples was measured by e-beam pulse radiolysis. A decrease in molecular weight of j-carrageenan would signify a decrease in viscosity, which expectedly would increase the diffusion rate of OH radicals and consequently increase the rate constant. Hydrolyzed j-carrageenan at an acidic solution (pH 2) gives higher k of OH reaction [17]. The current results on sonicated j-carrageenan, however, indicated a reverse trend than what was expected. Fig. 3 shows that the rate constants decreased from k = 9.9  108 M1 s1 (unsonicated j-carrageenan) to k = 9.4  108, 8.3  108 and 6.8  108 M1 s1 with decreasing molecular weights. Similarly, the rate constants of irradiated carrageenan at 25, 50 and 100 kGy (Mw = 188, 105, and 59 kDa, respectively shown in Table 1) decreased (from k = 1.1  109 to an average of

Table 1 Weight-average molecular weight, number average molecular weight and molecular weight distribution of sonicated and irradiated j-carrageenan. Mw

Mn

Mw/Mn

Sonicated samples Non-sonicated 5% Conc. 2.5% Conc. 1% Conc.

436,915 376,139 267,467 202,244

137,351 113,783 126,900 104,476

3.18 3.31 2.11 1.94

Absorbed dose (kGy) 0 25 50 100

449,941 188,261 104,833 59,336

158,386 66,547 37,373 22,493

2.84 2.83 2.81 2.63

Fig. 3. Determination of the rate constant reaction of OH radical with sonicated jcarrageenan.

k = 8.2  108 M1 s1) but did not vary with increasing radiation dose (Fig. 4). Two factors may affect the reaction rate of a polymer. First, a reduction of viscosity or molecular size leads to higher diffusion of OH in the polymer resulting in increased rate constant. Second, rate constant is directly proportional to the number of reactive sites for the OH interaction. Based on the results, the latter factor predominates in the sonication or irradiation of j-carrageenan. Cleavage of glycosidic linkage in j-carrageenan is the most likely effect in the sonication or irradiation of j-carrageenan as evidenced by a rapid decrease in molecular weight by either these two processes. But cleavage of glycosidic linkage alone would have increased the rate constant k of OH reaction similar to the one observed in the hydrolysis reaction j-carrageenan at pH = 2 [14]. Experiments on chitosan indicate that the rate constant of OH reaction with chitosan is increased with decreasing chain length [21]. Most likely, sonication or irradiation of j-carrageenan could have generated products that have reduced the reactive sites for OH reaction with j-carrageenan. Thus, decreasing rate constant of OH interaction with decreasing molecular weight was observed both in sonicated and irradiated j-carrageenan. The degradation mechanism of macromolecules by ultrasound is frequently attributed to cavitation (mechanical) effects and partially to the stress concentration on the segment of macromolecules [22]. At lower

2.0 1.8 1.6

A 0/A -1 / a.u.

Fig. 2. UV absorbance at 260 nm of j-carrageenan (0.25%) irradiated at different conditions.

Mw = 450 kDa (0kGy) Mw = 188 kDa (25kGy) Mw = 105 kDa (50kGy) Mw = 59 kDa (100kGy)

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.0 2.0e-10 4.0e-10 6.0e-10 8.0e-10 1.0e-9 1.2e-9 1.4e-9 1.6e-9 1.8e-9

[KC] / [K OH .] [SCN - ] / M -1s -1 Fig. 4. Determination of the rate constant reaction of OH radical with irradiated jcarrageenan.

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frequencies, 20–50 kHz range (the ‘low’ frequency domain reaches to 100 kHz), these effects are observed [23]. When ultrasound of a frequency >500 kHz is applied, an additional factor (radical reactions similar to radiolysis effect) may become more pronounced [24]. Since the frequency used for the sonication of j-carrageenan was only 80 kHz, it was expected that cleavage of glycosidic linkage could be caused only by vibrational effect. However, UV–vis spectra of sonicated j-carrageenan solutions, revealed otherwise as seen in Fig. 5a. The figure shows slightly increasing UV absorbance at 260 nm. Similar experiments done previously on sonicated chitosan (360 kHz) show two absorption bands (at 265 and at 297 nm), absent in the starting material, in the UV–vis spectroscopy. These are some transformations of chitosan-derived radicals that lead to the formation of carbonyl groups [21]. Ultrasound-induced degradation of chitosan in Ar-saturated solutions, both caused by OH or vibrational effects, is accompanied by side reactions. One source of such processes is a terminal radical formed as a result of glycosidic bond breakage. In the case of OH-mediated process, there are also non-terminal radicals located along the chain, which may not be capable of causing chain breakage, but may undergo other reactions [18,24]. The effect of sonochemical degradation of j-carrageenan may follow the same scheme as that of chitosan. Since the frequency is not so high, the formed carbonyl may simply be a terminal radical formed as a result of glycosidic bond breakage. As a consequence, diminishing reactive site for OH radical interaction was also observed as indicated by decreasing rate constant k with increasing concentration (decreasing molecular weight). In the case of the reaction of ionizing radiation

Absorbance

0.14 0.12

Mw == 436 436,195 kDa(non-sonicated) (non-sonicated)

0.10

Mw == 376,139 376 kDa Mw == 267,467 267 kDa Mw == 202,244 202 kDa

0.08 0.06 0.04

260nm

0.02 0.00

(a)

-0.02 200

300

400

500

600

Wavelength (nm) 0.8 450 kDa (0kGy) 188 kDa (25kGy) 105 kDa (50kGy) 59 kDa (100kGy)

0.6

Absorbance

260nm 0.4

0.2

0.0

(b) -0.2

200

300

400

500

600

Wavelength (nm) Fig. 5. UV–vis spectra of (a) sonicated and (b) irradiated j-carrageenan.

with j-carrageenan, reactions of OH and other radicals can produce carbonyl groups in several sites not only in the terminal groups. In fact Fig. 5b shows higher UV–vis absorbance peak at 260 nm than sonicated j-carrageenan. Reactions can be more severe than the sonication process especially under air condition where some peroxy radicals are generated. Oxidation reactions may take place with the formation of carbonyl groups/carboxyl groups and which can eventually lead to fragmentation patterns that may result in ring opening of the galacto-pyranose ring. This radiolysis reaction mechanism has been identified in many types of polysaccharides (chitosan, alginates, cellulose, etc.). Since OH acts on polysaccharides by H abstraction, formation of carbonyl in the polymer chain would result in the decrease of its reactive site (reduced H for abstraction). Two events – decrease in viscosity (increase in k) and decrease in reactive sites (decrease in k) with increasing absorbed dose may have occurred simultaneously producing an over-all effect of a consistently uniform rate constant for all doses. 3.3. The effect of sodium ion on the rate constant of reaction of OH radical with j-carrageenan It is known that OH radicals react with synthetic polyelectrolytes at rate constants dependent on the conformation of a macromolecule. When macromolecules are charged, they attain a linear conformation with a rate constant that is significantly higher than when they are in their neutral coiled conformation [25]. Extended chains fill up volume of the solution more uniformly than shrunk chains, which occupy only limited space leaving large voids of water devoid of the solute. The reaction rate constant of OH with macromolecules is dependent on the diffusion distance [26]. This effect can be observed for natural polymers and their derivatives. This phenomena is illustrated in the cellulose derivatives (CM-cellulose, CM-chitosan, and CM-chitin) [26,27]. It is also a known fact that carrageenans undergo thermoreversible ion-induced conformational transition from a disordered (coil) to an ordered (helix) form. The ability of some cations to promote helical formation folþ + + + lows this order: Li+, Na+, NRþ 4  NH4
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3.0

0.5 κ-carrageenan κ-carrageenan with 0.08M NaClO4

(Mw -1 – Mw0 -1) * 106

A 0 /A - 1 / a.u.

0.4

κ -carrageenan κ-carrageenan with NaClO4

2.5

0.3

0.2

0.1

2.0 1.5 1.0 0.5 0.0

0.0 0.0

1.0 e-9

2.0 e-9

[KC] / [K OH

3.0 e-9

4.0 e-9

5.0 e-9

0.0

0.5

Fig. 6. The effect of Na+ on the rate constant of OH reaction with j-carrageenan.

These two schools of thought for the conformational transition of j-carrageenan are illustrated in Fig. 7 [43]. Whether it is a single or double helix, the structure as seen from the figure becomes more rigid in the helical conformation. The single or double stranded helical conformation would have some polymers protected inside the strand, preventing exposure to the solvent, thus decreasing possible interaction with OH radical. As a consequence, k is decreased. In addition, increased viscosity in the helical form could result in lower mobility of OH radical and therefore decreased k. The effect of the conformational changes on the radiation degradation yield (Gd) of j-carrageenan was also investigated. Fig. 8 shows the effect of the addition of Na+ on the relationship of 1/ Mw  Mw0 with dose radiation dose. The Gd had substantially been decreased by the presence of Na+ from a value of 1.2 to 0.7  107 mol J1. This result clearly suggests that conformational change from coil to helix in the presence of metal ions (Na+) affects also the degradation yield of j-carrageenan. Since i-carrageenan forms a helical conformation to a lesser extent than j-carrageenan and k-carrageenan does not have this transitions, the Gd of i- and k-carrageenan were also investigated. Interestingly, the results coincided with their expected conformational state in the presence of Na+ as shown in Table 2. Gd of i-carrageenan was lowered but to a lesser extent than j-carrageenan (1.1 to 0.8  107 mol J1). k-Carrageenan decreased slightly its Gd (1.0 to 0.9  107 mol J1). This decrease in k-carrageenan

Double Helix

Coil

1.0

1.5

2.0

Dose (kGy)

.] [SCN - ] / M -1s -1

Fig. 8. The effect of Na+ on the reciprocals of Mw of j-carrageenan.

Table 2 The effect of Na+ on the radiation degradation yield of carrageenans. Irradiation condition (1% aqueous soln.) Without NaClO4 With NaClO4

Radiation degradation yield, Gd/107 mol J1

j

i

k

1.16 0.70

1.07 0.83

0.96 0.85

may be due to some deviations or traces of j-carrageenan that is normally present in the native k-carrageenan. The decrease in Gd of j- and i-carrageenan with the addition of sodium (Na+) can best be interpreted by the double stranded structure of j- and i-carrageenan helix rather than the single helix strand. Double stranded breaks can only happen from a single radical leading to the formation of scission on both strands or, more probably, from two separate strand breaks in close proximity [44]. A free radical attack on the single strand of a double helix may not result in a strand break due to interchain stabilization effects (metastable structure) of the double helix [45]. This stable state increases the probability of a given radical to undergo interstrand crosslinking. Thus, yield of crosslinking becomes greater than zero and a lower Gd was obtained for j- and i-carrageenan in their conformationally ordered structure. In a related study, the influence of the conformational state of j- and i- on the rate of acid hydrolysis has been investigated. Results indicate an increase by a factor of 200 and 10 for j- and i-carrageenan, respectively when passing above the conformational transition temperature. In another experiment, the Mw of the partially hydrolyzed samples decreased 1.5–3 times by inducing the disordered conformation of j- and i-carrageenan. These results are attributed to the stability properties of the multiple stranded structure of the ordered conformations of both j- and i-carrageenan [31,43]. 4. Conclusion

Single Helix

Fig. 7. Two models of conformational transition in j-carrageenan.

The Gd (1.7  107 mol J1) of 1% aqueous solution of j-carrageenan saturated with N2O was expectedly much higher than in aqueous j-carrageenan in air (1.3  107 mol J1). On the other hand, a lower Gd of 1.1  107 mol J1 was obtained from j-carrageenan solution saturated with N2. The rate constant of OH interaction with sonicated j-carrageenan decreased with decreasing molecular weight. On the other hand, the OH interaction with irradiated j-carrageenan decreased

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but did not vary significantly with decreasing molecular weight. The reduction may be due to decrease in reactive sites with the formation of carbonyl groups upon irradiation or sonication. Metal ion (Na+) induced conformational transition into helical form decreased the rate constant of OH reaction with j-carrageenan. Helical conformation leads to a more rigid structure which increases viscosity of j-carrageenan solution and therefore less mobility of OH. Similarly, the Gd in aqueous form was affected by the conformational state of j-carrageenan. The helical conformation gave a lower Gd (0.7  107 mol J1) than the coiled conformation (Gd = 1.2  107 mol J1). A helical structure has some interchain stabilization effects which increases the possibility of free radical interchain crosslinking.

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