In vitro antioxidant properties of red algal polysaccharides

Biomedicine & Preventive Nutrition 1 (2011) 161–167

Original article

In vitro antioxidant properties of red algal polysaccharides E.V. Sokolova ∗ , A.O. Barabanova , R.N. Bogdanovich , V.A. Khomenko , T.F. Solov’eva , I.M. Yermak Pacific Institute of Bioorganic Chemistry of Far East Branch of the Russian Academy of Sciences, Prospect 100-let Vladivostoku, 159, 690022, Vladivostok, Russia

a r t i c l e

i n f o

Article history: Received 5 March 2011 Accepted 22 June 2011 Keywords: Antioxidant Red seaweeds Carrageenan

a b s t r a c t The present study was devoted to in vitro antioxidant properties of lambda-, iks-, kappa-, kappa/betaand kappa/iota-carrageenans from red algae of Gigartinaceae and Tichocarpaceae families collected from the Russian Pacific Coast. In vitro antioxidant capacity of carrageenans with different structural types was assessed by measuring their ferric-reducing antioxidant capacity, relative antioxidant capacity (phosphomolybdenum assay), and their scavenging effect on hydroxyl radicals, superoxide anion, nitric oxide and hydrogen peroxide. Influence of pH value on potential antioxidant capacity was also been investigated by means of linoleic model system. The reducing capacity of the investigated carrageenans was not much expressed, and capacity of the commercial polysaccharide samples was negligible. Iks-carrageenan containing three sulphate groups per disaccharide unit and a 3,6-anhydrogalactose unit was among the most effective superoxide anion (54.83% at the concentration of 1 mg/mL) and nitric oxide scavengers (37.26% at the concentration of 0.25 mg/mL) and the only polysaccharide possessing iron ion chelating capacity. Antioxidant actions of lambda-, iks-, kappa-, kappa/beta- and kappa/iota-carrageenans against reactive oxygen/nitrogen species depend on polysaccharide concentration and such fine structural characteristics as presence of hydrophobic 3,6-anhydrogalactose unit, amount and position of sulphate groups, and an oxidant agent, on which sample antioxidant action is directed. The results indicated that carrageenans possess antioxidant capacity in vitro, and this action notably depends on the structure of the polysaccharide itself than the reducing capacity of the polysaccharides. © 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction The normal cellular metabolism is remarked by ongoing formation of pro-oxidants, which is to balance by antioxidant defence systems. When balance is interrupted the oxidative stress is occurred and can result in atherosclerosis, cataract formation, aging, carcinogenesis, chronic inflammation, ischemia, diabetes, septic and haemorrhagic shock, neurodegenerative disorders, etc. The negative effects of oxidative stress can be reduced by antioxidants [1–4]. Reactive oxygen metabolites such as superoxide anion, hydrogen peroxide, hypochlorous acid and free metal ions related to hydrogen peroxide formation causes a qualitative decay of foods, which leads to rancidity, toxicity and destruction of biochemical compounds important in physiologic metabolism [5]. As a result, potential negative impacts of oxidants are widely recognized, and modern science has increased tendencies to identification of dietary compounds from natural sources that applied in food, medicines and cosmetics. Most of the reported antioxidant

∗ Corresponding author. Tel.: +7 4232 311430; fax: +7 4232 314050. E-mail addresses: [email protected], [email protected] (E.V. Sokolova), [email protected] (A.O. Barabanova), [email protected] (I.M. Yermak). 2210-5239/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.bionut.2011.06.011

materials are water-insoluble. Water-soluble antioxidants play an important role in assisting their water insoluble counterparts in the removal of reactive oxygen species from the body [6]. Bioactive compounds produced by macroalgae are often stressinduced, tend to have a broad polarity range [7], and of immense industrial, human and agricultural value since ancient times, especially in the Orient [7,8]. Sulfated galactans of marine algae are among the most abundant non-mammalian sulfated polysaccharides found in nature [9]. The structure of algal sulfated polysaccharides varies according to algal species, environmental conditions, and life stage of seaweed, and every new one is a novel compound with potential altering biological properties [10–15] including antioxidant capacities [10,16–19]. Carrageenans are complex family of water soluble, linear, sulfated galactans of red seaweeds. These polysaccharides are composed of alternating ␣-(1–3) and ␤-(1–4) linked D-galactosyl residues and several types of carrageenan are identified on the basis of the modification of the disaccharide repeating unit by ester sulphate and by the presence of 3,6-anhydrogalactose as 4-linked residue, three types of which (lambda-, kappa- and iotacarrageenans) are commercially available. There are not much data about antioxidant capacity of carrageenans, and that properties were observed on commercial samples of carrageenans [20–22].

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We have isolated various types of carrageenans from Gigartinaceae and Tichocarpaceae families collected from the Sea of Japan of the Russian Coast and determined their structures earlier [23–26]. The aim of this work is to investigate in vitro antioxidant capacity of carrageenans with different structural types by measuring their ferric-reducing antioxidant capacity (FRAP), relative antioxidant capacity, and their scavenging effect on such specific radicals as hydroxyl radicals, superoxide anion, nitric oxide. pH influence on potential antioxidant capacity will also been investigated by means of linoleic model system. 2. Materials and methods

in aqueous solution (0.5 mL, 2.8% w/v) was next added, and the mixture was heated for 20 minutes in a boiling water bath. The amount of chromogen produced was measured at 532 nm. 2.5. Chelating capacity (site-specific hydroxyl radical production during deoxyribose assay) This assay was performed by the familiar mode as for non-site specific hydroxyl radical production but excluding addition of EDTA to the reaction mixture [32,33]. The data are quantified as 1/A, where A is optical absorbance measured at 532 nm. 2.6. Determination of nitric oxide scavenging capacity (microplate)

2.1. Materials Carrageenan were isolated from seaweeds of Chondrus armatus and C. pinnulatus (Gigartinaceae) and Tichocarpus crinitus (Tichocarpaceae) families harvested from the Sea of Japan of the Russian Coast, and their structures were determined as was described earlier [23–26]. Commercial lambda- (Fluka), kappa(Sigma) and iota- (Sigma) carrageenans and agarose (Sigma) were also used. 2.2. Ferric-reducing antioxidant capacity (FRAP) assay The ferric reducing antioxidant power was carried out based upon the methodology of Benzie and Strain [27] and Rupérez et al. [28]. Freshly prepared FRAP reagent (2.5 mL of 10 mM 2,4,6tripyridyl-2-triazine [TPTZ] in 40 mM HCl, 2.5 mL of 2 mM ferric chloride in water and 25 mL of 0.3 M sodium acetate buffer, pH 3.6, all the components were heated to 37◦ C) with a volume of 240 mkl, water (24 mkl) and sample solution (8 mkl, different concentrations) were mixed. The absorbance was measured at 595 nm after four minutes incubation at 37◦ C. The data obtained were expressed as mmoL of ascorbic acid equivalents/g of sample dried weight. 2.3. Phosphomolybdenum assay The phosphomolybdenum assay [29] was performed using ascorbic acid as a positive standard. An aliquot of sample solution (0.3 mL) containing a reducing species (in water) was combined with reagent solution (3 mL, 0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The test tubes were incubated in a water bath at 95◦ C for 90 minutes. After the samples had been cooled, the absorbance of the aqueous solution was measured at 695 nm against a blank. A typical blank solution contained reagent solution (1 mL) and the appropriate volume of the same solvent used for the sample, and was incubated under the same conditions as the rest of the samples. The water-soluble antioxidant capacity was expressed as mmoL of ascorbic acid equivalents/g of sample dried weight. 2.4. Hydroxyl radical scavenging capacity (non-site-specific hydroxyl radical production during deoxyribose assay) The deoxyribose assay for hydroxyl radical scavenging capacity [30,31] was performed using mannitol as a positive standard. To a mixture of FeCl3 (10 mM, 0.005 mL), EDTA (1 mM, 0.05 mL), 0.165 mL of 50 mM potassium phosphate buffer (pH 7.4), 2deoxyribose (10 mM, 0.18 mL), and H2 O2 (10 mM, 0.05 mL), were added 0.5 mL of carbohydrate samples or control compound (0.25–2.00 mg/mL), and 0.5 mL of ascorbic acid (1 mM). The mixture was then incubated for one hour at 37◦ C. A solution of thiobarbituric acid in 50 mM NaOH (0.5 mL, 1% w/v) and trichloroacetic acid

Measurement of nitric oxide scavenging capacity was performed on the basis of method described by Green et al. [34] with slight modifications. The reaction mixture containing sodium nitroprusside (10 mM, 75 mkl) in phosphate buffered saline (PBS) and carrageenans or reference compound at different concentrations (0.25–1.00 mg/mL, 75 mkl) were incubated at 25◦ C for 150 minutes. Then, the Griess reagent (1% sulphanilamide, 0.1% naphthylethylene diamine dihydrochloride in 2% H3 PO4 ) was added to the reaction mixture (1:1 = v:v). The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was measured at 546 nm. The percentage inhibition of nitric oxide generated was measured by comparing the absorbance values of the control and a sample. Ascorbic acid was used as a positive control. 2.7. Determination of superoxide anion radical scavenging capacity (microplate) Measurement of superoxide scavenging capacity was performed on the basis of method described by Nishimiki et al. [35] with slight modifications. The reaction mixture in a final volume of 275 mkl contained 156 mkm NBT in 0.1 M phosphate buffer with pH 7.4, 468 mkm NADPH, 60 mkm PMS in 0.1 M phosphate buffer with pH 7.4 and sample solution at various concentration (0.25–2.00 mg/mL). After incubation for five minutes at ambient temperature and shaking, the absorbance at 562 nm was measured to determine the quantity of formazan generated. Ascorbic acid was used as a positive control. 2.8. Determination of hydrogen peroxide scavenging capacity (microplate) The ability of samples to quench hydrogen peroxide was determined spectrophotometrically at UV region [36]. The samples of polysaccharides were dissolved in 262 mkl of 0.1 M pH 7.4 phosphate buffered saline (PBS) and mixed with 46 mkl of 2 mM solution of hydrogen peroxide. Ascorbic acid was used as a control. Absorbance of hydrogen peroxide at 230 nm was determined 10 minutes later in a spectrophotometer. For each concentration, a separated blank sample was used for background subtraction. 2.9. Determination of pH influence on antioxidant capacity Influence of pH on antioxidant capacity was determined using a diene conjugated formation method according to Decker et al. [37], Wills [38], Lingnert et al. [39]. Linoleic acid (10 mM) emulsified with an equal amount of Tween 20 (1% in 0.1 M potassium phosphate buffer of different pH level). The linoleic acid was dissolved in 95% ethanol (26.8 mg of linoleate/mL of ethanol) and then the system obtained was diluted with nine volumes of the buffered solutions

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(pH 3, 5, 7, 9 and 11). The Tween 20 final concentration of the emulsion consists of 1% (w/v) for stabilizing. Then the emulsion was added to sample solution (1 mg/mL) (5:1 = v:v, respectively) and incubated at 37◦ C for 20 hours. After ending incubation time, the sample/emulsion system was diluted with 25 V of buffer/ethanol (9:1 = v:v) system and the absorbance was measured at 234 nm. In 2.4 and 2.6–2.9 measurements obtained using methods above, the experiments were performed in triplicate. The scavenging capacity (inhibition percentage) was calculated as follows: scavenging capacity (%) = (1–Asample /Ablank ) × 100, where Ablank is the absorbance of the blank. 2.10. Statistical analysis All data were expressed as mean ± standard deviation. Statistical analysis was done by one-way Anova using the Statistika 6 computer software application. Differences were considered to be statistically significant if P < 0.05. 3. Results Carragenans differ by number and position of sulfated group and by presence or absence of 3,6-anhydrogalactose unit. The following carrageenan structural types were used during the study: kappa, kappa/beta-, kappa/iota-, lambda- and iks-types of carrageenans (Table 1). Agarose differs from beta-carrageenan only by L configuration of 4-linked galactopyranose unit instead of D configuration in carrageenans. 3.1. Ferric-reducing antioxidant capacity (FRAP) and phosphomolybdemun assay (relative antioxidant capacity) FRAP assay is based on reducing of ferric ions by investigated polysaccharides to ferrous ones which form colored complex

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with TPTZ at acidic pH to maintain iron stability. Kappa/iota- and kappa/beta-carrageenans were the most active reducing agents regarding ferric ions and had similar capacities (Table 2). Lambda, iks- and kappa-carrageenans were less effective with value of approximately 84.39, 66.61 and 58.50 mmoL of ascorbic acid equivalents/g of sample dry weight, respectively. Actions of commercial polysaccharide samples were negligible ones. A phosphomolybdenum method developed for the quantitative determination of antioxidant capacity is based on the reduction of Mo(VI) to Mo(V) by the sample and the subsequent formation of a green phosphate/Mo(V) complex at acidic pH. The capacity of the investigated samples did not strongly differ from each other at phosphomolybdenum assay, and their actions belong to interval from 278.52 (lambda-carrageenan) to 296.94 mmoL of ascorbic acid equivalents/g of sample dry weight (iks-carrageenan). Kappa/beta-carrageenan, during this assay, was the most effective one (360.1 mmoL of ascorbic acid equivalents/g of sample dry weight). (Table 2). Commercial carrageenans and agarose had not got high contribution to the phosphomolybdemun method and also were the least effective samples at FRAP assay. 3.2. Hydroxyl radical scavenging capacity (non-site-specific hydroxyl radical production during deoxyribose assay) The competition method in which the Fenton reaction is employed as an generator of hydroxyl radicals produced nonspecifically (with EDTA) and deoxyribose as a detecting molecule, degradation products of which formed pink chromogenic substances with thiobarbituric acid, has been used to investigate hydroxyl radical scavenging capacity. The hydroxyl radical scavenging capacities of polysaccharides strongly depended on their concentration, and structure of a sample was not important during expression this action (Fig. 1). Hence, their actions at the concentration of 2 mg/mL were similar, and three times less than mannitol

Table 1 The structures of carrageenans from algae of Gigartinaceae and Tichocarpaceae families represented as the letter code nomenclature [11]. Algal species

Carrageenan type

C. armatus T. crinitus C. armatus C. pinnulatus T. crinitus

lambda iks kappa kappa/iota kappa/beta

HO

CH2OH

Structure of disaccharide repeating unit 3-linked

4-linked

G2S G2S, 4S G4S G4S/G4S G4S/G

D2S, 6S DA DA DA/DA2S DA/DA

HO O O OH

G

CH2OH HO

O OH

CH2OH

O

O O

O

O

O

OH

G

D

DA OH

Table 2 Reducing capacity of the investigated polysaccharides determined by means of FRAP assay and phophomolybdenum method expressed as mmoL of ascorbic acid equivalent per gram of sample dry weight. Carrageenan source

Carrageenan type

FRAP assay

Phosphomolybdenum assay

mmoL of ascorbic acid equivalents per sample dry weight C. armatus T. crinitus C. armatus T. crinitus C. pinnulatus Fluka Sigma Sigma Sigma

lambda iks kappa kappa/beta kappa/iota lambda (com) kappa (com) iota (com) agarose

84.39 ± 30.48 66.61 ± 24.07 58.50 ± 25.27 89.47 ± 34.73 98.22 ± 44.88 effectless effectless effectless effectless

278.52 ± 44.66 296.94 ± 120.33 291.24 ± 83.74 360.10 ± 93.04 292.12 ± 77.53 effectless effectless effectless effectless

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Fig. 1. Hydroxyl radical scavenging capacity of the investigated samples expressed as inhibition percentage.

capacity. Commercial kappa-carrageenan and agarose sample were the least effective samples during this assay, and their capacity almost did not alter with concentration. 3.3. Chelating capacity (site-specific hydroxyl radical production during deoxyribose assay)

[34]. All the carrageenans exhibited nitric oxide scavenging capacity and were similar with that of the standard (Fig. 2). Of all the investigated samples, low sulphated kappa/beta- and commercial kappa-carrageenans had the lowest capacity at this assay. Agarose was an effectless scavenger. 3.5. Superoxide anion scavenging capacity

Another version of deoxyribose method is site-specific assay without adding EDTA to reaction mixture. In this instance, unchelated iron ions added to deoxyribose-containing reaction mixtures can become weakly associated with deoxyribose and sample capacity to compete for iron ions is measured [32,33]. This assay showed that iks-carrageenan possessed chelating capacity; the intense of its action was more expressed than ascorbic acid effect (mannitol: 5.08 ± 0.08, 5.28 ± 0.07, 5.58 ± 0.07, 6.08 ± 0.16 1/A and iks-carrageenan: 4.14 ± 0.16, 5.29 ± 0.11, 5.90 ± 0.01, 5.82 ± 0.02 1/A at the concentrations of 0.25, 0.5, 1.0 and 2.0 mg/mL respectively). The other carrageenans (lambda, kappa, kappa/beta and kappa/iota) were not effective samples on iron ions. 3.4. Nitric oxide scavenging capacity According to applied method without employing any enzymes used for reduction of nitrite occasionally formed to nitrite, were not included to the reaction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide

Measurement of superoxide scavenging capacity was performed on the basis of method described by Nishimiki et al. [35] with slight modifications. Superoxide anion derived from dissolved oxygen by the non-enzymatic phenazine methosulfatenicotinamide adenine dinucleotide phosphate (PMS/NADPH) coupling reaction reduces nitroblue tetrazolium (NBT), which forms a violet coloured formazan. A decrease in colour after addition of the antioxidant is a measure of its superoxide scavenging capacity. Lambda-, iks-, kappa-, kappa/beta- and kappa/iotacarrageenans were used during superoxide anion scavenging assay, and ascorbic acid was used as a reference (Fig. 3). The superoxide anion scavenging capacity of lambda-carrageenan was compared with capacity of ascorbic acid. The other carrageenans were more effective than ascorbic acid, and their capacity increased in the following row: kappa/iota- (45.95%) < kappa/beta- (47.67%) < kappa(53.92%), and iks-carrageenan was the most effective superoxide anion scavenger (54.82%) at 1.00 mg/mL. 3.6. Hydrogen peroxide scavenging capacity All the investigated polysaccharides (all the carrageenans and agarose) were inert regarding to hydrogen peroxide.

Fig. 2. Nitric oxide scavenging capacity of the investigated samples expressed as inhibition percentage.

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Fig. 3. Superoxide anion scavenging capacity of the investigated samples expressed as inhibition percentage.

Fig. 4. Influence of pH value on inhibition of linoleic acid oxidation by the carrageenans at the concentration of 1 mg/mL.

3.7. Determination of pH influence on antioxidant capacity Influence of pH on antioxidant capacity was determined using a diene conjugated formation method according to aforementioned assay [37–39]. Autoxidation of linoleic acid by allene rearrangement allows monitoring a strong UV absorbance at 234 nm (Amax of conjugated diene peroxides from linoleic acid oxidation) [40,41]. This modified hydrogen atom transfer (HAT) based antioxidant assay was used for investigating the influence of pH. The results at pH 7 can be employed to estimate the antioxidant capacity of the investigated carrageenans relatively to each other at the sample concentration of 1 mg/mL (Fig. 4). Concentration dependence of the samples to transfer the hydrogen atom was not performed as HAT-based assay gave only tentative information [41]. 4. Discussion Of late years, algal polysaccharides have been reported to be useful candidates in the search for an effective, non-toxic substance and have been demonstrated to play an important role as free radical scavengers in vitro and antioxidants for prevention of oxidative damage in living organisms [21]. The present study was devoted to in vitro antioxidant properties of carrageenans from red algae of Gigartinaceae and Tichocarpaceae families collected from the Russian Pacific Coast. Commercial samples of red algal polysaccharides (carrageenans and agarose) were used in some assays for the comparison. At this study, electron transfer

(ET) capacity was conducted, namely ferric reducing antioxidant power (FRAP) and relative antioxidant capacity assays. During FRAP assay, carrageenans from the Russian Pacific Coast seaweeds were not high effective ones, which was as the result of either slight precipitation formed for all the carrageenan types in the reaction mixture, contributing an error to the capacity or low ferric reducing capacity of the samples. To confirm the last presumption, another type of method similar to FRAP assay mechanism was performed. A reduction of Mo(IV) to Mo(V) during phosphomolybdenum method by samples, which have a less-positive redox potential under reaction conditions, obtain the confirmation of low relative reducing capacity of carrageenans. Commercial samples of carrageenans at both FRAP and phosphomolybdenum assays showed non-efficiency. Kappa/beta-carrageenan, the least sulfated polysaccharide after agarose, had the highest action at this assay. The other samples demonstrated near efficiency, and capacity of lambda-carrageenan with high sulphate ester contents was the least one. Apparently the more important factor during demonstration reducing capacity is not a presence of these functional groups but the structure of the polysaccharide itself. The ability of the samples tested to reduce formation of the conjugated bonds in linoleic acid systems with different pH values was measured. This modified hydrogen atom transfer (HAT) based antioxidant assay was used for investigating the influence of pH, and the results at pH 7 can be employed to estimate the antioxidant capacity of the investigated samples relatively to each other. As HAT reactions are typically fast and independent of pH

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conditions [42] the capacity of all the carrageenan types to transfer the hydrogen atom was observed and decreased with increase in the pH value (Fig. 4). Earlier was supposed that at an acidic pH, hydrolysis reaction should may have occurred resulting in the fragmentation of kappa-carrageenan [43,44]. So the more capacity of carrageenans to inhibit linoleic acid oxidation can be explained with autohydrolysis of the polysaccharides at acidic pH. However, all the carrageenans possessed high antioxidant capacity at pH 7. It should be mentioned that in the presence of water, linoleic acid will form micelles, which further complicate the assay as the antioxidant distribution between two phases can be critical [41] resulting in encapsulating linoleic acid micelles by amphiphilic antioxidant [45], and hydrogen atom transfer reaction can be difficult to distinguish. For the purpose of investigating the hydrogen peroxide scavenging capacity, the method based on the measurement of intrinsic absorbtion of hydrogen peroxide at 230 nm was applied. But absorbtion of the investigated samples requires conducting the subtraction of additional “blank”. All the polysaccharides (carrageenans and agarose) were inert regarding to hydrogen peroxide. The results obtained testify of no hydrogen peroxide scavenging capacity of the samples what make this methodology quite adequate for our study, and subtraction of background absorbtion appears to be harmless. Scavenging capacity of hydroxyl radicals generated nonenzymatically with the Fenton reaction was also determined by means of non-site specific deoxyribose assay. During this assay iks-carrageenan was the only polysaccharide possessing the chelating capacity. Lacking of influence of the carrageenans on hydrogen peroxide and iron ions excepting iks-carrageenan removes possible interference from the carrageenans with the development of hydroxyl radical production during Fenton reaction. All the carrageenan samples displayed moderate scavenging effect to hydroxyl radicals. The capacity of carrageenans of all the types to scavenge hydroxyl radicals is significantly determined with the polysaccharide concentration except commercial kappa-carrageenan. Agarose was the least active one regarding hydroxyl radicals and its concentration dependence was absence what can be explained with its chemical structure, which is a non sulfated polysaccharide forming much stronger gels than carrageenans. Commercial sample of iota-carrageenan exhibited the highest action at the concentration of 0.5 mg/ml during this assay, and the capacity of commercial lambda-carrageenan type was the same to that of lambda one from C. armatus with determined structure at the concentration of 0.5 mg/mL (Fig. 1). This is in accord with Rocha de Souza et al. [22] who showed that iota-carrageenan was a more effective hydroxyl radical scavenger than lambda- and kappa-carrageenans. The red algal polysaccharide behavior during this method was generally similar with that of nitric oxide scavenging capacity assay. The assay developed by Green et al. [34] was performed for estimating the nitric oxide scavenging capacity. At the concentration of 0.5 mg/mL, as for hydroxyl radicals, commercial iota-carrageenan was the most effective sample, and capacities of lambda-, iks-, and kappa/iota-carrageenans were inferior to ascorbic acid. Since kappa/beta-carrageenan, the least sulphated polysaccharide, was not a high active sample, and agarose, unsulphated polysaccharide, had not any scavenging action to nitric oxide, sulphated groups are leader constituents of these polysaccharides to scavenge nitric oxide (Fig. 2). But iks-carrageenan with three sulphated groups (Table 1) was more active at the lowest concentration and had the same action at the rest concentration values as lambda one with four groups, which could be explained with its fine structural characteristics. Iks-carrageenan type had also the highest scavenging action on superoxide anion independently on concentration. The assay

developed by Nishimiki et al. [35] was applied for investigating the superoxide scavenging capacity. The investigated samples being sulfated polysaccharides, which are able to affect proteins, the assay of superoxide anion scavenging capacity without applying enzymes was preferred. As electron transfer ability of the samples was low, the reducing of NBT by them was unlikely to intensive. As is shown from diagram (Fig. 3), all the investigated samples possessed superoxide anion scavenging capacity being compared with positive standard or more effective ones. Dependence on concentration was expressed weak and after the concentration of 1 mg/mL their action did not change. This was in agreement with Costa et al. [10] who found that there are no correlation between polysaccharide sulphate content and intensity of sample superoxide anion scavenging capacity. The low effectiveness of ascorbic acid can be explained with the fact that ascorbic acid was able to neutralize superoxide anion to hydrogen peroxide or to reduce NBT so that the results under comparison with reference obtained gave only rough data. Earlier, Rocha de Souza et al. [22] reported that sulfated polysaccharides from red and brown algae possessed scavenging action against superoxide anion, and fucoidan, and lambda-carrageenan were more effective than commercial kappa- and iota-carrageenans. In our case, the scavenging capacity of carrageenans was increase in the following row: lambda < kappa/iota < kappa/beta < kappa < iks, and such a parameter as sulphation degree appears not to be the only one under expressing this property. Superoxide anion scavenging capacity of the investigated carrageenans was substantially determined with fine structure characteristics of the carrageenan especially of hydrophobic 3,6-anhydrogalatose presence and high sulphate group number. Hence, carrageenans possess antioxidant capacity in vitro. HAT and ET based antioxidant actions of carrageenans depends on polysaccharide concentration, medium pH value and the polysaccharide fine structural characteristics. Antioxidant capacity against oxygen/nitrogen species (ROS/NOS) is defined with a combination of such fine structural peculiarities as presence of hydrophobic 3,6-anhydrogalactose unit, amount and position of sulphate and an oxidant agent, on which sample antioxidant action is directed, which is in accord to Ajisaka et al. [6]. The ROS/NOS antioxidant action of the investigated samples is of especially interest as polysaccharides were more effective during those assays, which testify about absent direct correlation between reducing capacity of the red algal polysaccharides and scavenging capacities against specific ROS/NOS radicals, which was especially noticeable for commercial polysaccharides. Although several studies have reported that in vitro antioxidant capacity was concomitant with a reducing capacity [10,46,47], the present study, unfortunately, did not elicit that evidence. The polysaccharide scavenging effect on hydrogen peroxide and chelating capacity of transition metal ions related with formation of hydroxyl radicals were absent with the exception of iks-carrageenan possessing chelating capacity of iron ions. The two antioxidant capacities are of great importance as they can participate in the formation of the most reactive oxidant species–hydroxyl radicals. But scavenging effect of polysaccharides investigated on superoxide anion radical and nitric oxide is much more impressed. As is well known, superoxide anion radical is capable of inactivating nitric oxide with formation of highly reactive peroxynitrite radical and dismutating to hydrogen peroxide.

Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.

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