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Evaluation of sea mustard (Undaria pinnatifida) sporophylls from South Korea as fucoidan source and its corresponding antioxidant activities

Evaluation of sea mustard (Undaria pinnatifida) sporophylls from South Korea as fucoidan source... Sporophylls from sea mustard, Undaria pinnatifida, which are by-products in seaweed production industries, were taken from Hansan Island, Tongyeong, and Gijang, Busan, and investigated for their fucoidan content and corresponding antioxidant activities. The extracted fucoidan yield from sporophylls of sea mustard samples from Tongyeong (TF) and Gijang (GF) were 12.1% and 13.6%, respectively. The antioxidant activities assessment of TF and GF extracts showed relative significant difference between samples, suggesting effect of location on bioactivities of fucoidan samples. GF was 50–68% more effective than TF against DPPH and superoxide radicals samples which may be attributed to the difference in their degree of sulfation and monosaccharide composition. Meanwhile, TF exhibited greater scavenging activity against hydroxyl radicals compared to GF which could be due to protein impurities. Keywords: Brown seaweed, Sea mustard, Undaria pinnatifida sporophyll, Fucoidan, Antioxidant activity Background brown seaweeds, and locally known as “Miyeok” in South With the advent of health awareness and health-conscious Korea or “Wakame” in Japan, sea mustard is also consid- buyers, consumption of seaweeds and related goods has ered a food commodity in China (Kim and Choi 1994). In progressively increased in the market. Seaweeds are seaweed processing industries, sea mustard by-products, nutrient-rich food source mainly composed of proteins, such as sporophylls, are found to have essentially the same amino acids, non-protein nitrogen, lipids, dietary fiber, vita- components to that of sea mustard, even after fermentation, mins, and minerals, as well as various bioactive compounds and are examined as potential alternative nutrient source which are significant for biomedical, pharmaceutical, and for broilers as animal feedstock (Shi et al. 2019). health products. Bioactive compounds derived from sea- Reactive oxygen species (ROS), which include super- weeds have been studied for anti-inflammatory, antiviral, oxide (O ) and hydroxyl radicals (OH), and hydrogen anti-thrombic, and anticoagulant activities, anticancer and peroxides (H O ) are considered double-edged swords. 2 2 antitumor properties, anti-obesity, anti-diabetic, anti- While playing a crucial role in several biological func- hypertensive, and anti-hyperlipidemic capabilities (Bedoux tions such as intracellular messaging, cellular differenti- et al. 2014;Smit 2004). However, the high demand in sea- ation, growth arrestment, apoptosis, immunity, and weed production has resulted to a substantial amount of defense against microorganisms, generated ROS may by-products, which usually end up being dumped back to also exceed their antioxidant capacity, disrupt the redox the ocean, during and after processing (Shi et al. 2019). balance, and, therefore, cause oxidative stress which, in One example of the commercially cultivated species is turn, leads to cellular dysfunction. Oxidative stress takes Undaria pinnatifida or sea mustards. From the family of a major part in the progression of some ailments like atherosclerosis, hypertension, aging, Alzheimer’s disease, kidney malfunction, and even cancer (Roberts and * Correspondence: bdchoi@gnu.ac.kr Department of Seafood Science and Technology/The Institute of Marine Sindhu 2009), and accumulated evidence also supported Industry, Gyeongsang National University, Tongyeong 53064, South Korea the relationship of increased oxidative stress with Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 2 of 7 diabetes and its complications (Henriksen et al. 2011). solution was filtered and the filtrate was neutralized to Antioxidant supplements have been reported to signifi- pH 7.0 before adding anhydrous ethanol at a final con- cantly reduce ROS generation and reinforce immune centration of 20% (v/v) ethanol-filtrate mixture to separ- system defense, consequently preventing and delaying ate alginic acid. The mixture was then centrifuged at cell damage. Compared to synthetic counterparts, the 5376×g for 30 min. The collected precipitate was dis- public prefer natural antioxidants with minimum side ef- solved in distilled water, dialyzed in deionized water for fects due to growing health safety concerns (Nishibori 48 h (MWCO 3600 Da), and lyophilized. The freeze- et al. 2012). As a result, exploration of prospective anti- dried crude fucoidan was further purified by fraction- oxidants from plant and marine sources has become a ation via anion-exchange chromatography method de- trend, in the hopes of treating degenerative diseases. scribed by Mak et al. (2013) with minor modifications. Over the decades, fucoidan, a bioactive polysaccharide Five grams of crude fucoidan was dissolved in 20 mL known for its antioxidant and other anti-proliferative ac- Tris-HCl buffer (0.05 M, pH 7.4) and passed through a tivities, has been isolated from various brown seaweed DEAE-Sephadex A-25 column (2.6 × 15 cm), which is species Ecklonia cava, Ascophylum nodusum, Laminaria equilibrated with Tris-HCl buffer and connected to a japonica, and U. pinnatifida (Mak et al. 2013; Wang fraction collector (Bio-Rad 2110, CA, USA). The first et al. 2009; Hu et al. 2010; Wijesinghe and Jeon 2012). fraction was eluted with Tris-HCl buffer, followed by a In this study, we investigated fucoidan content from spo- step-wise elution with 0–2.0 M NaCl solution at a flow- rophylls of sea mustard from two different locations, rate of 2 ml/min while eluates, were automatically col- Tongyeong and Gijang, in South Korea in order to pro- lected at 4 ml per tube. An aliquot from each eluted mote utilization of by-products from Miyeok cultivation sample was checked for presence of sugars using and processing, as potential bioactive compounds phenol-sulfuric acid method by Dubois et al. (1956)to source, to develop health-related natural products. The determine the changes in each elution. Elution was done isolated crude fucoidan extracts were tested for presence until no more sugars were present. Carbohydrate- of fucose, protein residue, amino sugars, sulfate (sulfated positive fractions were then pooled together, dialyzed in glycans), and uronic acid and evaluated for antioxidant deionized water for 48 h (MWCO 10.000 kDa), freeze- activities. dried, and stored in air-tight containers for further use in succeeding antioxidant activity assays. The crude Materials and methods fucoidan extracts were evaluated for total sugar content, Cultured sea mustards (U. pinnatifida)were collected protein residue, fucose, sulfate, amino sugars, and uronic from the coastal areas of Hansan Island in Tongyeong acids using the following methods, respectively: phenol- (TF) and Gijang in Busan (GF), respectively. Sea mustard sulfuric assay with D-galactose as standard (Dubois et al. sporophylls were washed, drained of excess water, and air- 1956); Lowry method with BSA as standard; fucose de- dried, protected from direct sunlight. The dried sporo- termination with cysteine-sulfuric acid reaction; meta- phylls were then pulverized using a grinder and kept in chromasia assay with DMMB with chondroitin-6-sulfate zip-locked bags at − 20 °C until further analysis. as standard (Barbosa et al. 2003); MBTH assay with N- Chemical and radical reagents including 1,1-diphenyl-2- acetyl-D-glucosamine as standard (Tsuji et al. 1969); and picrylhydrazyl (DPPH), nicotinamide adenine dinucleotide carbazole assay with D-glucuronic acid as standard (NADH), nitro blue tetrazolium (NBT), phenazine metho- (Cesaretti et al. 2003). sulfate (PMS), ethylenediaminetetraacetic acid (EDTA), 1, 9-dimethylmethylene blue (DMMB), 3-methyl-2-ben- DPPH radical scavenging activity zothiazolinone hydrazine hydrochloride (MBTH), and DPPH radical scavenging activity (RSA) of purified hydrogen peroxide (H O ) as well as fucoidan from Fucus fucoidan extracts were analyzed according to slightly 2 2 vesiculosus, D-galactose, bovine serum albumin (BSA), L- modified method by Shimada et al. (1992). Different fucose, chondroitin-6-sulfate, N-acetyl-D-glucosamine, concentrations (0.01–1 mg/mL) of sample and standard and D-glucuronic acid standards were all purchased from solutions were prepared in 40% methanol, and 4 mL ali- Sigma-Aldrich (St. Louis, MO, USA). Ascorbic acid and quots were mixed with 1 ml of 0.1 mM methanolic other reagents used were HPLC or analytical grade. DPPH. The mixtures were shaken vigorously and allowed to stand in the dark at room temperature for 35 Extraction, purification, and chemical composition of min. Absorbance was measured at 517 nm. Ascorbic acid fucoidan served as standard while 40% methanol was used as Crude fucoidan was obtained using hot water extraction blank. Fucoidan standard (Sigma-Aldrich, St. Louis, MO, method by dispersing the defatted sea mustard sporo- USA) was used as control for comparison. All determi- phyll powder in distilled water (1:30 w/w ratio) with nations were performed in triplicate and DPPH RSA was constant stirring for 3 h in a water bath at 80 °C. The computed using the following equation: DPPH radical Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 3 of 7 scavenging activity (%) = 1 − (A/A ) × 100, where A and Results and discussion A were the relative absorbances of DPPH solution with Sea mustard sporophyll’s fucoidan content and without standard or sample added, respectively. Brown seaweeds are rich in sulfated polysaccharides which Lower absorbance of reaction mixture indicated higher are potential functional or nutraceutical ingredients. radical scavenging activity. Seaweed-derived polysaccharides, including fucoidan, have been used in food, pharmaceutical, cosmetics, and other Superoxide radical scavenging activity industries (Wijesinghe and Jeon 2012). The extracted Superoxide RSA assay was performed based on method de- fucoidan from sporophylls of sea mustard, U. pinnatifida, scribed by Nishikimi et al. (1972)withsomeadjustments. from Tongyeong (TF) and Gijang (GF) were 12.1% and Superoxide radicals were generated in the PMS-NADH sys- 13.6%, respectively. These results were slightly different tem of 3 mL Tris-HCl buffer (16 mM, pH 8.0) containing from the study by Jeon et al. (2012), wherein they assessed 338 μMNADH, 72 μMNBT,30 μMPMS,and varyingcon- 29 alga species and reported yields of 6.2%, 9.2%, 8.8%, centrations (0.01–1 mg/mL) of sample or standard solutions. and 14.2% crude polysaccharide ethanolic extracts, for The mixture was incubated at room temperature for 5 min sporophyll of U. pinnatifida, U. pinnatifida, Sargassum and absorbance was read at 560 nm against Tris-HCl buffer fulvellum,and Sargassum thunbergii,accordingly, with as blank using microplate reader (Spectramax M2 Molecular higher yields (34.8%, 30.8%, and 30.6%, respectively) for Devices, Sunnyvale, CA, USA). Analysis was done in tripli- Dictyota dichotoma, Capsosiphon fulvescens,and Entero- cates, and ascorbic acid was used as standard and fucoidan morpha compressa. (Sigma-Aldrich, St. Louis, MO, USA) was used as control for Fucoidan content from seaweeds could vary from spe- comparison. Superoxide RSA was calculated using the fol- cies to species, extraction method, geographical location, lowing formula: Superoxide radical scavenging activity (%) = and seasonal variation (Rani et al. 2017). Aside from spe- 1 − (A/A ) × 100, where A and A were the relative absor- cies variety, the extraction method used by Jeon et al. 0 0 bances of radical solution with and without standard or sam- (2012) was not specific for fucoidan which could result ple added, respectively. Reaction mixture with lesser to other compounds, such as alginic acid, to be included absorbance exhibited greater superoxide anion scavenging and affect the yield. In another study, Lee YK, Lim, Lee activity. YH, and Park (2006) investigated three cultivars of U. pinnatifida in Korea with a lower yield (1.0–3.8%) of Hydroxyl radical scavenging activity fucoidan extracts while higher but comparable data Hydroxyl RSA of extracted fucoidan from sea mustard (13.71–26.34%) was observed for the same species in was measured using a modified method by Smironoff New Zealand by Mak et al. (2013). Although similar spe- and Cumbes (1989). An aliquot (1 mL) of sample and cies was used in these studies, the different fucoidan ex- standard solutions with different concentrations (0.01–1 traction yields may have resulted from different mg/mL) was mixed with 1 mL of sodium phosphate buf- extraction methods utilized and geographical location of fer (0.15 M, pH 7.4) containing 10 mM FeSO ,10mM the samples. Lee et al. (2006) used dilute acid extraction EDTA, and 2 mM sodium salicylate and 1 ml of 3% method which could have affected the extraction rate H O . The mixtures were incubated at 37 °C for 30 min, due to fucoidan’s reduced solubility at lower pH values 2 2 and absorbances were measured at 510 nm. Analysis was while Mak et al. (2013) employed calcium chloride done in triplicates and ascorbic acid was used as stand- extraction. ard, and fucoidan (Sigma-Aldrich, St. Louis, MO, USA) Bioactive sulfated polysaccharides from brown sea- was used as control for comparison. A sample blank was weeds increasingly gained attention in scientific fields, prepared by substituting sample solution with distilled particularly in pharmacology and biochemistry. Func- water and replacing H O with sodium phosphate buffer tional polysaccharides like fucans and alginic acid deriva- 2 2 (0.15 M, pH 7.4). Hydroxyl RSA was estimated using the tives from brown seaweeds have demonstrated a variety equation: Hydroxyl radical scavenging activity (%) = 1 of biological properties such as anticoagulant, anti- − (A/A ) × 100, where A and A were the relative absor- inflammatory, antiviral, antitumor, and antioxidant activ- 0 0 bances of sample and sample blank reaction mixtures, ities (Wijesinghe and Jeon 2012). Additionally, anti- respectively. apoptotic, anti-rheumatic, and anti-mutagenic properties of seaweeds were revealed to be mediated by their anti- Statistical analysis oxidant activities (Choi et al. 2006). The crude fucoidan All data were analyzed with analysis of variance using extracts were evaluated for total sugar, fucose, protein SAS software for Windows (SAS ver. 9.2, SAS Institute, residue, amino sugars, sulfated glycans, and uronic acid Cary, NC, USA). Duncan’s multiple-range test was used content, as shown in Table 1. to detect significant differences (p < 0.05) between means Total sugar, fucose, protein residue, amino sugars, sul- (n = 3). fate, and uronic acid content of fucoidan extract from Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 4 of 7 Table 1 Chemical composition of crude fucoidan from sea comparatively higher than TF at all concentrations. mustard sporophylls At 0.01 mg/ml, DPPH RSA of both TF (4.64%) and TF GF GF (9.20%) were higher than the control fucoidan (2.78%). However, as concentration of samples in- Total sugars (%) 13.27 ± 0.36 13.39 ± 0.36 creased to 1 mg/ml, DPPH RSA of control fucoidan Protein residue (%) 1.15 ± 0.04 1.20 ± 0.06 (47.15%) was higher than the crude fucoidan extracts Fucose (μg/mg) 285.57 ± 1.73 305.33 ± 1.72 (12.84% and 21.47%, respectively for TF and GF). Sulfated glycans (μg/mg) 708.16 ± 1.75 839.83 ± 1.71 Wang et al. (2009) observed similar results (5–25% Amino sugars (μg/mg) 253.94 ± 1.06 315.95 ± 1.17 DPPH RSA at 0.5–4.0 mg/ml) for synthesized fucoi- Uronic acid (μg/mg) 402.13 ± 1.53 470.41 ± 1.27 dan derivatives (oversulfated, acetylated, and benzoy- Values are presented as mean ± SD (n =3) lated) from L. japonica from Qingdao, China. In addition, relatively comparable DPPH scavenging ac- Tongyeong was composed of 13.27% total sugar, 1.15% tivities (4–35% RSA at 0.2–1.4 mg/ml) of desulfatedfu- protein, 285.57 μg/mg fucose, 708.16 μg/mg sulfated gly- coidan fractions from U. pinnatifida,alsofrom cans, 253.94 μg/mg amino sugar (hexosamines), and Qingdao, China, were reported by Hu et al. (2010). 402.13 μg/mg uronic acids. Meanwhile, components of Meanwhile, fucoidan fractions from U. pinnatifida GF were slightly higher (13.39% total sugar, 1.20% from New Zealand (Mak et al. 2013)exhibited higher, protein, 305.33 μg/mg fucose, 839.83 μg/mg sulfated gly- almost double, DPPH RSA than the observations in cans, 315.95 μg/mg amino sugar (hexosamines), and this study. This may be due to the variation in mo- 470.41 μg/mg uronic acids) than TF (13.27% total sugar, lecular weight, degree of sulfation, and monosacchar- 1.15% protein, 285.57 μg/mg fucose, 708.16 μg/mg sul- ide composition influencing fucoidan’s biological fated glycans, 253.94 μg/mg amino sugar (hexosamines), activities (Skriptsova et al. 2012). The fucoidan ex- and 402.13 μg/mg uronic acids). These data showed the tracts from this study have molecular weights roughly heterogeneous nature of the composition of crude fucoi- 10.000 kDa, which is lower compared to the fucoidan dan extracted from both locations which were relatively fractions from U. pinnatifida from New Zealand (mo- comparable to the data in brown seaweed species re- lecular weights ranging from 22 to 171 kDa), which ported by Mak et al. (2013), for U. pinnatifida, and Liu may explain the higher DPPH RSA compared to TF et al. (2016), for Sargassum pallidum. Additionally, the and GF samples. EC , an indication of the effective results confirmed presence of sulfated glycans with fu- concentration of the antioxidant to scavenge 50% of cose residue in the crude extracts. Structural characteris- the radicals, of the crude fucoidan extracts were also tics of fucoidan could be dependent on extraction computed. TF (5.60 mg/ml) was found to have higher technique, seaweed species, harvest season, geographical EC than GF (3.82 mg/ml), which implied that GF location, and algal maturity (Mak et al. 2013), which was more effective than TF against DPPH radicals. could explain the difference in data obtained from TF and GF samples. Presence of protein denoted the inher- Superoxide radical scavenging activity of sea mustard ent nature of fucoidan to retain salts and protein which fucoidan could be a part of the fucoidan structure or just a mere Superoxide anions are considered predecessors of singlet contaminant. Hayakawa and Nagamine (2009) suggested oxygen, hydrogen peroxide, and hydroxyl radicals, thus, that pure fucoidan should have 0.1% or less of protein indirectly triggering lipid peroxidation, as well as magni- content; therefore, the fucoidan extracts in this study fying oxidative cellular damage in lipids, proteins, and were in their crude forms. Nonetheless, these compo- DNAs and further intensifying ailments such as arthritis nents influence the bioactivity of the fucoidan extracts and Alzheimer’s disease (Wang et al. 2009). The scaven- from this study (Skriptsova et al. 2012). ging ability of sea mustard fucoidan samples on super- oxide radicals were examined in a concentration DPPH radical scavenging activity of sea mustard fucoidan dependent manner (Fig. 2), and the same trend was ob- DPPH is a stable free radical, typically used to evalu- served as with DPPH RSA; superoxide RSA of TF and ate antioxidant capacity of bioactive compounds in GF was significantly higher as concentration increased. different samples. Antioxidants with hydrogen- GF exhibited a notable increase in superoxide RSA from donating capability reduce DPPH into a stable dia- 0.1 mg/ml (29.3%) to 1 mg/ml (39.2%) with EC value of magnetic molecule, as indicated by the decrease in 1.63 mg/ml. Meanwhile, TF demonstrated superoxide absorbance at 517 nm (Oktay et al. 2003). As shown RSA of 22.6% at 0.1 mg/ml and 26.8% at 1 mg/ml with in Fig. 1, the scavenging activity of sea mustard fucoi- EC of 3.22 mg/ml. These results are slightly lower but dan extracts against DPPH radicals increase with in- almost similar to the data reported by Hu et al. (2010), creasing concentrations. DPPH RSA of GF was which range from 10 to 45% RSA at 0.6–1.6 mg/ml of U. Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 5 of 7 Fig. 1 DPPH radical scavenging activity of sea mustard-derived fucoidan. Data presented as means ± SD (n = 3). Bars with different superscripts significantly vary from each other (p < 0.05) pinnatifida fucoidan extract from China. TF and GF Hydroxyl radical scavenging activity of sea mustard were detected to have greater inhibitory effect on super- fucoidan oxide than DPPH radicals, as displayed by their EC Hydroxyl radicals, OH, are the most active and potent values. This could be due to superoxide anions being among reactive oxygen species which cause severe damage relatively weaker oxidants compared to DPPH and other to neighboring biomolecules like carbohydrates, proteins, radicals (Wang et al. 2009). Based on the resulting EC , lipids, and nucleic acids and initiate aging, cancer, and GF exhibited greater scavenging ability against super- other degenerative diseases (Chung et al. 1997). Hence, oxide radicals than TF, which may be credited to the inhibiting or scavenging hydroxyl radicals is vital for anti- higher sulfated glycan contents in GF (839.83 μg/mg) oxidant defense. TF and GF hydroxyl radical scavenging compared to TF (708.16 μg/mg) as electron-donating activity were also evaluated in a concentration-dependent constituents (like sulfate groups) increase RSA (Wang manner (Fig. 3). As the concentration increased, TF and et al. 2009). GF samples showed greater hydroxyl RSA (TF: 8.4%, Fig. 2 Superoxide radical scavenging activity of sea mustard-derived fucoidan. Data presented as means ± SD (n = 3). Bars with different letters significantly vary from each other (p < 0.05) Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 6 of 7 Fig. 3 Hydroxyl radical scavenging activity of sea mustard-derived fucoidan. Data presented as means ± SD (n = 3). Bars with different letters significantly vary from each other (p < 0.05) 13.4%, and 25.6%, and GF: 10.4%, 14.9%, and 16.6%, re- displayed higher RSA than GF against hydroxyl radical spectively, at 0.01 mg/ml, 0.1 mg/ml, and 1 mg/ml). These only which could be caused by greater interfering pro- results were more or less the same to the hydroxyl RSA of tein impurities in GF. Further characterization (structure crude fucoidan extracted from L. japonica (5–20% RSA at elucidation) and derivatization (acetylation, sulfation, 0.5–2.10 mg/ml) from Qingdao, China (Wang et al. 2009), etc.) is recommended to identify the mechanism of anti- and moderately lower than that of U. pinnatifida (12–35% oxidant action in each sample which may improve corre- RSA at 0.6–1.2 mg/ml), also from China (Hu et al. 2010). sponding bioactivities of TF and GF. Although the crude Previous studies reported two types of antioxidant mech- fucoidan extracts in this study, TF and GF, exhibited anism: inhibiting hydroxyl radical generation by formation lower antioxidant activities compared to ascorbic acid of metals complexes and scavenging the hydroxyl radicals and pure fucoidan, the results showed a prospect of util- generated which is related to iron chelating ability (Shon izing/exploiting sea mustard sporophylls, which are by- et al. 2003). The observed hydroxyl radical RSA of TF and products in seaweed processing, GF were lower than the control fucoidan (13.7%, 24.8%, Abbreviations and 40.2%, accordingly, at 0.01 mg/ml, 0.1 mg/ml, and 1 BSA: Bovine serum albumin; DEAE: Diethylaminoethyl; DMMB: 1,9- mg/ml). Moreover, TF (EC : 2.53 mg/ml) was found to 50 dimethylmethylene blue; DPPH: 1,1-diphenyl-2-picrylhydrazyl; EDTA: Ethylenediaminetetraacetic acid; GF: Gijang fucoidan; MBTH: 3-methyl- be more effective against hydroxyl radicals than GF (EC : 2-benzothiazolinone hydrazine hydrochloride; MWCO: Molecular weight 8.40 mg/ml) which could be due to the protein impurities cutoff; NADH: Nicotinamide adenine dinucleotide; NBT: Nitro blue in the extracts. GF (1.20%) was found to have slightly tetrazolium; PMS: Phenazine methosulfate; ROS: Reactive oxygen species; RSA: Radical scavenging activity; TF: Tongyeong fucoidan higher protein residue than TF (1.15%) which could have interfered with their hydroxyl RSA. Acknowledgements Not applicable. Conclusions Authors’ contributions Fucoidan extracted from sea mustard sporophylls from All authors helped in the conceptualization of the design and implementation Tongyeong (TF) and Gijang City (GF) both demon- of this study and they have read and approved the final manuscript. strated lower scavenging activity on DPPH, superoxide, and hydroxyl radicals when compared with ascorbic acid Funding Not applicable. as positive control and pure fucoidan standard as sample control. This could be caused by the impurities present Availability of data and materials in the crude fucoidan extracted in this study which was Please contact author for data requests. supported by the presence of protein residue greater Ethics approval and consent to participate than 0.1%. GF had greater total sugar, fucose, amino Not applicable. sugar, sulfated glycans, and uronic acids content than TF which resulted to GF exhibiting higher RSA against Consent for publication DPPH and superoxide radical. 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Carbohydr Polym. 2013;95(1):606–14. https://doi.org/10.1016/j.carbpol.2013. 02.047. Nishibori N, Sagara T, Hiroi T, Sawaguchi M, Itoh M, Her S, Morita K. Protective effect of Undaria pinnatifida sporophyll extract on iron induced cytotoxicity and oxidative stress in PC12 neuronal cells. Phytopharmacology. 2012;2(2): 271–84 Retrieved from http://inforesights.com/phytopharmacology/files/pp2 v2i13.pdf. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Fisheries and Aquatic Sciences Springer Journals

Evaluation of sea mustard (Undaria pinnatifida) sporophylls from South Korea as fucoidan source and its corresponding antioxidant activities

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Copyright © 2019 by The Author(s)
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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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10.1186/s41240-019-0141-4
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Abstract

Sporophylls from sea mustard, Undaria pinnatifida, which are by-products in seaweed production industries, were taken from Hansan Island, Tongyeong, and Gijang, Busan, and investigated for their fucoidan content and corresponding antioxidant activities. The extracted fucoidan yield from sporophylls of sea mustard samples from Tongyeong (TF) and Gijang (GF) were 12.1% and 13.6%, respectively. The antioxidant activities assessment of TF and GF extracts showed relative significant difference between samples, suggesting effect of location on bioactivities of fucoidan samples. GF was 50–68% more effective than TF against DPPH and superoxide radicals samples which may be attributed to the difference in their degree of sulfation and monosaccharide composition. Meanwhile, TF exhibited greater scavenging activity against hydroxyl radicals compared to GF which could be due to protein impurities. Keywords: Brown seaweed, Sea mustard, Undaria pinnatifida sporophyll, Fucoidan, Antioxidant activity Background brown seaweeds, and locally known as “Miyeok” in South With the advent of health awareness and health-conscious Korea or “Wakame” in Japan, sea mustard is also consid- buyers, consumption of seaweeds and related goods has ered a food commodity in China (Kim and Choi 1994). In progressively increased in the market. Seaweeds are seaweed processing industries, sea mustard by-products, nutrient-rich food source mainly composed of proteins, such as sporophylls, are found to have essentially the same amino acids, non-protein nitrogen, lipids, dietary fiber, vita- components to that of sea mustard, even after fermentation, mins, and minerals, as well as various bioactive compounds and are examined as potential alternative nutrient source which are significant for biomedical, pharmaceutical, and for broilers as animal feedstock (Shi et al. 2019). health products. Bioactive compounds derived from sea- Reactive oxygen species (ROS), which include super- weeds have been studied for anti-inflammatory, antiviral, oxide (O ) and hydroxyl radicals (OH), and hydrogen anti-thrombic, and anticoagulant activities, anticancer and peroxides (H O ) are considered double-edged swords. 2 2 antitumor properties, anti-obesity, anti-diabetic, anti- While playing a crucial role in several biological func- hypertensive, and anti-hyperlipidemic capabilities (Bedoux tions such as intracellular messaging, cellular differenti- et al. 2014;Smit 2004). However, the high demand in sea- ation, growth arrestment, apoptosis, immunity, and weed production has resulted to a substantial amount of defense against microorganisms, generated ROS may by-products, which usually end up being dumped back to also exceed their antioxidant capacity, disrupt the redox the ocean, during and after processing (Shi et al. 2019). balance, and, therefore, cause oxidative stress which, in One example of the commercially cultivated species is turn, leads to cellular dysfunction. Oxidative stress takes Undaria pinnatifida or sea mustards. From the family of a major part in the progression of some ailments like atherosclerosis, hypertension, aging, Alzheimer’s disease, kidney malfunction, and even cancer (Roberts and * Correspondence: bdchoi@gnu.ac.kr Department of Seafood Science and Technology/The Institute of Marine Sindhu 2009), and accumulated evidence also supported Industry, Gyeongsang National University, Tongyeong 53064, South Korea the relationship of increased oxidative stress with Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 2 of 7 diabetes and its complications (Henriksen et al. 2011). solution was filtered and the filtrate was neutralized to Antioxidant supplements have been reported to signifi- pH 7.0 before adding anhydrous ethanol at a final con- cantly reduce ROS generation and reinforce immune centration of 20% (v/v) ethanol-filtrate mixture to separ- system defense, consequently preventing and delaying ate alginic acid. The mixture was then centrifuged at cell damage. Compared to synthetic counterparts, the 5376×g for 30 min. The collected precipitate was dis- public prefer natural antioxidants with minimum side ef- solved in distilled water, dialyzed in deionized water for fects due to growing health safety concerns (Nishibori 48 h (MWCO 3600 Da), and lyophilized. The freeze- et al. 2012). As a result, exploration of prospective anti- dried crude fucoidan was further purified by fraction- oxidants from plant and marine sources has become a ation via anion-exchange chromatography method de- trend, in the hopes of treating degenerative diseases. scribed by Mak et al. (2013) with minor modifications. Over the decades, fucoidan, a bioactive polysaccharide Five grams of crude fucoidan was dissolved in 20 mL known for its antioxidant and other anti-proliferative ac- Tris-HCl buffer (0.05 M, pH 7.4) and passed through a tivities, has been isolated from various brown seaweed DEAE-Sephadex A-25 column (2.6 × 15 cm), which is species Ecklonia cava, Ascophylum nodusum, Laminaria equilibrated with Tris-HCl buffer and connected to a japonica, and U. pinnatifida (Mak et al. 2013; Wang fraction collector (Bio-Rad 2110, CA, USA). The first et al. 2009; Hu et al. 2010; Wijesinghe and Jeon 2012). fraction was eluted with Tris-HCl buffer, followed by a In this study, we investigated fucoidan content from spo- step-wise elution with 0–2.0 M NaCl solution at a flow- rophylls of sea mustard from two different locations, rate of 2 ml/min while eluates, were automatically col- Tongyeong and Gijang, in South Korea in order to pro- lected at 4 ml per tube. An aliquot from each eluted mote utilization of by-products from Miyeok cultivation sample was checked for presence of sugars using and processing, as potential bioactive compounds phenol-sulfuric acid method by Dubois et al. (1956)to source, to develop health-related natural products. The determine the changes in each elution. Elution was done isolated crude fucoidan extracts were tested for presence until no more sugars were present. Carbohydrate- of fucose, protein residue, amino sugars, sulfate (sulfated positive fractions were then pooled together, dialyzed in glycans), and uronic acid and evaluated for antioxidant deionized water for 48 h (MWCO 10.000 kDa), freeze- activities. dried, and stored in air-tight containers for further use in succeeding antioxidant activity assays. The crude Materials and methods fucoidan extracts were evaluated for total sugar content, Cultured sea mustards (U. pinnatifida)were collected protein residue, fucose, sulfate, amino sugars, and uronic from the coastal areas of Hansan Island in Tongyeong acids using the following methods, respectively: phenol- (TF) and Gijang in Busan (GF), respectively. Sea mustard sulfuric assay with D-galactose as standard (Dubois et al. sporophylls were washed, drained of excess water, and air- 1956); Lowry method with BSA as standard; fucose de- dried, protected from direct sunlight. The dried sporo- termination with cysteine-sulfuric acid reaction; meta- phylls were then pulverized using a grinder and kept in chromasia assay with DMMB with chondroitin-6-sulfate zip-locked bags at − 20 °C until further analysis. as standard (Barbosa et al. 2003); MBTH assay with N- Chemical and radical reagents including 1,1-diphenyl-2- acetyl-D-glucosamine as standard (Tsuji et al. 1969); and picrylhydrazyl (DPPH), nicotinamide adenine dinucleotide carbazole assay with D-glucuronic acid as standard (NADH), nitro blue tetrazolium (NBT), phenazine metho- (Cesaretti et al. 2003). sulfate (PMS), ethylenediaminetetraacetic acid (EDTA), 1, 9-dimethylmethylene blue (DMMB), 3-methyl-2-ben- DPPH radical scavenging activity zothiazolinone hydrazine hydrochloride (MBTH), and DPPH radical scavenging activity (RSA) of purified hydrogen peroxide (H O ) as well as fucoidan from Fucus fucoidan extracts were analyzed according to slightly 2 2 vesiculosus, D-galactose, bovine serum albumin (BSA), L- modified method by Shimada et al. (1992). Different fucose, chondroitin-6-sulfate, N-acetyl-D-glucosamine, concentrations (0.01–1 mg/mL) of sample and standard and D-glucuronic acid standards were all purchased from solutions were prepared in 40% methanol, and 4 mL ali- Sigma-Aldrich (St. Louis, MO, USA). Ascorbic acid and quots were mixed with 1 ml of 0.1 mM methanolic other reagents used were HPLC or analytical grade. DPPH. The mixtures were shaken vigorously and allowed to stand in the dark at room temperature for 35 Extraction, purification, and chemical composition of min. Absorbance was measured at 517 nm. Ascorbic acid fucoidan served as standard while 40% methanol was used as Crude fucoidan was obtained using hot water extraction blank. Fucoidan standard (Sigma-Aldrich, St. Louis, MO, method by dispersing the defatted sea mustard sporo- USA) was used as control for comparison. All determi- phyll powder in distilled water (1:30 w/w ratio) with nations were performed in triplicate and DPPH RSA was constant stirring for 3 h in a water bath at 80 °C. The computed using the following equation: DPPH radical Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 3 of 7 scavenging activity (%) = 1 − (A/A ) × 100, where A and Results and discussion A were the relative absorbances of DPPH solution with Sea mustard sporophyll’s fucoidan content and without standard or sample added, respectively. Brown seaweeds are rich in sulfated polysaccharides which Lower absorbance of reaction mixture indicated higher are potential functional or nutraceutical ingredients. radical scavenging activity. Seaweed-derived polysaccharides, including fucoidan, have been used in food, pharmaceutical, cosmetics, and other Superoxide radical scavenging activity industries (Wijesinghe and Jeon 2012). The extracted Superoxide RSA assay was performed based on method de- fucoidan from sporophylls of sea mustard, U. pinnatifida, scribed by Nishikimi et al. (1972)withsomeadjustments. from Tongyeong (TF) and Gijang (GF) were 12.1% and Superoxide radicals were generated in the PMS-NADH sys- 13.6%, respectively. These results were slightly different tem of 3 mL Tris-HCl buffer (16 mM, pH 8.0) containing from the study by Jeon et al. (2012), wherein they assessed 338 μMNADH, 72 μMNBT,30 μMPMS,and varyingcon- 29 alga species and reported yields of 6.2%, 9.2%, 8.8%, centrations (0.01–1 mg/mL) of sample or standard solutions. and 14.2% crude polysaccharide ethanolic extracts, for The mixture was incubated at room temperature for 5 min sporophyll of U. pinnatifida, U. pinnatifida, Sargassum and absorbance was read at 560 nm against Tris-HCl buffer fulvellum,and Sargassum thunbergii,accordingly, with as blank using microplate reader (Spectramax M2 Molecular higher yields (34.8%, 30.8%, and 30.6%, respectively) for Devices, Sunnyvale, CA, USA). Analysis was done in tripli- Dictyota dichotoma, Capsosiphon fulvescens,and Entero- cates, and ascorbic acid was used as standard and fucoidan morpha compressa. (Sigma-Aldrich, St. Louis, MO, USA) was used as control for Fucoidan content from seaweeds could vary from spe- comparison. Superoxide RSA was calculated using the fol- cies to species, extraction method, geographical location, lowing formula: Superoxide radical scavenging activity (%) = and seasonal variation (Rani et al. 2017). Aside from spe- 1 − (A/A ) × 100, where A and A were the relative absor- cies variety, the extraction method used by Jeon et al. 0 0 bances of radical solution with and without standard or sam- (2012) was not specific for fucoidan which could result ple added, respectively. Reaction mixture with lesser to other compounds, such as alginic acid, to be included absorbance exhibited greater superoxide anion scavenging and affect the yield. In another study, Lee YK, Lim, Lee activity. YH, and Park (2006) investigated three cultivars of U. pinnatifida in Korea with a lower yield (1.0–3.8%) of Hydroxyl radical scavenging activity fucoidan extracts while higher but comparable data Hydroxyl RSA of extracted fucoidan from sea mustard (13.71–26.34%) was observed for the same species in was measured using a modified method by Smironoff New Zealand by Mak et al. (2013). Although similar spe- and Cumbes (1989). An aliquot (1 mL) of sample and cies was used in these studies, the different fucoidan ex- standard solutions with different concentrations (0.01–1 traction yields may have resulted from different mg/mL) was mixed with 1 mL of sodium phosphate buf- extraction methods utilized and geographical location of fer (0.15 M, pH 7.4) containing 10 mM FeSO ,10mM the samples. Lee et al. (2006) used dilute acid extraction EDTA, and 2 mM sodium salicylate and 1 ml of 3% method which could have affected the extraction rate H O . The mixtures were incubated at 37 °C for 30 min, due to fucoidan’s reduced solubility at lower pH values 2 2 and absorbances were measured at 510 nm. Analysis was while Mak et al. (2013) employed calcium chloride done in triplicates and ascorbic acid was used as stand- extraction. ard, and fucoidan (Sigma-Aldrich, St. Louis, MO, USA) Bioactive sulfated polysaccharides from brown sea- was used as control for comparison. A sample blank was weeds increasingly gained attention in scientific fields, prepared by substituting sample solution with distilled particularly in pharmacology and biochemistry. Func- water and replacing H O with sodium phosphate buffer tional polysaccharides like fucans and alginic acid deriva- 2 2 (0.15 M, pH 7.4). Hydroxyl RSA was estimated using the tives from brown seaweeds have demonstrated a variety equation: Hydroxyl radical scavenging activity (%) = 1 of biological properties such as anticoagulant, anti- − (A/A ) × 100, where A and A were the relative absor- inflammatory, antiviral, antitumor, and antioxidant activ- 0 0 bances of sample and sample blank reaction mixtures, ities (Wijesinghe and Jeon 2012). Additionally, anti- respectively. apoptotic, anti-rheumatic, and anti-mutagenic properties of seaweeds were revealed to be mediated by their anti- Statistical analysis oxidant activities (Choi et al. 2006). The crude fucoidan All data were analyzed with analysis of variance using extracts were evaluated for total sugar, fucose, protein SAS software for Windows (SAS ver. 9.2, SAS Institute, residue, amino sugars, sulfated glycans, and uronic acid Cary, NC, USA). Duncan’s multiple-range test was used content, as shown in Table 1. to detect significant differences (p < 0.05) between means Total sugar, fucose, protein residue, amino sugars, sul- (n = 3). fate, and uronic acid content of fucoidan extract from Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 4 of 7 Table 1 Chemical composition of crude fucoidan from sea comparatively higher than TF at all concentrations. mustard sporophylls At 0.01 mg/ml, DPPH RSA of both TF (4.64%) and TF GF GF (9.20%) were higher than the control fucoidan (2.78%). However, as concentration of samples in- Total sugars (%) 13.27 ± 0.36 13.39 ± 0.36 creased to 1 mg/ml, DPPH RSA of control fucoidan Protein residue (%) 1.15 ± 0.04 1.20 ± 0.06 (47.15%) was higher than the crude fucoidan extracts Fucose (μg/mg) 285.57 ± 1.73 305.33 ± 1.72 (12.84% and 21.47%, respectively for TF and GF). Sulfated glycans (μg/mg) 708.16 ± 1.75 839.83 ± 1.71 Wang et al. (2009) observed similar results (5–25% Amino sugars (μg/mg) 253.94 ± 1.06 315.95 ± 1.17 DPPH RSA at 0.5–4.0 mg/ml) for synthesized fucoi- Uronic acid (μg/mg) 402.13 ± 1.53 470.41 ± 1.27 dan derivatives (oversulfated, acetylated, and benzoy- Values are presented as mean ± SD (n =3) lated) from L. japonica from Qingdao, China. In addition, relatively comparable DPPH scavenging ac- Tongyeong was composed of 13.27% total sugar, 1.15% tivities (4–35% RSA at 0.2–1.4 mg/ml) of desulfatedfu- protein, 285.57 μg/mg fucose, 708.16 μg/mg sulfated gly- coidan fractions from U. pinnatifida,alsofrom cans, 253.94 μg/mg amino sugar (hexosamines), and Qingdao, China, were reported by Hu et al. (2010). 402.13 μg/mg uronic acids. Meanwhile, components of Meanwhile, fucoidan fractions from U. pinnatifida GF were slightly higher (13.39% total sugar, 1.20% from New Zealand (Mak et al. 2013)exhibited higher, protein, 305.33 μg/mg fucose, 839.83 μg/mg sulfated gly- almost double, DPPH RSA than the observations in cans, 315.95 μg/mg amino sugar (hexosamines), and this study. This may be due to the variation in mo- 470.41 μg/mg uronic acids) than TF (13.27% total sugar, lecular weight, degree of sulfation, and monosacchar- 1.15% protein, 285.57 μg/mg fucose, 708.16 μg/mg sul- ide composition influencing fucoidan’s biological fated glycans, 253.94 μg/mg amino sugar (hexosamines), activities (Skriptsova et al. 2012). The fucoidan ex- and 402.13 μg/mg uronic acids). These data showed the tracts from this study have molecular weights roughly heterogeneous nature of the composition of crude fucoi- 10.000 kDa, which is lower compared to the fucoidan dan extracted from both locations which were relatively fractions from U. pinnatifida from New Zealand (mo- comparable to the data in brown seaweed species re- lecular weights ranging from 22 to 171 kDa), which ported by Mak et al. (2013), for U. pinnatifida, and Liu may explain the higher DPPH RSA compared to TF et al. (2016), for Sargassum pallidum. Additionally, the and GF samples. EC , an indication of the effective results confirmed presence of sulfated glycans with fu- concentration of the antioxidant to scavenge 50% of cose residue in the crude extracts. Structural characteris- the radicals, of the crude fucoidan extracts were also tics of fucoidan could be dependent on extraction computed. TF (5.60 mg/ml) was found to have higher technique, seaweed species, harvest season, geographical EC than GF (3.82 mg/ml), which implied that GF location, and algal maturity (Mak et al. 2013), which was more effective than TF against DPPH radicals. could explain the difference in data obtained from TF and GF samples. Presence of protein denoted the inher- Superoxide radical scavenging activity of sea mustard ent nature of fucoidan to retain salts and protein which fucoidan could be a part of the fucoidan structure or just a mere Superoxide anions are considered predecessors of singlet contaminant. Hayakawa and Nagamine (2009) suggested oxygen, hydrogen peroxide, and hydroxyl radicals, thus, that pure fucoidan should have 0.1% or less of protein indirectly triggering lipid peroxidation, as well as magni- content; therefore, the fucoidan extracts in this study fying oxidative cellular damage in lipids, proteins, and were in their crude forms. Nonetheless, these compo- DNAs and further intensifying ailments such as arthritis nents influence the bioactivity of the fucoidan extracts and Alzheimer’s disease (Wang et al. 2009). The scaven- from this study (Skriptsova et al. 2012). ging ability of sea mustard fucoidan samples on super- oxide radicals were examined in a concentration DPPH radical scavenging activity of sea mustard fucoidan dependent manner (Fig. 2), and the same trend was ob- DPPH is a stable free radical, typically used to evalu- served as with DPPH RSA; superoxide RSA of TF and ate antioxidant capacity of bioactive compounds in GF was significantly higher as concentration increased. different samples. Antioxidants with hydrogen- GF exhibited a notable increase in superoxide RSA from donating capability reduce DPPH into a stable dia- 0.1 mg/ml (29.3%) to 1 mg/ml (39.2%) with EC value of magnetic molecule, as indicated by the decrease in 1.63 mg/ml. Meanwhile, TF demonstrated superoxide absorbance at 517 nm (Oktay et al. 2003). As shown RSA of 22.6% at 0.1 mg/ml and 26.8% at 1 mg/ml with in Fig. 1, the scavenging activity of sea mustard fucoi- EC of 3.22 mg/ml. These results are slightly lower but dan extracts against DPPH radicals increase with in- almost similar to the data reported by Hu et al. (2010), creasing concentrations. DPPH RSA of GF was which range from 10 to 45% RSA at 0.6–1.6 mg/ml of U. Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 5 of 7 Fig. 1 DPPH radical scavenging activity of sea mustard-derived fucoidan. Data presented as means ± SD (n = 3). Bars with different superscripts significantly vary from each other (p < 0.05) pinnatifida fucoidan extract from China. TF and GF Hydroxyl radical scavenging activity of sea mustard were detected to have greater inhibitory effect on super- fucoidan oxide than DPPH radicals, as displayed by their EC Hydroxyl radicals, OH, are the most active and potent values. This could be due to superoxide anions being among reactive oxygen species which cause severe damage relatively weaker oxidants compared to DPPH and other to neighboring biomolecules like carbohydrates, proteins, radicals (Wang et al. 2009). Based on the resulting EC , lipids, and nucleic acids and initiate aging, cancer, and GF exhibited greater scavenging ability against super- other degenerative diseases (Chung et al. 1997). Hence, oxide radicals than TF, which may be credited to the inhibiting or scavenging hydroxyl radicals is vital for anti- higher sulfated glycan contents in GF (839.83 μg/mg) oxidant defense. TF and GF hydroxyl radical scavenging compared to TF (708.16 μg/mg) as electron-donating activity were also evaluated in a concentration-dependent constituents (like sulfate groups) increase RSA (Wang manner (Fig. 3). As the concentration increased, TF and et al. 2009). GF samples showed greater hydroxyl RSA (TF: 8.4%, Fig. 2 Superoxide radical scavenging activity of sea mustard-derived fucoidan. Data presented as means ± SD (n = 3). Bars with different letters significantly vary from each other (p < 0.05) Neri et al. Fisheries and Aquatic Sciences (2019) 22:24 Page 6 of 7 Fig. 3 Hydroxyl radical scavenging activity of sea mustard-derived fucoidan. Data presented as means ± SD (n = 3). Bars with different letters significantly vary from each other (p < 0.05) 13.4%, and 25.6%, and GF: 10.4%, 14.9%, and 16.6%, re- displayed higher RSA than GF against hydroxyl radical spectively, at 0.01 mg/ml, 0.1 mg/ml, and 1 mg/ml). These only which could be caused by greater interfering pro- results were more or less the same to the hydroxyl RSA of tein impurities in GF. Further characterization (structure crude fucoidan extracted from L. japonica (5–20% RSA at elucidation) and derivatization (acetylation, sulfation, 0.5–2.10 mg/ml) from Qingdao, China (Wang et al. 2009), etc.) is recommended to identify the mechanism of anti- and moderately lower than that of U. pinnatifida (12–35% oxidant action in each sample which may improve corre- RSA at 0.6–1.2 mg/ml), also from China (Hu et al. 2010). sponding bioactivities of TF and GF. Although the crude Previous studies reported two types of antioxidant mech- fucoidan extracts in this study, TF and GF, exhibited anism: inhibiting hydroxyl radical generation by formation lower antioxidant activities compared to ascorbic acid of metals complexes and scavenging the hydroxyl radicals and pure fucoidan, the results showed a prospect of util- generated which is related to iron chelating ability (Shon izing/exploiting sea mustard sporophylls, which are by- et al. 2003). The observed hydroxyl radical RSA of TF and products in seaweed processing, GF were lower than the control fucoidan (13.7%, 24.8%, Abbreviations and 40.2%, accordingly, at 0.01 mg/ml, 0.1 mg/ml, and 1 BSA: Bovine serum albumin; DEAE: Diethylaminoethyl; DMMB: 1,9- mg/ml). Moreover, TF (EC : 2.53 mg/ml) was found to 50 dimethylmethylene blue; DPPH: 1,1-diphenyl-2-picrylhydrazyl; EDTA: Ethylenediaminetetraacetic acid; GF: Gijang fucoidan; MBTH: 3-methyl- be more effective against hydroxyl radicals than GF (EC : 2-benzothiazolinone hydrazine hydrochloride; MWCO: Molecular weight 8.40 mg/ml) which could be due to the protein impurities cutoff; NADH: Nicotinamide adenine dinucleotide; NBT: Nitro blue in the extracts. GF (1.20%) was found to have slightly tetrazolium; PMS: Phenazine methosulfate; ROS: Reactive oxygen species; RSA: Radical scavenging activity; TF: Tongyeong fucoidan higher protein residue than TF (1.15%) which could have interfered with their hydroxyl RSA. Acknowledgements Not applicable. Conclusions Authors’ contributions Fucoidan extracted from sea mustard sporophylls from All authors helped in the conceptualization of the design and implementation Tongyeong (TF) and Gijang City (GF) both demon- of this study and they have read and approved the final manuscript. strated lower scavenging activity on DPPH, superoxide, and hydroxyl radicals when compared with ascorbic acid Funding Not applicable. as positive control and pure fucoidan standard as sample control. This could be caused by the impurities present Availability of data and materials in the crude fucoidan extracted in this study which was Please contact author for data requests. supported by the presence of protein residue greater Ethics approval and consent to participate than 0.1%. GF had greater total sugar, fucose, amino Not applicable. sugar, sulfated glycans, and uronic acids content than TF which resulted to GF exhibiting higher RSA against Consent for publication DPPH and superoxide radical. 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Fisheries and Aquatic SciencesSpringer Journals

Published: Nov 19, 2019

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