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International Journal of Biological Macromolecules 62 (2013) 265– 272

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

jo ur nal homep age: www.elsev ier .com/ locate / i jb iomac

ntioxidant activity of glycoprotein purified from Undaria pinnatifidaeasured by an in vitro digestion model

.M. Rafiquzzamana,1, Eun-Young Kima,1, Yu-Ri Kima, Taek-Jeong Namb, In-Soo Konga,∗

Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of KoreaDepartment of Food Science and Nutrition, Pukyong National University, Busan 608-737, Republic of Korea

r t i c l e i n f o

rticle history:eceived 27 June 2013eceived in revised form 24 August 2013ccepted 15 September 2013vailable online 20 September 2013

eywords:lycoproteinndaria pinnatifida

a b s t r a c t

The present study was performed to investigate the chemical composition and antioxidant activity ofglycoprotein purified from Undaria pinnatifida Harvey (UPGP). On SDS-PAGE, UPGP migrated as a singleband with a molecular weight of approximately 10 kDa and confirmed by staining with Schiff’s reagent asglycoprotein. It consists of a carbohydrate component (42.53%) and protein component (57.47%). Aminoacid profile, FT-IR spectrum and enzymatic glycosylation analysis suggested that protein is linked withcarbohydrate by O-glycosylation. UPGP showed dose-dependent antioxidant activities as detected bydifferent assays before and after in vitro digestion. The IC50 values of undigested UPGP were 0.25 ± 0.03,0.08 ± 0.005, 0.69 ± 0.12, and 0.25 ± 0.08 mg/mL for DPPH, ABTS, FRAP, and NO, respectively. Following

hemical compositionntioxidantNA protection

n vitro digestion model

in vitro digestion, the antioxidant activities of UPGP were decreased during the gastric phase compared tothose of undigested UPGP, with an increase occurring during the duodenal phase in all assays. However,the reducing power was unchanged after in vitro digestion. Furthermore, UPGP showed protective activityagainst oxidative DNA damage both undigested, after saliva and duodenal phase of digestion. Theseresults indicate that the antioxidant and DNA protection activities of UPGP may be pH-dependent andassay specific.

. Introduction

Reactive oxygen species (ROS), including hydroxyl radicals,uperoxide radicals, hydrogen peroxide, and nitrite oxide, areroduced as a consequence of the normal metabolism of livingrganisms [1]. ROS decrease the normal defensive systems ofrganisms, leading to various abnormalities, including myocardialschemia, carcinogenesis, inflammatory disease, and Alzheimer’sisease by attacking proteins, lipids (including those in cellularembranes), and DNA [2]. In addition, nitric oxide (NO) formed

y Ca+-independent inducible NOS may contribute to cytotoxic-ty, DNA damage, and carcinogenesis [3]. Consumption of naturalroduct which is rich in antioxidant compounds may help to makealance between ROS production and endogenous protection whenhe body is in under oxidative stress. In particular, the regulatedroduction of ROS is an essential prerequisite to maintain redoxomeostasis in humans [4].

Recent findings have demonstrated health benefits associ-ted with the consumption of seaweed, and that extracts ofifferent seaweeds exhibited different biofunctional activities,

∗ Corresponding author. Tel.: +82 051 629 5865.E-mail address: [email protected] (I.-S. Kong).

1 These authors contributed equally to this paper.

141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2013.09.009

© 2013 Elsevier B.V. All rights reserved.

including hypocholesterolemic, antithrombotic, antioxidant, andantidiabetic effects, reduction of coronary diseases, and estrogenmetabolism [5–7]. There is a long tradition in China, Japan, Korea,and Southeast Asia of consuming seaweeds as part of the regulardiet. The main species of seaweed cultivated in Korea are Porphyra(nori), Undaria (wakame in Japan and miyeok in Korea) and Lami-naria. Undaria pinnatifida is a widely eaten brown seaweed and isalso used as a traditional medicine in Korea. Interest in seaweed as asource of food is increasing, particularly because of the advantagesto the consumer beyond strictly nutritional benefits. Aquaculture ofseaweed is expanding at present as natural production cannot meetthe current demand. According to the Food and Agriculture Organi-zation of the United Nations, about 2.5 million tons of U. pinnatifidaare produced per year, of which 99% is from aquaculture.

It has been reported that U. pinnatifida is a rich source ofeicosapentaenoic acid, an omega-3 fatty acid, and that it con-tains high levels of calcium, iodine, thiamine, and niacin [8]. Inoriental medicine, U. pinnatifida has been used for blood purifi-cation, intestinal strength, and beautification of the skin and hair[9]. A number of studies have indicated that fucoidan from U.pinnatifida has a wide range of medicinal effects, including anti-

inflammatory, antiviral, and anticoagulant activities [10]. Moststudies have focused on evaluating the biological functions ofpolysaccharides and lipids from U. pinnatifida, although plant gly-coproteins are known to have more biological functions [11,12].

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ecent research in our laboratory has aimed to isolate and purifyhe glycoprotein from Saccharina japonica in order to characterizehe different biofunctional activities and reported that it has strongntioxidant, DNA protective, NO scavenging, and xanthine oxidasenhibition activity [12].

Two important aspects of the beneficial effects of any bioactiveompound are bioavailability and metabolic fate. The bioavail-bility of a dietary compound is dependent upon its digestivetability. It is important to determine the fate of bioactive com-ounds during human digestion to better understand the potentialenefits of such active compounds. Structural modifications andlterations in functional properties caused by the variation in pHlong the gastrointestinal tract could affect bioefficacy. Many stud-es have shown the significant influence of simulated digestionn the bioavailability of phytochemicals [13]. However, there areontradictory data regarding the stability of phenolic compoundsubjected to changes in pH, while other antioxidants such as Troloxnd tocopherol become more stable under gut conditions [14,15].n the past decade, in vitro digestion models useful for assessing theioavailability of compounds from foods have been developed. Inhe literature, there are no studies of the antioxidant and other bio-unctional activities of U. pinnatifida glycoprotein and its stabilityhen subjected to in vitro digestion.

In this study, we purified UPGP and evaluated its chemicalomposition, biological activities, including antioxidant and DNA-rotective activities. In addition, we investigated the bioavailabilityf the glycoprotein using an in vitro model that simulated severalhemical (pH, temperature and bile salt) and biological (gastricnd pancreatic enzyme) gastro-intestinal conditions. Moreover,hanges in antioxidant activity during digestion, as well as theigestive stability of the glycoprotein, were investigated.

. Materials and methods

.1. Purification and characterization of UPGP

.1.1. Purification of UPGPU. pinnatifida was obtained from a local market in Busan, Korea,

nd preserved in plastic boxes under dried conditions for exper-mental use. Glycoprotein purification from U. pinnatifida wasarried out as described previously with some modifications [16].riefly, 4 g of U. pinnatifida was mixed with 100 mL of distilledater (DW) and incubated for more than 4 h at room temperature

RT). The DW extract was filtered with gauze, and then precipi-ated by the addition of three volumes of ethanol for more than 6 ht RT. The precipitate was passed through Whatman 3MM paperGE Healthcare, Amersham, UK) to remove debris. The filtered sam-les were then concentrated in a rotatory evaporator followed byreeze-drying. The freeze-dried samples were dissolved in DW andialyzed overnight with the membrane. The dialyzed samples wereltered with Whatman paper and the glycoprotein concentrationas determined by measuring the absorbance at 280 nm.

.1.2. SDS-PAGETo check the purity of UPGP, the samples were subjected to 15%

olyacrylamide mini gel electrophoresis. The electrophoresis wasarried out at 110 V, 30 mA for 2.5 h. The gel was stained with silvertaining and periodic acid-Schiff (PAS), which specifically detectslycoprotein as a result of a redox reaction [17].

.1.3. Measurement of the carbohydrate and protein levels inPGP

The carbohydrate content of UPGP was determined by thehenol-sulfuric acid method using glucose as a reference [18]. Therotein moiety of UPGP was quantified according to the methodf Lowry et al. [19] using bovine serum albumin as a standard.

iological Macromolecules 62 (2013) 265– 272

The carbohydrate and protein levels in UPGP were determined bymeasuring the absorbance at 490 and 280 nm, respectively.

2.1.4. Analysis of chemical composition in UPGP2.1.4.1. Monosaccharides composition. The purified UPGP washydrolyzed using anhydrous methanol containing 2 M HCl at 80 ◦Cfor 20 h. Then the hydrolyzed products were neutralized withmethanol–KOH and dried. GC–MS analysis was performed byShimadzu GC/MS-QP2010. Hydrolyzed sample was injected at aconstant temperature of 260 ◦C in splitless mode and detectedat 300 ◦C. GC separation was performed on a DB-1MS col-umn (30 m × 0.25 mm × 0.25 �m) at a constant flow of helium(1 ml/min). Initial oven temperature was set at 105 ◦C at 4 ◦C/min,followed by 240 ◦C, 20 ◦C/min, 300 ◦C hold for 3.25 min.

2.1.4.2. Amino acid composition. The content of amino acid inpurified UPGP was determined using Amino Acid Analyzer (Mem-brapure Co. Germany). The purified UPGP sample was hydrolyzedwith 6 N HCl in evacuated sealed tubes for 24 h at 110 ◦C. Twentymicrolitres of hydrolysed UPGP was loaded and passed through thenarrow bore column (stainless steel 125 mm, ID 3 mm). Ninhydrinwas added to the HCl as an internal standard. The amount of eachamino acid was expressed in percentage of amino acid per 100 g ofamino acid.

2.1.4.3. FT-IR spectroscopy. The freeze dried UPGP was used forFourier transform infrared (FT-IR) measurement in the frequencyrange of 4000–650 cm−1a using Perkin Elmer (USA), Spectrum X.

2.1.4.4. Glycosylation analysis. Glycosidases are the useful tools forthe structural and functional analysis of oligosaccharides associ-ated with glycoproteins. The purified native 4 �l of UPGP (3 mg/ml)was incubated with 13 �l of reaction buffer (250 mM phosphatebuffer, pH 5.5) at 37 ◦C for 1 h. The sample was then incubatedwith 2 �l of O-glycosidase (2 m unit) purified from Streptococcuspneumoniae (EC. 3.2.1.97, Roche, Germany) for 3 h at 37 ◦C. Afterglycosidase treatment, deglycosylation of glycoprotein was mon-itored by Tricine-SDS-PAGE containing 12% acrylamide gel andvisualized by Coomassie Blue staining.

2.2. In vitro digestion model

The in vitro digestion model was used according a methodreported previously [20,21] with slight modifications. Initially, tosimulate saliva, glycoprotein solution was treated with the artifi-cial saliva solution (6.2 g/L NaCl, 2.2 g/L KCl, 0.22 g/L CaCl2, 1.2 g/LNaHCO3). The glycoprotein samples were then transferred to cleanamber bottles and mixed with saline. The samples were acidifiedto pH 2 with 1 mL of porcine pepsin preparation (0.04 g pepsinin 0.1 mol/L HCl) and incubated at 37 ◦C in a shaking water bathat 95 rpm for 1 h. After gastric digestion, the pH was increasedto 5.3 with 0.9 mol/L sodium bicarbonate, and then 200 �L of thebile salts glycodeoxycholate (0.04 g in 1 mL of saline), taurodeoxy-cholate (0.025 g in 1 mL of saline), and taurocholate (0.04 g in 1 mLof saline), and 100 �L of pancreatin (0.04 g in 500 mL saline), wereadded. The pH of each sample was increased to pH 7.4 with 1 mol/LNaOH and the samples were incubated at 37 ◦C in a shaking water

bath at 95 rpm for 2.5 h to complete the intestinal phase of thein vitro digestion process. After the intestinal digestion phase, 2 mLof each sample were extracted and stored at −20 ◦C. Samples wereanalyzed within 2 weeks.

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according to the method of Nevile and Glossmann [17], indicat-ing the purity of the glycoprotein. It consists of a carbohydratecomponent (42.53%) and protein component (57.47%).

S.M. Rafiquzzaman et al. / International Journ

.3. Antioxidant activity

.3.1. Di (phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH)adical scavenging activity

Antioxidants activities of the UPGP before and after in vitroigestion was analyzed by investigating their ability to scavengehe DPPH free radical and it was carried out according to the

ethod of Blois [22]. Briefly, a solution of 0.15 mM DPPH inthanol was prepared and mixed with 100 mL of DW. To eval-ate DPPH activity, the DPPH solution was mixed with UPGPt various concentrations. The reaction mixture was kept in theark at RT for 30 min. Ascorbic acid was used as a positiveeference. The ability to scavenge DPPH radicals was calcu-ated by the following equation: DPPH radical scavenging activity%) = [(Abscontrol − Abssample)]/(Abscontrol)] × 100, where Abscontrol ishe absorbance of DPPH radicals + methanol and Abssample is thebsorbance of DPPH radicals + sample extract/standard.

.3.2. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)ABTS) radical scavenging activity

Antioxidant activity of the UPGP before and after in vitro diges-ion was analyzed by measuring the ability to scavenge the ABTS+.his assay is based on decolorization that occurs when the radi-al cation of ABTS (ABTS+) is reduced to ABTS− [23]. The radicalas generated by the reaction of a 7 mM solution of ABTS in waterith 2.45 mM potassium persulfate (1:1). The mixture was held inarkness at RT for 16 h, as this is the time needed to obtain stablebsorbance values at 734 nm. The assay was performed with 980 �Lf ABTS and 20 �L of sample at various concentrations.

.3.3. Ferric reducing antioxidant power (FRAP) assayDetermination of the FRAP activity in UPGP before and after in

itro digestion was carried out according to a modified version ofhe method of Benzie and Strain [24]. The stock solutions included00 mM acetate buffer (3.1 g of C2H3NaO2·3H2O and 16 mL of2H4O2), pH 3.6, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution

n 40 mM HCl, and 20 mM FeCl3·6H2O solution. A fresh workingolution was prepared by mixing 25 mL of acetate buffer, 2.5 mLf TPTZ, and 2.5 mL of FeCl3·6H2O. UPGP (0.150 �L) was allowedo react with 2.85 mL of the FRAP solution for 30 min in the dark.eadings of the colored product (ferrous tripyridyltriazine com-lex) were taken at 593 nm.

.3.4. NO scavenging assayNO scavenging assay was performed according to a modi-

ed version of the method of Jagetia et al. [25]. Briefly, 50 �Lf UPGP at different concentrations was mixed with 450 �L ofodium nitroprusside (SNP, 10 mM) and incubated for 4 h. An equalmount of Griess reagent was then added and allowed to reactt RT for 10 min. The absorbance of the chromophore formedue to diazotization of the nitrite with sulfanilamide and subse-uent coupling with naphthyl ethylene diamine was measured at46 nm.

.3.5. Reducing power assayThe reducing power of UPGP before and after in vitro digestion

as evaluated using the method of Oyaizu [26]. Briefly, 750 �Lf UPGP at various concentrations was mixed with 750 �L ofhosphate buffer (0.2 M, pH 6.6) and 750 �L of potassium hex-cyanoferrate (1%, w/v), followed by incubation at 50 ◦C in aater bath for 20 min. The reaction was terminated by adding

50 �L of trichloroacetic acid solution (10%) and then centrifu-ation at 800 × g for 10 min. The supernatant was collected andixed with 1.5 mL of DW and 100 �L of ferric chloride solution

0.1%, w/v) and incubated at RT for 10 min. The absorbance of the

iological Macromolecules 62 (2013) 265– 272 267

reaction mixture at 700 nm was determined; greater absorbance ofthe reaction mixture indicated greater reducing power.

2.4. DNA-protective activity

The DNA protecting activity of glycoprotein before and afterin vitro digestion against hydroxyl radical induced damage wasevaluated according the method of Kim et al. [12] with slight modi-fications. Hydroxyl radicals were generated by the mixture of 30 �Lof ascorbic acid (1 mM final concentration) and 1 �L of copper sul-fate (II) (100 �M final concentration). Bacteriophage � DNA (40 �L,0.1 �g/mL) was exposed to this solution in the absence or pres-ence of UPGP (100 �L, 1 mg/mL). The mixture was then incubated at37 ◦C for 1 h after which the samples were loaded onto a 1% agarosegel and fragments were separated by electrophoresis.

2.5. Statistical analysis

The IC50 value (i.e., the concentration sufficient to obtain 50% ofthe maximum scavenging capacity) was determined according tothe method of Kim et al. [27]. All tests and analyses were repeatedat least three times with ascorbic acid as a reference. The results areexpressed as means ± SD. A one way analysis of variance (ANOVA)and Duncan test were used for multiple comparisons using theSPSS program (version 16.0 for windows, SPSS Inc.). Values wereconsidered to differ significantly if the P value was less than 0.05.

3. Results

3.1. Purification and characterization of UPGP

The isolation and purification of UPGP was performed asdescribed in the Section 2. As shown in Fig. 1, SDS-PAGE showeda single band with a molecular weight of approximately 10 kDa,and the gels were stained with silver staining and Schiff’s reagent

Fig. 1. Analysis of UPGP by 15% SDS-PAGE. The gels were stained with silver and Peri-odic acid-Schiff (PAS) staining. The numbered lanes represent M, molecular weightmarker; 1, silver staining; 2, PAS staining.

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Table 1IC50 values of UPGP and ascorbic acid (reference).

Activity UPGP/reference IC50

DPPH radicalscavenging

UPGPAscorbic acid 0.25 ± 0.030.04 ± 0.007

ABTS radicalscavenging

UPGPAscorbic acid 0.08 ± 0.0050.18 ± 0.05

FRAP UPGPAscorbic acid 0.69 ± 0.120.28 ± 0.09

NO scavenging UPGPAscorbic acid 0.25 ± 0.082.50 ± 0.44

68 S.M. Rafiquzzaman et al. / International Journ

.2. Analysis of chemical composition in UPGP

.2.1. Monosaccharides compositionGC–MS analysis of purified UPGP was carried out in order

o determine the monosaccharide composition. Individual com-onents were identified by comparison of their m/z values inhe Total Ion Content (TIC) with the previously reported data inibrary. Peak analysis showed some monosaccharides including-glucose, d-mannitol, d-glucitol, 2-deoxy-d-mannose, d-arabino-exopyranose.

.2.2. Amino acid compositionThe amino acid profile of purified UPGP was presented in Fig. 2.

he amino acid compositional analysis revealed that purified UPGPontains both essential and non-essential amino acids. This UPGPas rich in glutamic acid, alanine, aspartic acid, and poor in serine,

hreonine, valine, leucine, isoleucine, tyrosine, histidine and lysine.

.2.3. FT-IR spectroscopyThe FT-IR spectrum of purified UPGP reported six peaks between

280 cm−1 and 884 cm−1 and revealed the presence of various pro-ein backbone. The amide A band (3280 cm−1) originated from feriesonance and corresponded to N H stretching vibrations. Amide

band is important in determining the secondary structure. Themide I band corresponded to peak at 1615 cm−1 which is mainlyssociated with the stretching vibrations of C O and indicating �-heet structure of proteins. The other two peaks at 1405 cm−1 and074 cm−1 represented stretching vibrations of C O and C O thatre mainly generated by serine and threonine, respectively. Rock-ng vibrations of CH2 attributed two peaks namely 1034 cm−1 and84 cm−1.

.2.4. Glycosylation analysisThe simplest methods of assessing the extent of deglycosylation

s by mobility shifts on Tricine-SDS-PAGE gels. Following gel elec-

rophoresis, the protein band of enzyme digested UPGP was littlehifted downwards with compare to untreated purified UPGP. Thehifting of band pattern was due to removal of O-linked sugar by-glycosidase from the purified UPGP.

Fig. 2. Analysis of amino acid profile o

IC50 values for all activities are shown in mg/mL for UPGP and mM for ascorbic acidas a reference.

3.3. Antioxidant activity of UPGP

Most of the glycoprotein isolated thus far show antioxidantactivity. Therefore, we evaluated the antioxidant activity of purifiedUPGP using different in vitro antioxidant assays (DPPH, ABTS, FRAP,NO scavenging, and reducing power assays) and DNA-protectiveactivity against oxidative stress. To better understand the quanti-tative differences detected by these methods, it should be notedthat each method works via a different mechanism. Therefore, it isimportant to take into consideration that DPPH has been describedas being specific for lipophilic agents, FRAP for hydrophilic agents,and ABTS for both [28].

3.3.1. DPPH radical scavenging activityThe overall trend of DPPH radical scavenging activity was fol-

lowed the concentration of the UPGP before and after the in vitrodigestion model. Before the in vitro digestion, the IC50 values forUPGP and the standard (ascorbic acid) in this assay were 0.25 ± 0.03and 0.04 ± 0.007 mg/mL, respectively (Table 1). There was no signif-icant variation in terms of DPPH scavenging activity after the salivaphase of UPGP. However, as seen from the compiled data from dif-ferent phase of simulated digestion, DPPH scavenging activity of

UPGP after the gastric and duodenal phase showed approximately26.6% and 22% lower respectively, compared to undigested UPGP(Fig. 3).

f UPGP by amino acid analyzer.

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ig. 3. The DPPH scavenging activities of UPGP before and after in vitro digestion.cid was used as reference. The results were representative of three separate expeignificant difference (P ≤ 0.05).

.3.2. ABTS radical scavenging activityIn the ABTS assay, undigested UPGP showed dose-dependent

nhibition of ABTS with an IC50 of 0.08 ± 0.005 mg/mL, whereas thealue for ascorbic acid was 0.18 ± 0.05 mg/mL (Table 1). Radicalcavenging activities natively present in UPGP, were found to belightly higher compared to those displayed after the gastric phaseigestion but not compared to the duodenal phase of digestionFig. 4). In agreement with the DPPH assay, lowest ABTS scavengingctivities was recorded after the gastric phase of digestion.

.3.3. FRAP assayThe reducing antioxidant activity measured by FRAP assay

as also concentration-dependent. Before the in vitro digestion,PGP showed moderate scavenging activity against FRAP with

n IC50 value of 0.69 ± 0.12 mg/mL; that for ascorbic acid was.28 ± 0.09 mg/mL (Table 1). The reducing power of UPGP were8.63% and 100% at 0.1 and 4.0 mg/mL, respectively (Fig. 5). Theeducing power of UPGP after the gastric and duodenal phase of

ig. 4. The ABTS scavenging activities of UPGP before and after in vitro digestion. The pcid was used as reference. The results were representative of three separate experimenignificant difference (P ≤ 0.05).

ercentage of inhibition was plotted against the concentration of sample. Ascorbicts and data were expressed as mean ± SD. Difference letters superscripts indicate

digestion was 34.26% and 24.33%, lower respectively, than that ofundigested UPGP (Fig. 4).

3.3.4. NO scavenging assayDuring the evaluation of the NO scavenging ability of UPGP,

nitrite was measured using Griess reagent. UPGP stronglydecreased the NO level in a dose-dependent manner, with anIC50 value of 0.25 ± 0.08 mg/mL. This value is lower than that ofthe reference compound, ascorbic acid, which showed a value of2.5 ± 0.44 mg/mL. Following the in vitro digestion, there was signif-icantly variation in the NO scavenging capacity of UPGP (Fig. 5). Theactivity of UPGP at 1 mg/mL reached 100%, which was higher thanthat of ascorbic acid (Fig. 6).

3.3.5. Reducing power assay

In the reducing power assay, antioxidants caused the reduction

of Fe3+ to Fe2+, thereby changing the color of the solution to variousshades from green to blue depending on the reducing power of thetest compounds. Interestingly, no significant variation was found

ercentage of inhibition was plotted against the concentration of sample. Ascorbicts and data were expressed as mean ± SD. Difference letters superscripts indicate

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Fig. 5. The FRAP assay of UPGP before and after in vitro digestion. The percentage of inhibition was plotted against the concentration of sample. Ascorbic acid was used asreference. The results were representative of three separate experiments and data were expressed as mean ± SD. Difference letters superscripts indicate significant difference(P ≤ 0.05).

Fig. 6. The reducing power assay of UPGP before and after in vitro digestion. The percentage of inhibition was plotted against the concentration of sample. Ascorbic acid wasused as reference. The results were representative of three separate experiments and data were expressed as mean ± SD. Difference letters superscripts indicate significantdifference (P ≤ 0.05).

Fig. 7. The NO scavenging activities of UPGP before and after in vitro digestion. The percentage of inhibition was plotted against the concentration of sample. Ascorbic acid wasused as reference. The results were representative of three separate experiments and data were expressed as mean ± SD. Difference letters superscripts indicate significantdifference (P ≤ 0.05).

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Fig. 8. Agarose gel electrophoretic separation of damaged DNA and the protectiveeffect of UPGP before and after in vitro digestion. The numbered lane represent 1, �DNA alone; 2, DNA plus Cu (II)-ascorbic acid; 3, DNA plus Cu (II)-ascorbic acid andundigested UPGP; 4, DNA plus Cu (II)-ascorbic acid and UPGP after saliva phase; 5,DaD

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NA plus Cu (II)-ascorbic acid and UPGP after gastric phase; 6, DNA plus Cu (II)-scorbic acid and UPGP after duodenal phase; S, supercoiled DNA strands; N, nickedNA strands.

uring the measurement of reducing power before and after in vitroimulated digestion model (Fig. 7). The results of this assay indicatehat the reducing power of UPGP was similar to that of ascorbic acid.

.4. DNA-protective activity

Glycoprotein have been reported to protect DNA from damagey neutralizing charged radicals and through the induction of con-ormational changes in DNA. To validate these effects, we evaluatedhe protective effect of UPGP. To this end, � DNA was incubatedn the presence of Cu (II) sulfate and ascorbic acid with or with-ut UPGP. The undigested glycoprotein-treated sample showed alear band of � DNA, whereas the untreated � DNA showed only

smear (Fig. 8). There was little variation in its DNA-protectivectivity after in vitro digestion. The glycoprotein after gastric phaseid not protect DNA damage whereas UPGP after saliva and duode-al phase showed DNA protecting activity against oxidative stressFig. 7).

. Discussion

ROS, which are constantly generated in living systems, causearious diseases, including degenerative diseases and cell lysis [2].o solve these problems, it is necessary to search for natural ratherhan chemical antioxidants because of the possibility of adverseffects of the latter on health. Over the past few decades, manylycoprotein have been isolated from fungi, yeasts, algae, lichens,nd plants and their biological activities have been evaluated inerms of anticancer, antioxidant, and immunomodulatory effects11,12,29]. However, there have been no studies to date regardinghe biofunctional activities of glycoprotein from U. pinnatifida. Thus,e isolated and purified glycoprotein from U. pinnatifida to evalu-

te the biopharmacological activities and its bioaccessibility underimulated in vitro digestion model. As shown in Fig. 1, purified UPGPigrated as a single band with an approximate molecular weight of

0 kDa on SDS-PAGE, which consists of carbohydrate (42.53%) androtein (57.47%). After electrophoresis, the gels were stained withchiff’s reagent which confirm it as glycoprotein. This is consistentith the results of Kim et al. [12], who purified a glycoprotein of

imilar molecular weight from S. japonica.In order to characterize the purified UPGP, we performed several

nalyses including determination of amino acid and monosaccha-ide compositions, FT-IR spectroscopy and glycosylation analysis.

iological Macromolecules 62 (2013) 265– 272 271

Amino acid analysis and FT-IR spectrum provided the informa-tion regarding amino acid profile, protein backbone and secondarystructure which is important for structural characterization of theglycoprotein [30]. Both analyses showed that UPGP contains ser-ine and thereonine indicating the presence of O-linked glycan.Serine and thereonine residues are essential for O-glycosylationwhile asparagine is essential for N-glycosylation [31]. However,asparagines residue in UPGP was not identified by FT-IR and aminoacid profile analysis. The presence of O-linked glycan was furthersupported by the observations of enzymatic glycosylation analy-sis. This analysis clearly showed that the band of enzyme digestedUPGP downshifted with compare to undigested UPGP which is inagreement with findings of Verena et al. [32]. Generally, character-ization of glycoprotein structure is a complex process with severalsteps. Therefore, further studies are required to determine the exactstructure of UPGP.

Regarding antioxidant activity, it has been found that UPGPshowed promising antioxidant activity as detected by DPPH, ABTSand reducing power, which is comparable with that of the well-known antioxidant, ascorbic acid, consistent with the findingsof what they have reported before [11,12,33]. In contrast, previ-ous studies involving the evaluation of antioxidant activities bymethanol, ethanol, sulfated or desulfated polysaccharide, and enzy-matic extracts of U. pinnatifida showed low antioxidant activitycompared to the glycoprotein in this study [34–36]. Similarly, wealso found that UPGP can successfully scavenge NO via inhibitingnitrite formation by directly competing with oxygen. This result isconsistent with the findings reported by Kim et al. [12]. In this study,� DNA was damaged by hydroxyl radical generation. The additionof UPGP to the reaction mixture showed protective effect againstthe damage of native DNA (Fig. 8) which is in agreement with thefindings of Kim et al. [12] and Albishi et al. [37]. The DNA-protectiveactivity of UPGP is a reflection of its positive effects against manydiseases in human body.

Concerning determination of the bioavailability of UPGP, weused an in vitro digestion model because it gives an indication ofthe bioavailability of different biofunctional activities in a biologicalsystem, since the model is designed to simulate in vivo digestion.During the simulated saliva stage, there was no significant varia-tion in antioxidant and DNA-protective activity. However, after invitro digestion, the overall antioxidant activities of UPGP tended tobe lower during the gastric phase than in the undigested and duo-denal phases. Similarly, in the case of DPPH-scavenging activity,UPGP showed significant variation in activity at different stages ofin vitro digestion. This significant variation in scavenging activitymay have been due to the variation in pH because the viabilityof the DPPH method is dependent upon pH [38] (Fig. 3). How-ever, ABTS scavenging activity was almost unchanged followingin vitro digestion (Fig. 4). This result is consistent with the findingsof Han et al. [34], who reported that the ABTS assay gives consistentresults over a wide pH range. After the gastric phase, UPGP showedslightly reduced ABTS activities compared with the duodenal phase.The observed differences between the gastric and duodenal phasesof digestion are in agreement with previous reports [39,40]. Kimet al. [12] found that glycoprotein from S. japonica retained 80%DPPH scavenging activity at pH 4–6 and that the activity was lowerbeyond this pH range. The results of the present study strongly sup-port these findings and suggest that the chemical bond betweenglycoprotein and protons may be unstable under weak acidic, neu-tral, and weak alkaline conditions.

Measuring the reducing power of UPGP after in vitro digestionis important for evaluating antioxidant power. The reducing power

of UPGP was measured using the methods of Oyaizu [26] and FRAPassay. Using the FRAP assay, we demonstrated that the reducingpower of UPGP was significantly lower during the gastric and duo-denal phases compared to undigested UPGP. Wootton-Beard et al.

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39] reported that the FRAP values of tea samples were signifi-antly decreased following in vitro digestion. In contrast, Peter et al.41] found that FRAP values were increased after the gastric phasend remained high following the duodenal phase. Interestingly, theeducing activity of UPGP, as measured using the methods of Oyaizu26], remained stable after in vitro digestion. In terms of explaininghis discrepancy, we think that the pH of the buffer solution (3.6n the FRAP assay, 6.6 in the Oyaizu method) might be one rea-on, which is supported by Bouayed et al. [42]. Another importantbservation in this study is the pH dependency of the scavengingctivity of UPGP. We speculate that intestinal cells may be bet-er protected against oxidative stress by UPGP, which has highercavenging activity at the pH in the intestine, compared to thatn the stomach. Regarding the DNA-protective activity analysis, it

as found that UPGP after duodenal and saliva phase can protectNA against oxidative damage but after gastric phase, UPGP doesot protect DNA damage. This result indirectly confirmed that DNArotective activity is also pH-dependent, in agreement with thendings of changing different antioxidant activity during simulatedigestion in our present study.

Our data suggest that UPGP acted as a bioavailable free-radicalcavenger and thereby prevented oxidative stress. The biologicalctivity may be due to the structural diversity of glycoprotein. In theresent study, glycoprotein treated with NaIO4 (a carbohydrate-eactivating agent) showed only 32% DPPH scavenging activityompared with undigested UPGP (data not shown). This result sug-ests that the glycoprotein needs both its carbohydrate and proteinomponents to show optimal activity, in agreement with previousndings [12,28]. Generally glycoprotein differs from pure proteinsnd nucleic acids in terms of its large tertiary structure [43].

. Conclusion

The chemical analyses of UPGP have been suggested that proteins attached with carbohydrate by O-glycosylation. Regarding bio-ogical activities, based on the data obtained from different phasesf digestion, UPGP was almost stable in terms of antioxidant andNA-protective activities during the digestion process. There was

ittle difference between its antioxidant and DNA protecting activi-ies during the gastric and duodenal phases of digestion and those ofndigested UPGP. Overall, our results indicate that the antioxidantnd DNA protecting activities of UPGP may be pH-dependent andssay-specific, and suggest higher antioxidant and DNA protectingctivity in the intestine than in the stomach. Furthermore, UPGPhowed protective activity against DNA damage due to oxidativetress both before and after in vitro duodenal digestion. Therefore,PGP could be an important natural and bioavailable antioxidantnd DNA-protective agent. However, further study is required tolucidate the structure of the glycoprotein and the mechanisms ofts biofunctional activities at the molecular and biochemical levels.

cknowledgements

This work was supported by a Research Grant of Puky-ng National University (2013 year). The authors would like to

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iological Macromolecules 62 (2013) 265– 272

acknowledge to the Korea Basic Science Institute for the amino acidanalysis.

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