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

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International Journal of Biological Macromolecules

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

Antioxidant activity of glycoprotein purified from Undaria pinnatifida

measured by an in vitro digestion model
S.M. Rafiquzzaman a,1 , Eun-Young Kim a,1 , Yu-Ri Kim a , Taek-Jeong Nam b , In-Soo Kong a,∗
a
Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea
b
Department of Food Science and Nutrition, Pukyong National University, Busan 608-737, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: The present study was performed to investigate the chemical composition and antioxidant activity of
Received 27 June 2013 glycoprotein purified from Undaria pinnatifida Harvey (UPGP). On SDS-PAGE, UPGP migrated as a single
Received in revised form 24 August 2013 band with a molecular weight of approximately 10 kDa and confirmed by staining with Schiff’s reagent as
Accepted 15 September 2013
glycoprotein. It consists of a carbohydrate component (42.53%) and protein component (57.47%). Amino
Available online 20 September 2013
acid profile, FT-IR spectrum and enzymatic glycosylation analysis suggested that protein is linked with
carbohydrate by O-glycosylation. UPGP showed dose-dependent antioxidant activities as detected by
Keywords:
different assays before and after in vitro digestion. The IC50 values of undigested UPGP were 0.25 ± 0.03,
Glycoprotein
Undaria pinnatifida 0.08 ± 0.005, 0.69 ± 0.12, and 0.25 ± 0.08 mg/mL for DPPH, ABTS, FRAP, and NO, respectively. Following
Chemical composition in vitro digestion, the antioxidant activities of UPGP were decreased during the gastric phase compared to
Antioxidant those of undigested UPGP, with an increase occurring during the duodenal phase in all assays. However,
DNA protection the reducing power was unchanged after in vitro digestion. Furthermore, UPGP showed protective activity
In vitro digestion model against oxidative DNA damage both undigested, after saliva and duodenal phase of digestion. These
results indicate that the antioxidant and DNA protection activities of UPGP may be pH-dependent and
assay specific.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction including hypocholesterolemic, antithrombotic, antioxidant, and

antidiabetic effects, reduction of coronary diseases, and estrogen
Reactive oxygen species (ROS), including hydroxyl radicals, metabolism [5–7]. There is a long tradition in China, Japan, Korea,
superoxide radicals, hydrogen peroxide, and nitrite oxide, are and Southeast Asia of consuming seaweeds as part of the regular
produced as a consequence of the normal metabolism of living diet. The main species of seaweed cultivated in Korea are Porphyra
organisms [1]. ROS decrease the normal defensive systems of (nori), Undaria (wakame in Japan and miyeok in Korea) and Lami-
organisms, leading to various abnormalities, including myocardial naria. Undaria pinnatifida is a widely eaten brown seaweed and is
ischemia, carcinogenesis, inflammatory disease, and Alzheimer’s also used as a traditional medicine in Korea. Interest in seaweed as a
disease by attacking proteins, lipids (including those in cellular source of food is increasing, particularly because of the advantages
membranes), and DNA [2]. In addition, nitric oxide (NO) formed to the consumer beyond strictly nutritional benefits. Aquaculture of
by Ca+ -independent inducible NOS may contribute to cytotoxic- seaweed is expanding at present as natural production cannot meet
ity, DNA damage, and carcinogenesis [3]. Consumption of natural the current demand. According to the Food and Agriculture Organi-
product which is rich in antioxidant compounds may help to make zation of the United Nations, about 2.5 million tons of U. pinnatifida
balance between ROS production and endogenous protection when are produced per year, of which 99% is from aquaculture.
the body is in under oxidative stress. In particular, the regulated It has been reported that U. pinnatifida is a rich source of
production of ROS is an essential prerequisite to maintain redox eicosapentaenoic acid, an omega-3 fatty acid, and that it con-
homeostasis in humans [4]. tains high levels of calcium, iodine, thiamine, and niacin [8]. In
Recent findings have demonstrated health benefits associ- oriental medicine, U. pinnatifida has been used for blood purifi-
ated with the consumption of seaweed, and that extracts of cation, intestinal strength, and beautification of the skin and hair
different seaweeds exhibited different biofunctional activities, [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]. Most
∗ Corresponding author. Tel.: +82 051 629 5865.
studies have focused on evaluating the biological functions of
E-mail address: [email protected] (I.-S. Kong).
polysaccharides and lipids from U. pinnatifida, although plant gly-
1
These authors contributed equally to this paper. coproteins are known to have more biological functions [11,12].

0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2013.09.009
266 S.M. Rafiquzzaman et al. / International Journal of Biological Macromolecules 62 (2013) 265–272

Recent research in our laboratory has aimed to isolate and purify The carbohydrate and protein levels in UPGP were determined by
the glycoprotein from Saccharina japonica in order to characterize measuring the absorbance at 490 and 280 nm, respectively.
the different biofunctional activities and reported that it has strong
antioxidant, DNA protective, NO scavenging, and xanthine oxidase
inhibition activity [12]. 2.1.4. Analysis of chemical composition in UPGP
Two important aspects of the beneficial effects of any bioactive 2.1.4.1. Monosaccharides composition. The purified UPGP was
compound are bioavailability and metabolic fate. The bioavail- hydrolyzed using anhydrous methanol containing 2 M HCl at 80 ◦ C
ability of a dietary compound is dependent upon its digestive for 20 h. Then the hydrolyzed products were neutralized with
stability. It is important to determine the fate of bioactive com- methanol–KOH and dried. GC–MS analysis was performed by
pounds during human digestion to better understand the potential Shimadzu GC/MS-QP2010. Hydrolyzed sample was injected at a
benefits of such active compounds. Structural modifications and constant temperature of 260 ◦ C in splitless mode and detected
alterations in functional properties caused by the variation in pH at 300 ◦ C. GC separation was performed on a DB-1MS col-
along the gastrointestinal tract could affect bioefficacy. Many stud- umn (30 m × 0.25 mm × 0.25 ␮m) at a constant flow of helium
ies have shown the significant influence of simulated digestion (1 ml/min). Initial oven temperature was set at 105 ◦ C at 4 ◦ C/min,
on the bioavailability of phytochemicals [13]. However, there are followed by 240 ◦ C, 20 ◦ C/min, 300 ◦ C hold for 3.25 min.
contradictory data regarding the stability of phenolic compounds
subjected to changes in pH, while other antioxidants such as Trolox
and tocopherol become more stable under gut conditions [14,15]. 2.1.4.2. Amino acid composition. The content of amino acid in
In the past decade, in vitro digestion models useful for assessing the purified UPGP was determined using Amino Acid Analyzer (Mem-
bioavailability of compounds from foods have been developed. In brapure Co. Germany). The purified UPGP sample was hydrolyzed
the literature, there are no studies of the antioxidant and other bio- with 6 N HCl in evacuated sealed tubes for 24 h at 110 ◦ C. Twenty
functional activities of U. pinnatifida glycoprotein and its stability microlitres of hydrolysed UPGP was loaded and passed through the
when subjected to in vitro digestion. narrow bore column (stainless steel 125 mm, ID 3 mm). Ninhydrin
In this study, we purified UPGP and evaluated its chemical was added to the HCl as an internal standard. The amount of each
composition, biological activities, including antioxidant and DNA- amino acid was expressed in percentage of amino acid per 100 g of
protective activities. In addition, we investigated the bioavailability amino acid.
of the glycoprotein using an in vitro model that simulated several
chemical (pH, temperature and bile salt) and biological (gastric
and pancreatic enzyme) gastro-intestinal conditions. Moreover, 2.1.4.3. FT-IR spectroscopy. The freeze dried UPGP was used for
changes in antioxidant activity during digestion, as well as the Fourier transform infrared (FT-IR) measurement in the frequency
digestive stability of the glycoprotein, were investigated. range of 4000–650 cm−1 a using Perkin Elmer (USA), Spectrum X.

2. Materials and methods

2.1.4.4. Glycosylation analysis. Glycosidases are the useful tools for
2.1. Purification and characterization of UPGP the structural and functional analysis of oligosaccharides associ-
ated with glycoproteins. The purified native 4 ␮l of UPGP (3 mg/ml)
2.1.1. Purification of UPGP was incubated with 13 ␮l of reaction buffer (250 mM phosphate
U. pinnatifida was obtained from a local market in Busan, Korea, buffer, pH 5.5) at 37 ◦ C for 1 h. The sample was then incubated
and preserved in plastic boxes under dried conditions for exper- with 2 ␮l of O-glycosidase (2 m unit) purified from Streptococcus
imental use. Glycoprotein purification from U. pinnatifida was pneumoniae (EC. 3.2.1.97, Roche, Germany) for 3 h at 37 ◦ C. After
carried out as described previously with some modifications [16]. glycosidase treatment, deglycosylation of glycoprotein was mon-
Briefly, 4 g of U. pinnatifida was mixed with 100 mL of distilled itored by Tricine-SDS-PAGE containing 12% acrylamide gel and
water (DW) and incubated for more than 4 h at room temperature visualized by Coomassie Blue staining.
(RT). The DW extract was filtered with gauze, and then precipi-
tated by the addition of three volumes of ethanol for more than 6 h
at RT. The precipitate was passed through Whatman 3MM paper 2.2. In vitro digestion model
(GE Healthcare, Amersham, UK) to remove debris. The filtered sam-
ples were then concentrated in a rotatory evaporator followed by The in vitro digestion model was used according a method
freeze-drying. The freeze-dried samples were dissolved in DW and reported previously [20,21] with slight modifications. Initially, to
dialyzed overnight with the membrane. The dialyzed samples were simulate saliva, glycoprotein solution was treated with the artifi-
filtered with Whatman paper and the glycoprotein concentration cial saliva solution (6.2 g/L NaCl, 2.2 g/L KCl, 0.22 g/L CaCl2 , 1.2 g/L
was determined by measuring the absorbance at 280 nm. NaHCO3 ). The glycoprotein samples were then transferred to clean
amber bottles and mixed with saline. The samples were acidified
2.1.2. SDS-PAGE to pH 2 with 1 mL of porcine pepsin preparation (0.04 g pepsin
To check the purity of UPGP, the samples were subjected to 15% in 0.1 mol/L HCl) and incubated at 37 ◦ C in a shaking water bath
polyacrylamide mini gel electrophoresis. The electrophoresis was at 95 rpm for 1 h. After gastric digestion, the pH was increased
carried out at 110 V, 30 mA for 2.5 h. The gel was stained with silver to 5.3 with 0.9 mol/L sodium bicarbonate, and then 200 ␮L of the
staining and periodic acid-Schiff (PAS), which specifically detects bile salts glycodeoxycholate (0.04 g in 1 mL of saline), taurodeoxy-
glycoprotein as a result of a redox reaction [17]. cholate (0.025 g in 1 mL of saline), and taurocholate (0.04 g in 1 mL
of saline), and 100 ␮L of pancreatin (0.04 g in 500 mL saline), were
2.1.3. Measurement of the carbohydrate and protein levels in added. The pH of each sample was increased to pH 7.4 with 1 mol/L
UPGP NaOH and the samples were incubated at 37 ◦ C in a shaking water
The carbohydrate content of UPGP was determined by the bath at 95 rpm for 2.5 h to complete the intestinal phase of the
phenol-sulfuric acid method using glucose as a reference [18]. The in vitro digestion process. After the intestinal digestion phase, 2 mL
protein moiety of UPGP was quantified according to the method of each sample were extracted and stored at −20 ◦ C. Samples were
of Lowry et al. [19] using bovine serum albumin as a standard. analyzed within 2 weeks.
 S.M. Rafiquzzaman et al. / International Journal of Biological Macromolecules 62 (2013) 265–272 267

2.3. Antioxidant activity reaction mixture at 700 nm was determined; greater absorbance of
the reaction mixture indicated greater reducing power.
2.3.1. Di (phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH)
radical scavenging activity
2.4. DNA-protective activity
Antioxidants activities of the UPGP before and after in vitro
digestion was analyzed by investigating their ability to scavenge
The DNA protecting activity of glycoprotein before and after
the DPPH free radical and it was carried out according to the
in vitro digestion against hydroxyl radical induced damage was
method of Blois [22]. Briefly, a solution of 0.15 mM DPPH in
evaluated according the method of Kim et al. [12] with slight modi-
ethanol was prepared and mixed with 100 mL of DW. To eval-
fications. Hydroxyl radicals were generated by the mixture of 30 ␮L
uate DPPH activity, the DPPH solution was mixed with UPGP
of ascorbic acid (1 mM final concentration) and 1 ␮L of copper sul-
at various concentrations. The reaction mixture was kept in the
fate (II) (100 ␮M final concentration). Bacteriophage ␭ DNA (40 ␮L,
dark at RT for 30 min. Ascorbic acid was used as a positive
0.1 ␮g/mL) was exposed to this solution in the absence or pres-
reference. The ability to scavenge DPPH radicals was calcu-
ence of UPGP (100 ␮L, 1 mg/mL). The mixture was then incubated at
lated by the following equation: DPPH radical scavenging activity
37 ◦ C for 1 h after which the samples were loaded onto a 1% agarose
(%) = [(Abscontrol − Abssample )]/(Abscontrol )] × 100, where Abscontrol is
gel and fragments were separated by electrophoresis.
the absorbance of DPPH radicals + methanol and Abssample is the
absorbance of DPPH radicals + sample extract/standard.
2.5. Statistical analysis
2.3.2. 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
(ABTS) radical scavenging activity The IC50 value (i.e., the concentration sufficient to obtain 50% of
Antioxidant activity of the UPGP before and after in vitro diges- the maximum scavenging capacity) was determined according to
tion was analyzed by measuring the ability to scavenge the ABTS+ . the method of Kim et al. [27]. All tests and analyses were repeated
This assay is based on decolorization that occurs when the radi- at least three times with ascorbic acid as a reference. The results are
cal cation of ABTS (ABTS+ ) is reduced to ABTS− [23]. The radical expressed as means ± SD. A one way analysis of variance (ANOVA)
was generated by the reaction of a 7 mM solution of ABTS in water and Duncan test were used for multiple comparisons using the
with 2.45 mM potassium persulfate (1:1). The mixture was held in SPSS program (version 16.0 for windows, SPSS Inc.). Values were
darkness at RT for 16 h, as this is the time needed to obtain stable considered to differ significantly if the P value was less than 0.05.
absorbance values at 734 nm. The assay was performed with 980 ␮L
of ABTS and 20 ␮L of sample at various concentrations. 3. Results

2.3.3. Ferric reducing antioxidant power (FRAP) assay 3.1. Purification and characterization of UPGP
Determination of the FRAP activity in UPGP before and after in
vitro digestion was carried out according to a modified version of The isolation and purification of UPGP was performed as
the method of Benzie and Strain [24]. The stock solutions included described in the Section 2. As shown in Fig. 1, SDS-PAGE showed
300 mM acetate buffer (3.1 g of C2 H3 NaO2 ·3H2 O and 16 mL of a single band with a molecular weight of approximately 10 kDa,
C2 H4 O2 ), pH 3.6, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution and the gels were stained with silver staining and Schiff’s reagent
in 40 mM HCl, and 20 mM FeCl3 ·6H2 O solution. A fresh working according to the method of Nevile and Glossmann [17], indicat-
solution was prepared by mixing 25 mL of acetate buffer, 2.5 mL ing the purity of the glycoprotein. It consists of a carbohydrate
of TPTZ, and 2.5 mL of FeCl3 ·6H2 O. UPGP (0.150 ␮L) was allowed component (42.53%) and protein component (57.47%).
to react with 2.85 mL of the FRAP solution for 30 min in the dark.
Readings of the colored product (ferrous tripyridyltriazine com-
plex) were taken at 593 nm.

2.3.4. NO scavenging assay

NO scavenging assay was performed according to a modi-
fied version of the method of Jagetia et al. [25]. Briefly, 50 ␮L
of UPGP at different concentrations was mixed with 450 ␮L of
sodium nitroprusside (SNP, 10 mM) and incubated for 4 h. An equal
amount of Griess reagent was then added and allowed to react
at RT for 10 min. The absorbance of the chromophore formed
due to diazotization of the nitrite with sulfanilamide and subse-
quent coupling with naphthyl ethylene diamine was measured at
546 nm.

2.3.5. Reducing power assay

The reducing power of UPGP before and after in vitro digestion
was evaluated using the method of Oyaizu [26]. Briefly, 750 ␮L
of UPGP at various concentrations was mixed with 750 ␮L of
phosphate buffer (0.2 M, pH 6.6) and 750 ␮L of potassium hex-
acyanoferrate (1%, w/v), followed by incubation at 50 ◦ C in a
water bath for 20 min. The reaction was terminated by adding
750 ␮L of trichloroacetic acid solution (10%) and then centrifu-
gation at 800 × g for 10 min. The supernatant was collected and
Fig. 1. Analysis of UPGP by 15% SDS-PAGE. The gels were stained with silver and Peri-
mixed with 1.5 mL of DW and 100 ␮L of ferric chloride solution odic acid-Schiff (PAS) staining. The numbered lanes represent M, molecular weight
(0.1%, w/v) and incubated at RT for 10 min. The absorbance of the marker; 1, silver staining; 2, PAS staining.
268 S.M. Rafiquzzaman et al. / International Journal of Biological Macromolecules 62 (2013) 265–272

3.2. Analysis of chemical composition in UPGP Table 1

IC50 values of UPGP and ascorbic acid (reference).

3.2.1. Monosaccharides composition Activity UPGP/reference IC50

GC–MS analysis of purified UPGP was carried out in order DPPH radical UPGPAscorbic acid 0.25 ± 0.03
to determine the monosaccharide composition. Individual com- scavenging 0.04 ± 0.007
ponents were identified by comparison of their m/z values in ABTS radical UPGPAscorbic acid 0.08 ± 0.005
the Total Ion Content (TIC) with the previously reported data in scavenging 0.18 ± 0.05
FRAP UPGPAscorbic acid 0.69 ± 0.12
library. Peak analysis showed some monosaccharides including
0.28 ± 0.09
d-glucose, d-mannitol, d-glucitol, 2-deoxy-d-mannose, d-arabino- NO scavenging UPGPAscorbic acid 0.25 ± 0.08
hexopyranose. 2.50 ± 0.44

IC50 values for all activities are shown in mg/mL for UPGP and mM for ascorbic acid
3.2.2. Amino acid composition as a reference.
The amino acid profile of purified UPGP was presented in Fig. 2.
The amino acid compositional analysis revealed that purified UPGP
contains both essential and non-essential amino acids. This UPGP 3.3. Antioxidant activity of UPGP
was rich in glutamic acid, alanine, aspartic acid, and poor in serine,
threonine, valine, leucine, isoleucine, tyrosine, histidine and lysine. Most of the glycoprotein isolated thus far show antioxidant
activity. Therefore, we evaluated the antioxidant activity of purified
3.2.3. FT-IR spectroscopy UPGP using different in vitro antioxidant assays (DPPH, ABTS, FRAP,
The FT-IR spectrum of purified UPGP reported six peaks between NO scavenging, and reducing power assays) and DNA-protective
3280 cm−1 and 884 cm−1 and revealed the presence of various pro- activity against oxidative stress. To better understand the quanti-
tein backbone. The amide A band (3280 cm−1 ) originated from feri tative differences detected by these methods, it should be noted
resonance and corresponded to N H stretching vibrations. Amide that each method works via a different mechanism. Therefore, it is
I band is important in determining the secondary structure. The important to take into consideration that DPPH has been described
amide I band corresponded to peak at 1615 cm−1 which is mainly as being specific for lipophilic agents, FRAP for hydrophilic agents,
associated with the stretching vibrations of C O and indicating ␤- and ABTS for both [28].
sheet structure of proteins. The other two peaks at 1405 cm−1 and
1074 cm−1 represented stretching vibrations of C O and C O that 3.3.1. DPPH radical scavenging activity
are mainly generated by serine and threonine, respectively. Rock- The overall trend of DPPH radical scavenging activity was fol-
ing vibrations of CH2 attributed two peaks namely 1034 cm−1 and lowed the concentration of the UPGP before and after the in vitro
884 cm−1 . digestion model. Before the in vitro digestion, the IC50 values for
UPGP and the standard (ascorbic acid) in this assay were 0.25 ± 0.03
3.2.4. Glycosylation analysis and 0.04 ± 0.007 mg/mL, respectively (Table 1). There was no signif-
The simplest methods of assessing the extent of deglycosylation icant variation in terms of DPPH scavenging activity after the saliva
is by mobility shifts on Tricine-SDS-PAGE gels. Following gel elec- phase of UPGP. However, as seen from the compiled data from dif-
trophoresis, the protein band of enzyme digested UPGP was little ferent phase of simulated digestion, DPPH scavenging activity of
shifted downwards with compare to untreated purified UPGP. The UPGP after the gastric and duodenal phase showed approximately
shifting of band pattern was due to removal of O-linked sugar by 26.6% and 22% lower respectively, compared to undigested UPGP
O-glycosidase from the purified UPGP. (Fig. 3).

Fig. 2. Analysis of amino acid profile of UPGP by amino acid analyzer.

 S.M. Rafiquzzaman et al. / International Journal of Biological Macromolecules 62 (2013) 265–272 269

Fig. 3. The DPPH scavenging activities of UPGP before and after in vitro digestion. The percentage of inhibition was plotted against the concentration of sample. Ascorbic
acid was used as reference. The results were representative of three separate experiments and data were expressed as mean ± SD. Difference letters superscripts indicate
significant difference (P ≤ 0.05).

3.3.2. ABTS radical scavenging activity digestion was 34.26% and 24.33%, lower respectively, than that of
In the ABTS assay, undigested UPGP showed dose-dependent undigested UPGP (Fig. 4).
inhibition of ABTS with an IC50 of 0.08 ± 0.005 mg/mL, whereas the
value for ascorbic acid was 0.18 ± 0.05 mg/mL (Table 1). Radical
3.3.4. NO scavenging assay
scavenging activities natively present in UPGP, were found to be
During the evaluation of the NO scavenging ability of UPGP,
slightly higher compared to those displayed after the gastric phase
nitrite was measured using Griess reagent. UPGP strongly
digestion but not compared to the duodenal phase of digestion
decreased the NO level in a dose-dependent manner, with an
(Fig. 4). In agreement with the DPPH assay, lowest ABTS scavenging
IC50 value of 0.25 ± 0.08 mg/mL. This value is lower than that of
activities was recorded after the gastric phase of digestion.
the reference compound, ascorbic acid, which showed a value of
2.5 ± 0.44 mg/mL. Following the in vitro digestion, there was signif-
icantly variation in the NO scavenging capacity of UPGP (Fig. 5). The
activity of UPGP at 1 mg/mL reached 100%, which was higher than
3.3.3. FRAP assay
that of ascorbic acid (Fig. 6).
The reducing antioxidant activity measured by FRAP assay
was also concentration-dependent. Before the in vitro digestion,
UPGP showed moderate scavenging activity against FRAP with 3.3.5. Reducing power assay
an IC50 value of 0.69 ± 0.12 mg/mL; that for ascorbic acid was In the reducing power assay, antioxidants caused the reduction
0.28 ± 0.09 mg/mL (Table 1). The reducing power of UPGP were of Fe3+ to Fe2+ , thereby changing the color of the solution to various
18.63% and 100% at 0.1 and 4.0 mg/mL, respectively (Fig. 5). The shades from green to blue depending on the reducing power of the
reducing power of UPGP after the gastric and duodenal phase of test compounds. Interestingly, no significant variation was found

Fig. 4. The ABTS scavenging activities of UPGP before and after in vitro digestion. The percentage of inhibition was plotted against the concentration of sample. Ascorbic
acid was used as reference. The results were representative of three separate experiments and data were expressed as mean ± SD. Difference letters superscripts indicate
significant difference (P ≤ 0.05).
270 S.M. Rafiquzzaman et al. / International Journal of Biological Macromolecules 62 (2013) 265–272

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 as
reference. 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 was
used as reference. 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. 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 was
used as reference. The results were representative of three separate experiments and data were expressed as mean ± SD. Difference letters superscripts indicate significant
difference (P ≤ 0.05).
 S.M. Rafiquzzaman et al. / International Journal of Biological Macromolecules 62 (2013) 265–272 271

Amino acid analysis and FT-IR spectrum provided the informa-

tion regarding amino acid profile, protein backbone and secondary
structure which is important for structural characterization of the
glycoprotein [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-glycosylation
while asparagine is essential for N-glycosylation [31]. However,
asparagines residue in UPGP was not identified by FT-IR and amino
acid profile analysis. The presence of O-linked glycan was further
supported by the observations of enzymatic glycosylation analy-
sis. This analysis clearly showed that the band of enzyme digested
UPGP downshifted with compare to undigested UPGP which is in
agreement with findings of Verena et al. [32]. Generally, character-
ization of glycoprotein structure is a complex process with several
steps. Therefore, further studies are required to determine the exact
structure of UPGP.
Fig. 8. Agarose gel electrophoretic separation of damaged DNA and the protective Regarding antioxidant activity, it has been found that UPGP
effect of UPGP before and after in vitro digestion. The numbered lane represent 1, ␭ showed promising antioxidant activity as detected by DPPH, ABTS
DNA alone; 2, DNA plus Cu (II)-ascorbic acid; 3, DNA plus Cu (II)-ascorbic acid and
and reducing power, which is comparable with that of the well-
undigested UPGP; 4, DNA plus Cu (II)-ascorbic acid and UPGP after saliva phase; 5,
DNA plus Cu (II)-ascorbic acid and UPGP after gastric phase; 6, DNA plus Cu (II)- known antioxidant, ascorbic acid, consistent with the findings
ascorbic acid and UPGP after duodenal phase; S, supercoiled DNA strands; N, nicked of what they have reported before [11,12,33]. In contrast, previ-
DNA strands. ous studies involving the evaluation of antioxidant activities by
methanol, ethanol, sulfated or desulfated polysaccharide, and enzy-
during the measurement of reducing power before and after in vitro matic extracts of U. pinnatifida showed low antioxidant activity
simulated digestion model (Fig. 7). The results of this assay indicate compared to the glycoprotein in this study [34–36]. Similarly, we
that the reducing power of UPGP was similar to that of ascorbic acid. also found that UPGP can successfully scavenge NO via inhibiting
nitrite formation by directly competing with oxygen. This result is
3.4. DNA-protective activity consistent with the findings reported by Kim et al. [12]. In this study,
␭ DNA was damaged by hydroxyl radical generation. The addition
Glycoprotein have been reported to protect DNA from damage of UPGP to the reaction mixture showed protective effect against
by neutralizing charged radicals and through the induction of con- the damage of native DNA (Fig. 8) which is in agreement with the
formational changes in DNA. To validate these effects, we evaluated findings of Kim et al. [12] and Albishi et al. [37]. The DNA-protective
the protective effect of UPGP. To this end, ␭ DNA was incubated activity of UPGP is a reflection of its positive effects against many
in the presence of Cu (II) sulfate and ascorbic acid with or with- diseases in human body.
out UPGP. The undigested glycoprotein-treated sample showed a Concerning determination of the bioavailability of UPGP, we
clear band of ␭ DNA, whereas the untreated ␭ DNA showed only used an in vitro digestion model because it gives an indication of
a smear (Fig. 8). There was little variation in its DNA-protective the bioavailability of different biofunctional activities in a biological
activity after in vitro digestion. The glycoprotein after gastric phase system, since the model is designed to simulate in vivo digestion.
did not protect DNA damage whereas UPGP after saliva and duode- During the simulated saliva stage, there was no significant varia-
nal phase showed DNA protecting activity against oxidative stress tion in antioxidant and DNA-protective activity. However, after in
(Fig. 7). vitro digestion, the overall antioxidant activities of UPGP tended to
be lower during the gastric phase than in the undigested and duo-
4. Discussion denal phases. Similarly, in the case of DPPH-scavenging activity,
UPGP showed significant variation in activity at different stages of
ROS, which are constantly generated in living systems, cause in vitro digestion. This significant variation in scavenging activity
various diseases, including degenerative diseases and cell lysis [2]. may have been due to the variation in pH because the viability
To solve these problems, it is necessary to search for natural rather of the DPPH method is dependent upon pH [38] (Fig. 3). How-
than chemical antioxidants because of the possibility of adverse ever, ABTS scavenging activity was almost unchanged following
effects of the latter on health. Over the past few decades, many in vitro digestion (Fig. 4). This result is consistent with the findings
glycoprotein have been isolated from fungi, yeasts, algae, lichens, of Han et al. [34], who reported that the ABTS assay gives consistent
and plants and their biological activities have been evaluated in results over a wide pH range. After the gastric phase, UPGP showed
terms of anticancer, antioxidant, and immunomodulatory effects slightly reduced ABTS activities compared with the duodenal phase.
[11,12,29]. However, there have been no studies to date regarding The observed differences between the gastric and duodenal phases
the biofunctional activities of glycoprotein from U. pinnatifida. Thus, of digestion are in agreement with previous reports [39,40]. Kim
we isolated and purified glycoprotein from U. pinnatifida to evalu- et al. [12] found that glycoprotein from S. japonica retained 80%
ate the biopharmacological activities and its bioaccessibility under DPPH scavenging activity at pH 4–6 and that the activity was lower
simulated in vitro digestion model. As shown in Fig. 1, purified UPGP beyond this pH range. The results of the present study strongly sup-
migrated as a single band with an approximate molecular weight of port these findings and suggest that the chemical bond between
10 kDa on SDS-PAGE, which consists of carbohydrate (42.53%) and glycoprotein and protons may be unstable under weak acidic, neu-
protein (57.47%). After electrophoresis, the gels were stained with tral, and weak alkaline conditions.
Schiff’s reagent which confirm it as glycoprotein. This is consistent Measuring the reducing power of UPGP after in vitro digestion
with the results of Kim et al. [12], who purified a glycoprotein of is important for evaluating antioxidant power. The reducing power
similar molecular weight from S. japonica. of UPGP was measured using the methods of Oyaizu [26] and FRAP
In order to characterize the purified UPGP, we performed several assay. Using the FRAP assay, we demonstrated that the reducing
analyses including determination of amino acid and monosaccha- power of UPGP was significantly lower during the gastric and duo-
ride compositions, FT-IR spectroscopy and glycosylation analysis. denal phases compared to undigested UPGP. Wootton-Beard et al.
272 S.M. Rafiquzzaman et al. / International Journal of Biological Macromolecules 62 (2013) 265–272

[39] reported that the FRAP values of tea samples were signifi- acknowledge to the Korea Basic Science Institute for the amino acid
cantly decreased following in vitro digestion. In contrast, Peter et al. analysis.
[41] found that FRAP values were increased after the gastric phase
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