Food Research International 44 (2011) 33–38

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Binding of proanthocyanidins to soybean (Glycine max) seed ferritin inhibiting

protein degradation by protease in vitro
Jianjun Deng a, Meiliang Li a, Tuo Zhang a, Bin Chen b,⁎, Xiaojing Leng a, Guanghua Zhao a,⁎
a
CAU & ACC Joint-Laboratory of Space Food, College of Food Science and Nutritional Engineering, Key Laboratory of Functional Dairy, Ministry of Education, China Agricultural University,
Beijing 100083, PR China
b
State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing 100094, PR China

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

Article history: Biofortification with phytoferritin is considered as a promising approach to the eradication of iron deficiency
Received 8 August 2010 anemia. However, phytoferritin is not stable enough to be against degradation by protease(s) in the
Accepted 14 November 2010 gastrointestinal tract, thereby leading to its low bioaccessibility. Fortunately, binding of proanthocyanidins
(PAs) to protein offers the opportunity to prevent phytoferritin from degradation by the protease(s). To test
Keywords: this idea, the interaction of PAs from grape seeds with soybean seed ferritin (SSF) was studied using a
Phytoferritin
combination of fluorescence, CD spectra, stopped-flow light scattering (SLS), and dynamic light scattering
Proanthocyanidins
Association
(DLS). Results showed that PAs can indeed bind to SSF in a dose-dependent manner. Consequently, such
Digestion binding can significantly inhibit the degradation of SSF by the protease(s) in simulated gastric fluid (SGF) at
Light scattering pH 4.0 when the mass ratio of PAs to SSF is more than 1:1. Similarly, the stability of SSF in simulated intestinal
fluid (SIF) was also increased upon treatment with PAs. These findings raise the possibility that the
bioaccessibility of phytoferritin to the gastrointestinal tract was improved in the presence of proanthocyanidins.
© 2010 Elsevier Ltd. All rights reserved.

1. Introduction alternated by these dietary components that are capable of capturing

Iron deficiency anemia (IDA) is a major public health problem in
the world, affecting more than 2 billion people (WHO/FAO 2006).
Women who are pregnant or lactating and young children are the
most affected, especially in the developing countries. The major
consequences of IDA are poor pregnancy outcome, reduced cognitive
and motor development in infants, decreased immune function and
tiredness and reduced work capacity (Beard 2001). Inadequate
dietary intakes and low bioavailability of iron are the major factors
contributing to IDA. Iron supplementation, such as ferrous iron salts
(ferrous sulfate and ferrous gluconate), is considered the most
common strategy to combat IDA currently. However, since these
iron supplements have some toxic and side effects to human body
(Theil 2004), an alternative strategy needs to be developed to solve
the problem of IDA.
Nutritional iron is classified as two types: heme iron of animal
origin and non-heme iron of plant origin. The former is absorbed as
the stable porphyrin complex unaffected by other food components
such as phytates and tannins, whereas the latter is easier to be
⁎ Corresponding authors. Zhao is to be contacted at Tel./fax: + 86 10 62738737.
E-mail addresses: chenb12@yahoo.com.cn (B. Chen), gzhao1000@yahoo.com
(G. Zhao).
iron from the non-heme iron in plant foods and form insoluble
compounds in the intestinal lumen, resulting in inhibition of iron
absorption (Theil 2004). However, phytoferritin iron, as a novel and
alternative dietary iron source, is masked by a protein coat, so it is less
sensitive to chelators (phytates and tannis) existing in the diet,
representing an alternative strategy to the eradication of global iron
deficiency in the 21st century (Beard, Burton, and Theil 1996; Murray-
Kolb, Welch, Theil, and Beard 2003).
Phytoferritin is a broad superfamily of iron storage protein, and
plays a crucial role in the germination and early growth of seedlings
(Lobréaux and Briat 1991). The three-dimensional structure is very
well conserved throughout the animal, the plant, and microbial
kingdoms. All ferritins have 24 subunits that arranged in 432
symmetry to form a hollow protein shell (outside diameter is
12–13 nm, inside 7–8 nm) that can store up to ~ 4500 Fe3+ atoms in
its inner cavity in the form of an iron oxyhydroxide-phosphate
mineral (Theil 2004; Harrison and Arosio 1996). In legume seeds,
more than 90% of iron in the form of ferritin is stored in legume seeds
(Theil 2004). Therefore, ferritins, especially plant ferritins, have
recently attracted great attention due to their considerable potential
for development of novel and natural iron supplements (Theil 2004;
Lönnerdal 2009). However, ferritin is not stable enough against
degradation by proteases existing in the gastrointestinal tract.
Therefore, it is of special interest to find edible compounds which
have the ability to prevent phytoferritin from degradation by the
protease(s), the focus of this work.

0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2010.11.020
34 J. Deng et al. / Food Research International 44 (2011) 33–38
Proanthocyanidins (PAs), as a class of naturally occurring phenols,
are widely distributed in the plant kingdom. Grape seeds are a
particularly rich source of PAs which take the form of oligomers or
polymers of polyhydroxy flavan-3-ol units, consisting of (+)-catechin,
(−)-epicatechin, (−)-epicatechin-O-gallate, and (−)-epigallocatechin,
linked to each other by C4–C8 or C4–C6 B-type bonds. PAs might have
various biological functions including antioxidant, antiviral, anti-tumor,
anti-inflammatory, antiallergic, vasodilatory actions and insulin-like
activity (Bagchi et al. 2000). It has been established that tannins interact
with proteins, leading to a decrease in protein digestibility (Guyot,
Pellerin, Brillouet, and Cheynier 1996; Horigome, Kumar, and Okamota
1988). In the present study, we investigated the interaction of grape
seed PAs with SSF under physiological conditions. Also, the stability of
SSF in the absence and presence of PAs was estimated in simulated
digestive fluid (SDF).
2. Materials and methods
2.1. Materials
Dried soybean (Glycine max) seeds were obtained from the local
market. Grape seeds (Cabernet sauvignon) were supplied by
Qinhuangdao Hwaseong Winery (Qinhuangdao, China). Sodium
dithionite, 2, 2′-bipyridyl, N′, N′-bis-methyleneacrylamide, pepsin,
trypsin, sodium dodecyl sulfate (SDS), tris(hydroxymethyl)amino-
methane (Tris), TEMED, β-mercaptoethanol, and coomassie brilliant
blue R250 were obtained from Sigma-Aldrich Co. (Beijing, China).
Native and SDS electrophoresis marker were purchased from
GE Healthcare Bio-Sciences AB (Beijing, China). Acetonitrile and
3-(N-morpholino) propanesulfonic acid (Mops) were obtained from
Amersco (Beijing, China). All other reagents used were of analytical
grade or purer.
2.2. Preparation of SSF and PAs
HoloSSF was purified as recently described (Deng et al. 2010).
Apoferritin was prepared using sodium dithionite in 100 mM Mops
buffer (pH 7.9) under anaerobic conditions (Treffry, Hirzmann,
Yewdall, and Harrison 1992) followed by dialysis against the working
buffers indicated in figure captions. Protein concentrations were
determined according to the Lowry method (Lowry, Rosebrough, Farr,
and Randall 1951) with bovine serum albumin (BSA) as standard. PAs
from grape seed were extracted and purified as reported methods (Ku
and Mun 2007). Resulting samples were lyophilized, powdered, and
frozen at − 80 °C until used.
2.3. HPLC–MS analysis of PAs
HPLC–MS analysis was performed by an Alliance 2695 Separations
Module (Waters, Milford, MA, USA) coupled to a Micromass Quattro
Micro triple–quadrupole mass spectrometer (Micromass, Manchester,
UK) with MassLynx software. The lyophilized powders were dissolved
in ultrapure water with a concentration 50 mg/L, and then loaded
onto a reversed phase MP-C18 column (250 mm × 4.6 mm, 5 μm,
Venusil, Agela, USA) which was equilibrated eluted with 2% acetic acid
in water and acetonitrile. Eluting condition was as follows: the
percentage of acetonitrile was raised linearly from 10 to 40% within
60 min and then kept 60% in the following 10 min. HPLC conditions
were as follows: column temperature, 30 °C; flow rate, 0.4 mL/min;
detection wavelength, 210–350 nm; sample size, 20 μL. The MS
instrument was operated in the negative ion mode, scanning from
m/z 200 to 1500 at a scanning rate of 2.0 s cycle. The mass parameters
were as follows: capillary voltage 4.2 kV; cone voltage, 50 V; source
temperature, 110 °C; desolvation temperature, 300 °C; desolvation
gas flow, 600 L N2/h; cone gas flow, 40 L N2/ h.
2.4. Protein gel electrophoresis
Determination of the molecular weight of the native SSF was done
using a 4–20% polyacrylamide gradient gel run at 25 V for 13 h at 4 °C.
The buffer system of the gel was Tris–HCl (0.025 M, pH 8.3). Gels were
stained with coomassie blue R-250. Electrophoresis of proteins under
denaturing conditions was done in 15% SDS-polyacrylamide gel.
Tested protein samples (~20 μg) were suspended in 50 μL water. To
the solution was added 100 μL sample buffer containing 25% glycerol,
12.5% 0.5 M Tris–HCl, pH 6.8, 2% SDS, 1% bromophenol blue, and 5%
β-mercaptoethanol. After the solution was boiled for 10 min, the
supernatant was isolated by centrifugation at 10,000 ×g for 10 min.
2.5. Fluorescence quenching titration
Fluorescence quenching titration experiments were performed
using the Cary Eclipse spectrophotometer (Varian, Polo Alto, USA).
The concentration of the apoSSF was 1 μM in 100 mM NaCl and
100 mM Mops, pH 7.0, at 25 °C. The titrations were conducted by
adding 2 μL increments of PAs (5 mg/mL) to 1 mL apoSSF. After each
PAs addition, fluorescence spectra were scanned. The excitation
wavelength was 280 nm, and the emission wavelength was read at
290–450 nm.
2.6. Circular dichroism (CD)
Protein sample were dissolved in 0.8 mM Mops buffer, pH 7.0. CD
spectra were recorded with a PiStar-180 spectrometer (Applied Photo-
physics, UK), using quartz cuvette of 1 mm optical path length at 25 °C. CD
spectra were scanned at the far UV range (190–260 nm) with 5 replicates
at 50 nm/min, bandwidth=1 nm. The CD data was expressed in terms of
mean residual ellipticity, (θ), in degrees cm2 dmol− 1. The induced
ellipticity was defined as the ellipticity of the PAs-apoSSF mixture,
minus the ellipticity of PAs alone at the same conditions. Percentage of
secondary structure was calculated using the web-based program K2D
(http://www.emblheidelberg.de/~andrade/k2d).
2.7. Stopped-flow light scattering (SLS) and dynamic light scattering
(DLS) experiments
The SLS measurements were performed with a pneumatic drive
Hi-Tech SFA-20 M apparatus in conjunction with a Cary Eclipse
spectrofluorimeter (Varian, Polo Alto, USA) as previously described
(Ivanova, Jowitt, and Lu 2008). To observe the binding of SSF to PAs,
equal 140 μL volumes of PAs (22.4–74.6 μg/mL) and buffered SSF
(224 μg/mL) were mixed at 25 °C in the thermostated sample
compartment containing a 280 μL quartz stopped-flow cuvette. All
quoted concentrations are final concentrations after mixing the two
reagents. The DLS measurements were performed at 25 °C using a
Viscotek model 802 dynamic light-scattering instrument (Viscotek
Europe Ltd.) as recently reported (Li et al. 2009). The hydrodynamic
radius RH was calculated with the regularization histogram method
using the spheres model, from which an apparent molecular mass was
estimated according to a standard curve calibrated from known
globular proteins. OmniSIZE 2.0 software was used to calculate the
size distribution of aggregated protein from the addition of PAs.
2.8. Simulated gastric fluid (SGF) digestion stability assay
SGF was prepared as described in the United States Pharmacopoeia
(Anonymous 1995), which consists of 3.2 mg/mL pepsin in 30 mM
NaCl at pH 2 (100 mM KCl–HCl buffer) or pH 4 (100 mM HAc–NaAc
buffer). Aliquots (200 μL) of SGF were placed in 1.5 mL microcen-
trifuge tubers and incubated in a water bath at 37 °C. 40 μL of SSF
(1 mg/mL in 5 mM Mops buffer and 30 mM NaCl at pH 7.0) in the
presence of PAs (0–10 mg/mL) was added to each of the SGF vials to
 J. Deng et al. / Food Research International 44 (2011) 33–38 35

start the digestion reaction. The ratio of pepsin to SSF was about 16:1 900
(w/w). 1 h later, 60 μL of 1 M NaOH was added to each vial to stop the
a
reaction. Then, SDS-PAGE was run to analyze the stability of SSF. 750

Fluorescence intensity
600
2.9. Simulated intestinal fluid (SIF) digestion stability assay

SIF was prepared as described in the United States Pharmacopoeia
(Anonymous 1995) and consists of 10 mg/mL trypsin in 100 mM
KH2PO4–NaOH buffer, pH 7.5. Aliquots (200 μL) of SIF was placed in
1.5 mL microcentrifuge tubers and incubated in a water bath at 37 °C.
40 μL of SSF (1 mg/mL in 5 mM Mops buffer and 30 mM NaCl at pH
7.0) in the presence of PAs (0–10 mg/mL) was added to each of the SIF
vials to start the digestion reaction. The ratio of trypsin to SSF was
about 50:1 (w/w). 1 h later, the reaction was immediately stopped by
placing the tube in a boiling water bath for 10 min. Then, the samples
were analyzed by SDS-PAGE.
2.10. Statistical analysis
The data were analyzed using the Statistical Analysis System (SAS
9.0) package software for analysis of variance, Duncan's test. All
experiments were carried out in triplicate. The significance was
established at p ≤ 0.05.
3. Results and discussion
3.1. SSF and PA analysis
Nondenaturing gel electrophoresis (native PAGE) revealed puri-
fied SSF as a single complex, estimated to be about 560 kDa
(Supplemental materials, Fig. S1A). SDS-polyacrylamide gel electro-
phoresis analysis indicated that ferritin complex contains two kinds of
subunits (28.0 and 26.5 kDa) (Supplemental materials, Fig. S1B).
Densitometric analysis indicated that the ratio of 28.0 kDa subunit to
the 26.5 kDa one is about 1:1, a result consistent with previous
observation (Masuda, Goto, and Yoshihara 2001; Deng et al. 2010).
PAs separated from grape seeds were analyzed by HPLC and 5
fractions were obtained, the retention time of which were 8.02, 9.37,
10.67, 11.37, and 12.87 min, respectively (Supplemental materials,
Fig. S2A). ESI-MS spectrum of PAs in negative mode showed 5 peaks at
m/z 289, 577, 729, 865, and 1017, respectively (Supplemental
materials, Fig. S2B), which correspond to a catechin/epicatechin
monomer, dimer, dimer gallates, trimers, and trimer gallates as
suggested previously (De Freitas, Glories, Bourgeois, and Vitry 1998).
450
300 e
150
0
300 330 360 390 420 450
Wavelength (nm)
Fig. 1. Fluorescence quenching spectra of apoSSF at different concentrations of PAs. (a) 1 μM
apoSSF; (b)–(d) 1 μM apoSSF in the presence of 10, 20, and 30 μg/mL PAs; (e) 30 μg/mL PAs.
Conditions: λEx =280 nm, λEm =290 nm, slits for excitation and emission of 2.5 and 10 nm,
respectively, [NaCl]=100 mM, [Mops]=100 mM, pH 7.0, 25 °C.
To further obtain information about such interaction in detail, CD
experiments were carried out in far-UV region, and results were
shown in Fig. 2. The secondary structure of apoSSF was determined by
CD spectra. ApoSSF contains about 37% α-helix, 26% β-sheet, and 38%
random coil estimated by using program K2D, and the addition of PAs
does not change the secondary structure of apoSSF to a significant
extent (Fig. 2, inset), a result indicating that the interaction of PAs
with SSF cannot cause the secondary structure changes of the protein.
Previous studies showed that PAs have very high affinities for
proline-rich proteins and polymers while tightly coiled globular
proteins have much lower affinities for PAs (Hagerman and Butler
1981; Hümmer and Schreier, 2008). Additionally, it was also reported
that the interaction of salivary proteins and PAs (De Freitas and
Mateus 2001) resulted in insoluble aggregates by hydrogen bonding
and hydrophobic interactions between the two species (Artz, Bishop,
Keith Dunker, Schanus, and Swanson 1987). Similarly, SSF is also rich
in proline residues, and belonging to globular protein family, what
will take place when meeting PAs? To answer this question, stopped-
flow light scattering (SLS) experiments were undertaken, and
resulting kinetic curves in the absence and presence of PAs were
given in Fig. 3A. It was observed that light scattering intensity of SSF
had almost no change upon mixing with 100 mM Mops buffer (pH
40
apoSSF
Secondary structure fraction (%)

The profile resulted by ESI-MS demonstrated that the separated PAs 35

apoSSF + PAs

consist of at least 5 compounds as previously mentioned. We failed to 30

detect other ingredients such as epicatechin gallate in the present
4x104 25
experimental conditions.
20
3x104
15
3.2. Interaction of SSF with PAs
(deg. cm2 dmol-1)

10
2x104
A combination of fluorescence, CD spectra, SLS, and DLS was used 5

to investigate whether SSF interacts with PAs at pH 7.0. The 1x104 0

α-Helix
fluorescence spectra of apoSSF in the absence and presence of β -sheet random coil

different concentrations of PAs in 100 mM Mops buffer (pH 7.0) are 0

shown in Fig. 1. ApoSSF exhibits a strong fluorescence peak at 324 nm
on excitation at 280 nm, indicating that most of the observed -1x104 apoSSF
fluorescence is contributed by the sole Trp residue at position 243 apoSSF + PAs
of the H-2 subunit (Masuda, Goto, and Yoshihara 2001), whereas PAs -2x104
have no fluorescence under the experimental conditions. With
200 210 220 230 240 250 260
increasing concentration of PAs, the fluorescence intensity of apoSSF
decreases significantly accompanied by a slightly blue shift of Wavelength (nm)
maximum emission wavelength, suggesting that the microenviron- Fig. 2. CD spectra of apoSSF and apoSSF plus PAs. Inset: estimated secondary structural
ment around Trp is changed due to the addition of PAs. These results fractions of apoSSF and apoSSF plus PAs. Conditions: [apoSSF] = 84 μg/mL in buffer
indicated that there is an interaction between PAs and SSF. containing 0.8 mM Mops (pH 7.0), 0 or 1.4 μg/mL PAs, 25 °C.
36 J. Deng et al. / Food Research International 44 (2011) 33–38

7.9 nm
A 1.0
A
400
g
0.5
Scattered light intensity

320 125 nm
f
0.0

Relative Intensity
e
240 68.1 nm
1.0 B
160 d 0.5
275 nm
11.3 nm
80 0.0
c
b 140 nm 757 nm
1.0
0 a C
0 20 40 60 80 100 120 0.5
14.4 nm
Time (s)
0.0
10 100 1000
B RH (nm)
70

60 Fig. 4. Relative scattered light intensity distribution curves for 112 μg/mL SSF (A), 112 μg/mL
SSF in the presence of 1.12 μg/mL (B) or 2.24 μg/mL PAs (C). Conditions: [NaCl]=100 mM,
[Mops]=100 mM, pH 7.0, 25 °C.
50
Initial rate ( ΔI/s)

40
represents a small amount of aggregates. After addition of PAs to
30
SSF, the size distribution is significantly altered toward large
aggregates with increasing PAs concentrations (Fig. 4B and C).
20 These results again indicated that the cross-linking of SSF and PAs
leads to the association of SSF, a finding in accord with the SLS results
10 of Fig. 3.

0
10 15 20 25 30 35 40
PAs (µg/mL)
Fig. 3. (A) Scatter light intensity (SSF aggregation) induced by PAs as a function of time. The
SSF aggregation was initiated by mixing SSF with different concentrations of PAs (a, 0 μg/mL;
b, 11.2 μg/mL; c, 16 μg/mL; d, 18.7 μg/mL; e, 22.4 μg/mL; f, 28 μg/mL; g, 37.3 μg/mL), and
association was followed by the intensity of scattered light at a 90° to the incident beam. The
cure represents an average of three experimental measurements. Conditions: [SSF]=112 μg/
mL in 100 mM Mops (pH 7.0), 25 °C. (B) A plot of initial rate (ν0) as the change in scattered
light intensity per second against the PA concentration.
7.0), whereas a rapid increase in intensity was observed when PAs
was shot against SSF. With increasing PAs concentration, the light
scattering intensity increased remarkably. All kinetic curves are
hyperbolic, and the light scattering intensity reaches maximum
within 20 s when 3:1 of SSF to PAs was used.
These results indicated that PAs induce protein–protein associa-
tion. The initial rate (ν0) of light scattering intensity as a function of
PAs concentration was shown in Fig. 3B. With increasing PAs
concentration, the initial rate increased in a biphasic manner. When
more than 5:1 of SSF to PAs was added, SSF aggregates are formed
more rapidly by a process that appears to be first order in PAs
concentration. Also, with the extension of time, no decrease in the
light scattering intensity was observed (not shown), indicating that
the aggregation reaction of SSF induced by PAs is irreversible, and that
resulting protein aggregates are stable.
To further confirm the previously mentioned observation, SSF
association caused by PAs was also characterized by dynamic light
scattering (DLS). All samples were allowed to stand 10 min at room
temperature before DLS measurements. Two populations with RH
values of 7.9 and 125 nm are evident in the scattered light intensity
distribution curve of the SSF sample (Fig. 4A). The population
centered at 7.9 nm is rich in monomers, comprising about 98% of
the total, whereas the second population having RH = 125 nm
3.3. Effects of PAs on stability of SSF in simulated digestive fluid
The previously mentioned results revealed that PAs of grape seed
do bind to SSF. Previous studies showed that the binding of PAs to a
number of proteins such as salivary proteins (De Freitas and Mateus
2001) and BSA (Artz, Bishop, Keith Dunker, Schanus, and Swanson
1987) inhibited their degradation by proteases. This raises a question
as to whether the binding of PAs to ferritin presented in this work
likewise prevents ferritin from degradation by proteases in vitro. To
answer this question, SSF was mixed with PAs with different mass
ratios (1:10 to 10:1) at pH 2.0 and 4.0, respectively, and then resulting
mixture was digested 1 h in simulated digestive fluid (SDF) at 37 °C.
SDS-PAGE analysis was shown in Fig. 5. SSF is almost completely
degraded into small peptides (which cannot be detected with SDS-
PAGE) by pepsin in SGF at pH 2.0 (Fig. 5A), a result being consistent
with previous finding with pea seed ferritin (Bejjani, Pullakhandam,
Punjal, and Nair 2007). Even the mass ratio of PAs to SSF reaches 10:1;
the protective effect was still very little, suggesting that PAs might
have no ability to protect SSF from degradation by pepsin at pH 2.0, a
condition similar to that of the adult SGF.
In parallel experiments, the stabilities of SSF in the absence and
presence of PAs were also investigated in SGF at pH 4.0 as shown in
Fig. 5B. The two subunits of SSF were degraded into many bands by
pepsin with a control sample (lane 2), while the addition of PAs
markedly inhibited the degradation of SSF to a great extent under the
same experimental conditions. SSF was almost not digested by pepsin
in a PAs to SSF mass ratio of 5:1. The condition of pH 4.0 may reflect
that of the stomach contents of infants and young children (Agunod,
Yamaguchi, Lopez, Luhby, and Glass 1969), so PAs might be used as a
natural food additive to prevent SSF from degradation by pepsin in
their stomach. These results raise the possibility that ferritin could
escape from the stomach in the form of an intact molecule in the
presence of PAs at pH 4.0, consistent with recent findings showing
that whole ferritin molecules can be taken up via a receptor-mediated
 J. Deng et al. / Food Research International 44 (2011) 33–38
Fig. 5. SDS-PAGE analysis of the stability of SSF (1.0 mg/mL) with PAs at different
concentrations (0.1–10 mg/L) in SDF. SGF digestion stability assay at pH 2.0 (A) or pH 4.0
(B): lane M, protein markers and their corresponding molecular masses; lane 1, SSF in the
absence of pepsin; lane 2, SSF + pepsin; lane 3, SSF:PAs (w/w) = 1:10; lanes 4–8, mass
ratio of SSF to PAs from 1:10 to 10:1 in the presence of pepsin; lane 9, pepsin. (C) SIF (pH
7.5) digestion stability assay: lane 1, SSF in the absence of trypsin; lane 2, SSF + trypsin;
lane 3, SSF:PAs (w/w) = 1:10; lanes 4–8, mass ratio of SSF to PAs from 1:10 to 10:1 in the
presence of trypsin; lane 9, trypsin.
endocytotic process in the intestine (Kalgaonkar and Lönnerdal 2009;
San Martin et al. 2008).
Subsequently, the stabilities of SSF with or without PAs were also
estimated by SDS-PAGE in SIF at pH 7.5, and the results were shown in
Fig. 5C. It was observed that SSF was significantly digested by trypsin
in a control sample (lane 2), whereas the presence of PAs inhibited
SSF degradation to different extents depending on dose. Such
inhibitory effect is much significant when the mass ratio of PAs to
SSF is more than 1:1. Thus, it is possible that most of ferritin molecules
keep intact in the presence of PAs in the intestinal, once they escaped
from stomach.
4. Conclusion
The present study demonstrates that grape seed PAs, as a class of
naturally plant polyphenols, can bind to SSF for the first time. The
binding exhibited a pronounced inhibitory effect for SSF degradation
by pepsin at pH 4.0 as well as by trypsin at 7.5. This may represent an
alternative method to improve ferritin iron bioaccessibility in vivo.
37
Acknowledgement
This project was supported by State Key Laboratory of Space
Medicine Fundamentals and Application, China Astronaut Research
and Training Center (SMFA09A012).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10.1016/j.foodres.2010.11.020.
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WHO/FAO (2006). Guidelines on food fortification with micronutrients Geneva. PAs: proanthocyanidins
SDF: simulated digestive fluid
SDS: sodium dodecyl sulfate
Glossary SGF: simulated gastric fluid
SIF: simulated intestinal fluid
BSA: bovine serum albumin SLS: stopped-flow light scattering
CD: circular dichroism SSF: soybean seed ferritin
DLS: dynamic light scattering Tris: tris(hydroxymethyl)aminomethane