Food Sci. Technol. Res.

, ++ (,), ,,,ῌ,-*, ,**/

Isolation and Properties of Antioxidative Peptides from Water-Soluble Royal Jelly Protein
Hydrolysate

Hang GUO+, Yoshiaki KOUZUMA, and Masami YONEKURA,῎

+
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo +2-ῌ2/*3, Japan
,
Laboratory of Food Functionality, School of Agriculture, Ibaraki University, Ami, Ibaraki -**ῌ*-3-, Japan

Received March +., ,**/ ; Accepted May +1, ,**/

Several novel antioxidative peptides from the hydrolysate of water-soluble royal jelly protein (WSRJP)
were isolated, identified, and characterized. WSRJP was extracted from royal jelly and hydrolyzed with
protease N, and the resulting hydrolysate was fractionated by ultrafiltration with cuto# membranes of +
and - kDa. Three fractions (ῌ+ kDa, +ῌ- kDa, and῍- kDa) were separated. The antioxidative activity of
the fractions against the peroxidation of linoleic acid was determined in vitro, using the +,--diethyl-,-
thiobarbituric acid method. Theῌ+kDa fraction, which exhibited the highest antioxidative activity, was
further purified using anion-exchange and reverse phase high performance liquid chromatography. Four-
teen antioxidative peptides were identified using a protein sequencer and electron spray ionization-mass
spectrometry. Among these, four dipeptides (Phe-Asp, Trp-Val, Leu-Trp, and Trp-Leu) were revealed to
have potent antioxidative activity against lipid peroxidation. Moreover, three of these antioxidative
dipeptides (Phe-Asp, Trp-Val, and Leu-Trp) were found to protect against oxidative stress-induced cell
death in human cultured cells.

Keywords : antioxidative peptide, royal jelly protein, hydrolysate, oxidative stress, human cultured cell

Introduction
Royal jelly (RJ) from the honeybee, Apis mellifera L., is
rich in protein (+,ῌ+/῍), sugars (+*ῌ+0῍), lipids (-ῌ0῍),
free amino acids, minerals, and vitamins, and has tradi-
tionally been used as a health food (Howe et al., +32/). RJ
has been reported to show antitumor activity (Townsend
et al., +3/3), vasodilative and hypotensive activities
(Shinoda et al., +312 ; Matsui et al., ,**,), antibacterial ac-
tivity (Fujiwara et al., +33*), anti-inflammatory activity
(Tamura et al., +32/ ; Fujii et al., +33*), and proliferation
activity in human monocytes (Watanabe et al., +330 ; +332)
and rat hepatocytes (Fujii et al., +330). RJ contains five
major proteins belonging to a protein family designated
MRJPs (major royal jelly proteins ; Schmitzova et al.,
+332). The family consists of five main members (MRJP+,
MRJP,, MRJP-, MRJP., MRJP/), which have been chara-
cterized by the cloning and sequencing of their respective
cDNAs. However, to date, the biological function of MRJPs
remains unknown.
People use health foods for a variety of reasons, one of
which is the alleviation of oxidative stress-induced
damage. It has been demonstrated that oxidative stress
can cause cancers and many lifestyle-related diseases,
such as arterial sclerosis, high blood pressure, myocardial
infarction, cerebral apoplexy, dementia, diabetes, and cat-
aracts (Joyce, +321). It has been suggested that some
dietary antioxidants, including vitamin E and vitamin C,
E-mail : yonekura@mx.ibaraki.ac.jp
may protect DNA, protein, and cell membranes from ox-
idative stress-induced damage by reactive oxygen species
(ROS), which include the superoxide anion (O,ῌ), hydro-
gen peroxide (H,O,), peroxyl radicals (ROOῌ), and the
hydroxyl radical (ῌOH) (Sikka et al., +33/). In recent
years researchers have been focusing on antioxidants
obtained from food sources, which include vitamins, poly-
phenols and peptides. A recent study by Inoue et al.
(,**-) reported that dietary RJ may increase the average
life span of C-H/HeJ mice, possibly through the mecha-
nism of reduced oxidative damage.
Bioactive peptides may play important roles as antiox-
idants because dietary proteins are absorbed by the intes-
tine as peptides and amino acids (Kitts and Weiler, ,**- ;
Meisel and FitzGerald, ,**-). Several antioxidative pep-
tides have been found in the enzymatic hydrolysate of
soybean protein (Chen et al., +33/ ; +330 ; +332), egg albu-
min (Tsuge et al., +33+), casein (Suetsuna et al., ,***), and
other proteins (Suetsuna, ,*** ; Jao and Ko, ,**, ; Kudoh
et al., ,**+ ; Kim et al., ,**+). A more recent study has
demonstrated that RJ protein shows high antioxidative
activity (Nagai and Inoue, ,**.). However, it is still not
clear whether antioxidative peptides can be obtained
from RJ protein.
To investigate whether RJ contains bioactive peptides
with antioxidative roles, we carried out the present study,
which resulted in the identification and isolation of anti-
oxidative peptides from water-soluble royal jelly protein
(WSRJP) hydrolysates. Using an in vitro system, we also
Antioxidative Peptides from Royal Jelly Protein
investigated whether these antioxidative peptides can
protect cultured human cells against oxidative stress-
induced cell death.
Materials and Methods
Reagents Protease N and S, papain W-.*, and brome-
lain F were obtained from Amano Enzyme Company
(Nagoya, Japan). Trypsin, pepsin, +,--diethyl-,-thiobarbi-
turic acid (DETBA), and tert-butyl hydroperoxide (t-BOOH)
were purchased from Sigma-Aldrich Fine Chemicals (St.
Louis, MO, USA). Fetal bovine serum (FBS) was from
Nichirei (Tokyo, Japan), and penicillin and trypan blue
were from GIBCO (Grand Island, NY, USA). Four syn-
thetic dipeptides were purchased from Kokusan Chemical
Company (Tokyo, Japan). All other reagents were obtained
from Wako Pure Chemical Industries (Osaka, Japan).
Royal jelly was obtained from API Company (Nagoya,
Japan).
Preparation of water-soluble royal jelly protein (WSRJP)
Royal jelly was lyophilized and extracted twice with 1*ῌ
(v/v) ethanol. The precipitate was lyophilized for +0
hours, and designated as crude RJ protein. Fifty grams
of crude RJ protein was added to + L of distilled water and
neutralized with sodium hydroxide with gentle stirring
for + h. The extracts were centrifuged at .*,***ῑg for -*
min at .῕, and the supernatants were pooled. Finally,
the solution was dialyzed against distilled water for ,. h,
and then lyophilized.
Enzymatic hydrolysis To obtain peptides, WSRJP
was dissolved in *.+ M phosphate bu#er (pH 1.*) and the
mixture was adjusted to the optimal pH of six di#erent
types of proteases. Enzymatic hydrolyses were performed
at pH 1.* and //῕ for protease N, at pH 2.* and 1*῕ for
protease S, at pH 2.* and ./῕ for papain W-.*, at pH 3.*
and 0/῕ for bromelain F, at pH ,.* and -1῕ for pepsin,
and at pH 1.2 and -1῕ for trypsin. Proteolytic reactions
were carried out for ,. h by adding a protease at a concen-
tration of *.*-ῌ (w/w). Samples taken at ,. h from each
proteolytic mixture were immediately heated in a boiling
water bath for / min, and centrifuged at +2,/**ῑg in a
microcentrifuge for ,* min. The supernatant was lyophi-
lized for use in antioxidative activity assays. The degree
of hydrolysis was measured using the o-phthaldialdehyde
method, by measuring the absorbance at -.* nm (Church
et al., +32-).
Measurement of antioxidative activity The antioxida-
tive activity of WSRJP hydrolysates in inhibiting per-
oxidation of linoleic acid was determined according to the
DETBA method (Suda et al., +33.). For oxidation, peptide
samples (*.+,/ mg) were dissolved in + ml of 2*ῌ (v/v)
ethanol. An aliquot (,* ml) of each sample was mixed
with ,* ml of linoleic acid (*., mg/ml in +**ῌ ethanol), and
heated at 2*῕ for 0* min. After the mixture was cooled
in an ice bath, *., ml of ,* mM butylated hydroxytoluene
and *.0 ml of ,.01ῌ (w/v) sodium dodecyl sulfate was
added to the reaction mixture. After mixing, -., ml of
+,./ mM DETBA in sodium phosphate bu#er (*.+,/ M, pH
-.*, warmed to /*῕) was added, followed by mixing and
heating at 3/῕ for +/ min. The mixture was cooled in an
223
ice bath, and . ml of ethyl acetate was added to each tube,
followed by vortexing and centrifugation at 1/*ῑg for +*
min. The supernatant was extracted for the next step.
A control sample containing linoleic acid and other addi-
tives without antioxidants, representing +**ῌ lipid per-
oxidation, was also prepared. The blanks were prepared
as described above, but without linoleic acid. The fluore-
scence intensities of the samples were measured against
their blanks at an excitation wavelength of /+/ nm and an
emission wavelength of /// nm in a spectrophotofluoro-
meter. Antioxidative activity was expressed as an inhi-
bition percentage of lipid peroxidation using the follow-
ing equation :
Inhibition percentage of lipid peroxidationῒ῎+ῐῌAsampleῐ
Ablank῍ῌῌAcontrolῐAblank῍῏ῑ+** ῌA : absorbance῍
All samples and the reference substance were assayed
at least in triplicate, and the results were averaged. An
inhibition percentage versus concentration curve was
drawn, and the concentration of sample required for /*ῌ
inhibition was determined by linear interpolation and
expressed as the IC/* value.
Cell viability assay Human monocytic leukemia cell
line U3-1 was cultured in RPMI +0.* medium supple-
mented with +*ῌ FBS, penicillin (+** units/ml) and strep-
tomycin (+** ng/ml) at -1῕ in a humidified atmosphere
containing /ῌ CO, and 3/ῌ air. Cells were plated into a
30-well microplate at a concentration of .ῑ+*/ cells/ml,
followed by treatment with or without a peptide sample
for ,. h. In order to induce oxidative stress, after ,. h of
culture, the U3-1 cells were incubated with or without /**
mM t-BOOH at -1῕ for , h. To test the e#ect of the
sample on t-BOOH-induced oxidative stress, cell viability
was assayed by the trypan blue exclusion method.
Isolation of antioxidative peptides The protease N
hydrolysate (,* ml) was fractionated by ultrafiltration,
using membranes with two di#erent molecular weight
cut-o#s of + and - kDa (Amicon, Cambridge, MA, USA).
Three fractions (ΐ+ kDa, +ῌ- kDa,῔- kDa) were separated.
The antioxidative activities of the three fractions were
determined by the DETBA method.
Theΐ+ kDa fraction, which exhibited the highest anti-
oxidative activity, was further purified using anion-ex-
change high performance liquid chromatography (HPLC).
Two grams dry weight of theΐ+ kDa fraction was dis-
solved in + ml of ,* mM Tris-HCl bu#er (pH 2.*), and was
applied to a TSKgel DEAE-/PW column (column size 1./ῑ
1/ mm, Tosoh, Tokyo, Japan) that had been equilibrated
with the same bu#er. The peptide fraction was eluted in
a linear gradient of NaCl from * to *.,/ M in ,* mM
Tris-HCl bu#er over /* min, and the eluted peaks were
detected by UV absorbance at ,2* nm ; the flow rate was
*.1 ml/min.
Fraction III was collected and lyophilized for reverse-
phase (RP)-HPLC. Chromatography on the RP-HPLC system
was carried out at ambient room temperature (,*ῌ,/῕).
Fraction III was separated by RP-HPLC on a Wakosil II /
C-+2 column (column size ..0ῑ,/* mm, Wako Pure Chem-
ical Industries). It was equilibrated with *.+ῌ (v/v) tri-
fluoroacetic acid (TFA) in water (solvent A), and a linear
224
gradient was developed using solvent A and solvent B
(*.+ῌ (v/v) TFA in 2*ῌ (v/v) acetonitrile). For the sepa-
ration of peptides, elution was achieved with a linear
gradient from solvent A to .2ῌ (v/v) solvent B for 0* min
at a flow rate of *.2 ml/min, and the eluted peaks were
detected by UV absorbance at ,+0 nm. Each of the pep-
tides was manually collected, and evaporated to dryness
in a centrifugal concentrator (CC-+2+, Tomy Seiko Compa-
ny, Tokyo, Japan) under vacuum. The antioxidative ac-
tivities of the purified peptides were determined by the
DETBA method.
Amino acid sequence analysis The antioxidative pep-
tides were subjected to N-terminal amino acid sequencing
on an Applied Biosystems .3. protein sequencer (Perkin
Elmer, Foster City, USA). Edman degradation was per-
formed according to the standard program supplied by
Applied Biosystems.
Electron spray ionization-mass spectrometry (ESI-MS)
The masses of the antioxidant peptides collected by RP-
HPLC were determined by atmospheric pressure ionization-
electrospray ionization (API-ESI). The original method
was performed on a Perkin-Elmer SCIEX API -** triple-
quadrupole mass spectrometer. API-ESI was performed
in positive mode, and mass spectroscopy was carried out
in full scanning mode. Operating conditions for the API
-** were optimized by flow injections of peptides at a flow
rate of *.- ml/min, and were determined as follows :
nebulizing gas pressure, 0 psi ; curtain gas, 2 psi. Multi-
ple reaction monitoring experiments in positive ioniza-
tion mode were performed using a dwell time of +.* ms by
scanning the quadrupole in the +**ῌ+,** Da mass range at
Fig. +. Antioxidative activitites of the hydrolysates of water-soluble royal jelly protein (WSRJP) digested with six
di#erent proteases for ,. h. Antioxidative activity of sample (0,./ mg/ml) was evaluated by the +,--diethyl-,-thiobarbituric
acid method. Control, undigested WSRJP. Each column with vertical bar represents meanῌSD of three determinations.
** represents significant di#erence from the control at p῍*.*+.
H. GUO et al.
a scan rate of +.+ s/scan for ,* min.
Database search for peptide identification The SWISS-
PROT protein database was searched with the sequence
tag information to find the precursor for the antioxidative
peptides.
Statistical analysis All data are expressed as meanῌ
standard deviation. Di#erences in mean values among
the experimental groups were analyzed by Student’s
t-test. Di#erences were considered statistically signifi-
cant at p῍*.*/, p῍*.*+, and p῍*.**+.
Results and Discussion
Antioxidative activity of WSRJP hydrolysates WSRJP
was hydrolyzed with six di#erent proteases for ,. h, and
the e#ects of the resulting samples on lipid peroxidation
are shown in Fig. +. Compared with WSRJP, the WSRJP
hydrolysates exhibited a remarkable increase in inhibi-
tion of lipid peroxidation. The WSRJP hydrolysate pro-
duced by protease N, which inhibited lipid peroxidation
by 20ῌ, had the strongest activity. The trypsin and
bromelain F hydrolysates had strong inhibitory e#ects on
lipid peroxidation ; the hydrolysates of protease S, papain
W-.*, and pepsin showed a moderate level of antioxida-
tive activity (.*ῌ/*ῌ). In all cases, antioxidative activity
was increased with increasing hydrolysate concentration
(data not shown). The IC/* values of the six enzymatic
hydrolysates were as follows : protease N, +. mg/ml ; tryp-
sin, ,* mg/ml ; bromelain F, 1+ mg/ml ; protease S, 23 mg/ml ;
papain W-.*, ++2 mg/ml ; and pepsin, ,,3 g/ml.
The WSRJP hydrolyzed with protease N showed the
highest degree of hydrolysis (Fig. ,), while trypsin, brome-
Antioxidative Peptides from Royal Jelly Protein
Fig. ,. Degree of hydrolysis of WSRJP hydrolysates digested with six di#erent proteases for ,. h. Absorbance at -.* nm
by the o-phthaldialdehyde method represents the degree of hydrolysis of WSRJP hydrolysates. Control, undigested
WSRJP. Each column with vertical bar represents meanῌSD of three determinations. *** represents significant
di#erence from the control at p῍*.**+.
lain F, protease S, papain W-.*, and pepsin exhibited low
degrees of hydrolysis. It can be seen, therefore, there is
no significant correlation between the inhibition rate of
lipid peroxidation and the degree of hydrolysis ; the anti-
oxidative activity of hydrolysates may depend on their
peptide composition and their structure. Similar results
for the antioxidative activity of soybean protein hydro-
lysates have been reported by Chen et al. (+330). This
study focused on the isolation and identification of anti-
oxidative peptides from the protease N hydrolysate of
WSRJP, which had the highest antioxidative activity and
degree of hydrolysis among the enzymatic hydrolysates.
Isolation and structural analysis of antioxidative pep-
tides Because the protease N hydrolysate of WSRJP ex-
hibited the strongest antioxidative activity, the anti-
oxidative peptides were isolated from this hydrolysate.
The hydrolysate was first filtered with ultrafiltration
membranes of molecular weight cut-o#s + kDa and - kDa,
and the lipid peroxidation inhibition ability of each fi-
ltrate was measured by the DETBA method (Fig. -). It
was found that the fraction of molecular weight below +
kDa (F+) exhibited the same high level of antioxidative
activity as the protease N hydrolysate.
This fraction (ῌ+ kDa) was further purified using anion-
exchange chromatography followed by separation into
six fractions (Fig. .). The antioxidative activities of the
six fractions could not be measured because the fractions
contained sodium chloride, which could disturb lipid
peroxidation. Therefore, one major fraction (fraction III)
of the six was further separated by RP-HPLC. As shown
in Fig. /, fraction III revealed a total of twenty cleanly
separated peaks. In order to measure antioxidative ac-
tivity, each fraction was separately collected through
225
repeated chromatography.
The antioxidative activities of peptides isolated by RP-
HPLC were measured using an aliquot of the RP-HPLC
fraction without quantification. As shown in Table +,
fourteen peptides showed antioxidative activities higher
than -*ῌ. From the amino acid sequences of these four-
teen antioxidative peptides, analyzed by Edman sequenc-
ing, and their masses as determined by ESI-MS, their
structures were determined (Table +). Schmitzova et al.
(+332) reported the primary structures of five MRJPs based
on cDNA clone sequences. Using the SWISS-PROT data-
base, it was found that the fourteen antioxidative pep-
tides isolated from WSRJP hydrolysate were derived from
these MRJPs (Table +).
In general, dietary proteins are first digested by various
proteinases in the gastrointestinal tract to produce
oligopeptides and di- and tripeptides, some of which may
have biological activity, and then absorbed in the intesti-
nal epithelium. It has been reported that dipeptides and
tripeptides are actively transported via a specific peptide
transporter in the intestinal epithelial cells (Shimizu, ,**.).
Therefore, this study focused on functional dipeptides.
Among the fourteen peptides isolated from WSRJP
hydrolysate, four chemically synthesized dipeptides (Phe-
Asp, Trp-Val, Leu-Trp, Trp-Leu) were used in experiments
in which their antioxidative activities were compared
with those of other antioxidants such as vitamins C and E
and carnosine (b-Ala-His). The results showed that these
four dipeptides exhibit antioxidative activity of more
than 2*ῌ (Fig. 0).
Antioxidative peptides have been found in the enzymatic
hydrolysate of soybean protein (Chen et al., +33/ ; +330 ;
+332), egg albumin (Tsuge et al., +33+), casein (Suetsuna et
226 H. GUO et al.

Fig. -. Antioxidative activities of the protease N hydrolysate of WSRJP and its fractions separated by ultrafiltration.
Antioxidative activity of sample (0,./ mg/ml) was evaluated by the +,--diethyl-,-thiobarbituric acid method. Control,
undigested WSRJP ; Hydrolysate, protease N hydrolysate of WSRJP ; F+, MW῍+ kDa ; F,, MW + kDa - - kDa ; F-, MW῎-
kDa. Each column with vertical bar represents meanῌSD of three determinations. *** represents significant di#erence
from the control at p῍*.**+.

Fig. .. Elution profile of the MW῍+ kDa fraction from the protease N hydrolysate of WSRJP separated by
anion-exchange high-performance liquid chromatography on a TSKgel DEAE-/PW column. , Absorbance at ,2* nm ; ,
NaCl concentration (ῌ).
Antioxidative Peptides from Royal Jelly Protein
Fig. /. Elution profile of fraction III (Fig. .) from the protease N hydrolysate of WSRJP separated by reverse phase
high-performance liquid chromatography on a Wakosil II /C+2 column.
concentration (ῌ).
al., ,***), and other proteins (Suetsuna, ,*** ; Jao and Ko,
,**, ; Kudoh et al., ,**+ ; Kim et al., ,**+). The fourteen
antioxidative peptides we isolated from WSRJP hydro-
lysate are all novel peptides. Among these, four dipeptides
(Leu-Trp, Phe-Asp, Trp-Leu, Trp-Val) were found to have
potent antioxidative activity on lipid peroxidation in
vitro. Dipeptides containing Leu, Trp, Val or Ile residues
at the N-terminal and Met, His, Tyr or Trp residues in the
sequence have been shown to have antioxidative capacity
(Yamaguchi et al., +31/ ; +310 ; Kawashima et al., +313).
We therefore reasoned that the Trp and Leu residues of
Leu-Trp, Trp-Leu, and Trp-Val might be associated with
antioxidative activity. However, the reason for the high
antioxidative activity of Phe-Asp is not clear, because
phenylalanine and aspartic acid do not exhibit strong
antioxidative activity against peroxidation of linoleic
acid (Yamaguchi et al., +310).
Cell survival assay Carnosine, a dipeptide found in
skeletal muscles, is a known antioxidant and has been
found to suppress the secretion of inflammatory cytokines
by intestinal epithelial cells exposed to oxidative stress
(Shimizu, ,**. ; Son et al., ,**.). We therefore decided to
examine the e#ects of the four new synthetic antioxida-
tive dipeptides and carnosine on t-BOOH-induced oxida-
tive stress in U3-1 cells. The trypan blue dye exclusion
assay was used to measure cell viability. As shown in
Fig. 1, cell viability was significantly decreased in the
control (-3.,ῌ) and in the sample treated with carnosine
(.+ῌ) after t-BOOH treatment. In contrast, when Phe-
Asp, Trp-Val, and Leu-Trp were added to the medium, cell
viability was significantly increased to 1*.1ῌ, /-.2ῌ, and
./.,ῌ, respectively. Trp-Leu had no e#ect on rates of
cell death. These data suggest that Phe-Asp, Trp-Val,
and Leu-Trp isolated from the protease N hydrolysate of
WSRJP can protect U3-1 cells against t-BOOH-induced
227
, Absorbance at ,+0 nm ; , acetonitrile
oxidative stress in vitro. Phe-Asp was shown to inhibit
oxidative stress-induced cell death, but in the same con-
centrations neither phenylalanine nor aspartic acid had
the same e#ect (Fig. 2). Similar results were obtained for
Trp-Val and Leu-Trp (data not shown). It is possible that
the antioxidative mechanisms of Phe-Asp, Trp-Val, and
Leu-Trp in cells are di#erent from those of their constitu-
ent amino acids.
It has been reported that t-BOOH promotes peroxida-
tion of cell membrane lipids and elevation of cytosolic
calcium ion concentration, and is responsible for mitochon-
drial damage caused by tert-butoxyl radicals (Guidarelli
et al., +330 ; +331). Several peptides have been shown to
have high radical scavenging activities (Chen et al., +332 ;
Saito et al., ,**-). It is possible that the antioxidative
mechanism of the three dipeptides Phe-Asp, Trp-Val, and
Leu-Trp in oxidant-induced cell death involves scaveng-
ing of t-BOOH-derived radicals.
In conclusion, this study demonstrates that the prote-
ase N hydrolysate of WSRJP has strong antioxidative
activity on the peroxidation of linoleic acid. Fourteen
novel antioxidative peptides from WSRJP hydrolysate
were newly identified. Furthermore, three of these anti-
oxidative dipeptides (Phe-Asp, Trp-Val, Leu-Trp) were
found to protect cultured human cells against oxidative
stress-induced cell death. It is thought that these anti-
oxidative peptides derived from royal jelly proteins may
prevent cell damage induced by oxidative stress in vivo
and therefore reduce the risk of lifestyle-related diseases
caused by reactive oxygen radicals. However, further
study is needed to clarify the antioxidative-stress mecha-
nism of these peptides in vivo.
228 H. GUO et al.

Table +. Identification of antioxidative peptides isolated from the protease N hydrolysate of water-soluble
royal jelly protein by reverse phase high performance liquid chromatography.
Antioxidative Peptides from Royal Jelly Protein 229

Fig. 0. Antioxidative activities of four dipeptides, carnosine, ascorbic acid, and a-tocopherol. The sample concentration
was +** mM. Control, undigested WSRJP (0,./ mg/ml). Each column with vertical bar represents meanῌSD of three
determinations. *** represents significant di#erence from the control at p῍*.**+.

Fig. 1. E#ect of four dipeptides and carnosine on the cell
viability of U3-1 cells treated with t-BOOH. Control, no
addition of sample. Cells (.῍+*/ cells/ml) were incubated
with + mg/ml of sample at -1῏ for ,. h, and then treated
with /** mM t-BOOH at -1῏ for , h. Cell viability was
determined using the trypan blue exclusion method. , t-BOOH at -1῏ for , h. Cell viability was determined
with t-BOOH ; , without t-BOOH. Each column with
vertical bar represents meanῌSD of three determinations.
* and ** represent significant di#erence from the control
treated with t-BOOH at p῎*.*/ and p῎*.*+, respectively.
Fig. 2. E#ect of phenylalanyl-aspartic acid (Phe-Asp) and
its constituent amino acids on the cell viability of U3-1
cells treated with t-BOOH. Control, no addition of sample.
Cells (.῍+*/ cells/ml) were incubated with -./ mM of
sample at -1῏ for ,. h, and then treated with /** mM
using the trypan blue exclusion method. , with t-BOOH ;
, without t-BOOH. Each column with vertical bar
represents meanῌSD of three determinations. *** repre-
sents significant di#erence from the control treated with
t-BOOH at p῎*.**+.
230
References
Chen, H.M., Muramoto, K. and Yamauchi, F. (+33/). Structural
analysis of antioxidative peptides from soybean b-conglycinin.
J. Agric. Food Chem., .-, /1.ῌ/12.
Chen, H.M., Muramoto, K., Yamauchi, F. and Nokihara, K. (+330).
Antioxidant activity of designed peptides based on the anti-
oxidative peptide isolated from digests of a soybean protein. J.
Agric. Food Chem., .., ,0+3ῌ,0,-.
Chen, H.M., Muramoto, K., Yamauchi F., Fujimoto, K. and Noki-
hara,K. (+332). Antioxidative properties of histidine-containing
peptides designed from peptide fragments found in the digests
of a soybean protein. J. Agric. Food Chem., .0, .3ῌ/-.
Church, F.C., Swaisgood, H.E., Porter, D.H. and Catignani, G.C.
(+32-). Spectrophotometric assay using o-phthaldialdehyde for
determination of proteolysis in milk and isolated milk pro-
teins. J. Dairy Sci., 00, +,+3ῌ+,,1.
Fujii, A., Kobayashi, S., Kuboyama, N., Furukawa, Y., Kaneko, Y.,
Ishihama, S., Yamamoto, H. and Tamura, T. (+33*). Augmenta-
tion of wound healing by royal jelly (RJ) in streptozotocin-
diabetic rats. Jpn. J. Pharmacol., /-, --+ῌ--1.
Fujii, M., Yonekura, M., Higuchi, T., Morimitsu, K., Yoshino, I.,
Mukai, S., Aoki, T., Fukunaga, T., Inoue, Y., Sato, M. and
Kanaeda, J. (+330). E#ect of -/* kDa glycoprotein in royal jelly
on primary culture of rat hepatocytes. Food Sci. Technol. Int., ,,
,,-ῌ,,/.
Fujiwara, S., Imai, J., Fujiwara, M., Yaeshima, T., Kawashima, T.
and Kobayashi, K. (+33*). A potent antibacterial protein in
royal jelly. Purification and determination of the primary
structure of royalisin. J. Biol. Chem., ,0/, ++---ῌ++--1.
Guidarelli, A., Brambilla, L., Rota, C., Tomasi, A., Cattabeni, F. and
Cantoni, O. (+330). The respiratory-chain poison antimycin A
promotes the formation of DNA single-strand breaks and
reduces toxicity in U3-1 cells exposed to t-butylhydroperoxide.
Biochem. J., -+1, -1+ῌ-1/.
Guidarelli, A., Cattabeni, F. and Cantoni, O. (+331). Alternative
mechanisms for hydroperoxide-induced DNA single strand
breakage. Free Rad. Res., ,0, /-1ῌ/.1.
Howe, S.R., Dimick, P.S. and Benton, A.W. (+32/). Composition of
freshly harvested and commercial royal jelly. J. Apic. Res., ,.,
/,ῌ0+.
Inoue, S., Koya-Miyata, S., Ushio, S., Iwaki, K., Ikeda, M. and
Kurimoto, M. (,**-). Royal jelly prolongs the life span of C-H/
HeJ mice : correlation with reduced DNA damage. Exp. Gerontol.,
-2, 30/ῌ303.
Jao, C.L. and Ko, W.C. (,**,). +,+-Diphenyl-,-picrylhydrazyl (DPPH)
radical scavenging by protein hydrolyzates from tuna cooking
juice. Fish. Sci., 02, .-*ῌ.-/.
Joyce, D.A. (+321). Oxygen radicals in disease. Adverse Drug Reac-
tion Bull., +,1, .10ῌ.13.
Kawashima, K., Itoh, H., Miyoshi, M. and Chibata, I. (+313). Antiox-
idant properties of branched-chain amino acid derivatives.
Chem. Pharm. Bull., ,1, +3+,ῌ+3+0.
Kim, S.K., Kim, Y.T., Byun, H.G., Nam, K.S., Joo, D.S. and Shahidi,
F. (,**+). Isolation and characterization of antioxidative pep-
tides from gelatin hydrolysate of Alaska pollack skin. J. Agric.
Food Chem., .3, +32.ῌ+323.
Kitts, D.D. and Weiler, K. (,**-). Bioactive proteins and peptides
from food sources. Applications of bioprocesses used in isola-
tion and recovery. Curr. Parm. Des., 3, +-*3ῌ+-,-.
Kudoh, Y., Matsuda, S., Igoshi, K. and Oki, T. (,**+). Antioxidative
peptide from milk fermented with Lactobacillus delbrueckii
subsp. bulgaricus IFO+-3/-. Nippon Shokuhin Kagaku Kogaku
Kaishi, .2, ..ῌ/* (in Japanese).
H. GUO et al.
Matsui, T., Yukiyoshi, A., Doi, S., Sugimoto, H., Yamada, H. and
Matsumoto, K. (,**,). Gastrointestinal enzyme production of
bioactive peptides from royal jelly protein and their anti-
hypertensive ability in SHR. J. Nutr. Biochem., +-, 2*ῌ20.
Meisel, H. and FitzGerald, R. J. (,**-). Biofunctional peptides from
milk proteins : mineral binding and cytomodulatory e#ects.
Curr. Pharm. Des., 3, +,23ῌ+,3/.
Nagai, T. and Inoue, R. (,**.). Preparation and the functional
properties of water extract and alkaline extract of royal jelly.
Food Chem., 2., +2+ῌ+20.
Saito, K., Jin, D.H., Ogawa, T., Muramoto, K., Hatakeyama, E.,
Yasuhara, T. and Nokihara, K. (,**-). Antioxidative properties
of tripeptide libraries prepared by the combinatorial chemis-
try. J. Agric. Food Chem., /+, -002ῌ-01..
Schmitzova, J., Klaudiny, J., Albert, S., Schroder, W., Schreck-
engost, W., Hanes, J., Judova, J. and Simuth, J. (+332). A family
of major royal jelly proteins of the honeybee Apis mellifera L.
CMLS, Cell. Mol. Life Sci., /., +*,*ῌ+*-*.
Shimizu, M. (,**.). Food-derived peptides and intestinal func-
tions. BioFactors, ,+, .-ῌ.1.
Shinoda, M., Nakajin, S., Oikawa, T., Sato, K., Kamogawa, A. and
Akiyama, Y. (+312). Biochemical studies on vasodilative factor
in royal jelly. Yakugaku Zasshi, 32, +-3ῌ+./ (in Japanese).
Sikka, S.C., Rajasekaran, M. and Hellstrom, W. J.G. (+33/). Role of
oxidative stress and antioxidants in male infertility. J. Androl.,
+0, .0.ῌ.02.
Son, D.O., Satsu, H., Kiso, Y. and Shimizu, M. (,**.). Characteriza-
tion of carnosine uptake and its physiological function in
human intestinal epithelial Caco-, cells. BioFactors, ,+, -3/ῌ-32.
Suda, I., Furuta, S. and Nishiba, Y. (+33.). Fluorometric determina-
tion of a +,--diethyl-,-thiobarbituric acid-malondialdehyde adduct
as an index of lipid peroxidation in plant materials. Biosci.
Biotechnol. Biochem., /2, +.ῌ+1.
Suetsuna, K. (,***). Antioxidant peptides from the protease digest
of prawn (Penaeus japonicus) muscle. Mar. Biotechnol., ,, /ῌ+*.
Suetsuna, K., Ukeda, H. and Ochi, H. (,***). Isolation and charac-
terization of free radical scavenging activities peptides derived
from casein. J. Nutr. Biochem., ++, +,2ῌ+-+.
Tamua, T., Fujii, A. and Kuboyama, N. (+32/). Study on muta-
genicity of royal jelly. Honeybee Sci., 0, 1ῌ+, (in Japanese).
Townsend, G.F., Morgan, J.F. and Hazlett, B. (+3/3). Activity of +*-
hydroxydecenoic acid from royal jelly against experimental
leukemia and ascitic tumors. Nature, +2-, +,1*ῌ+,1+.
Tsuge, N., Eikawa, Y., Nomura, Y., Yamamoto, M. and Sugisawa,
K. (+33+). Antioxidative activity of peptides prepared by en-
zymatic hydrolysis of egg-white albumin. Nippon Nogeikagaku
Kaishi, 0/, +0-/ῌ+0.+ (in Japanese).
Watanabe, K., Shinmoto, H., Kobori, M., Tsushida, T., Shinohara,
K., Kanaeda, J. and Yonekura, M. (+330). Growth stimulation
with honey royal jelly DIII protein of human lymphocytic cell
lines in a serum-free medium. Biotechnol. Techniques, +*, 3/3ῌ
30,.
Watanabe, K., Shinmoto, H., Kobori, M., Tsushida, T., Shinohara,
K., Kanaeda, J. and Yonekura, M. (+332). Stimulation of cell
growth in the U-3-1 human myeloid cell line by honey royal
jelly protein. Cytotechnology, ,0, ,-ῌ,1.
Yamaguchi, N., Yokoo, Y. and Fujimaki, M. (+31/). Studies on
antioxidative activities of amino compounds on fats and oils.
Part II. Antioxidative activities of dipeptides and their syn-
ergistic e#ects of tocopherol. Nippon Shokuhin Kogyo Gakkaishi,
,,, .,/ῌ.-* (in Japanese).
Yamaguchi, N. (+310). E#ect of the food components on oxidative
stabilities of fat and oil. Yu Kagaku, ,/, ,.3ῌ,/0 (in Japanese).