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1939

Journal of Food Protection, Vol. 67, No. 9, 2004, Pages 19391944


Copyright Q, International Association for Food Protection

Antioxidant Activity of Peptides Derived from Egg White


Proteins by Enzymatic Hydrolysis
VALOS, M. MIGUEL, B. BARTOLOME,
A. DA AND PEZ-FANDIN
R. LO O*

Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

MS 03-586: Received 19 December 2003/Accepted 21 March 2004

ABSTRACT
This work reports the antioxidant activity of peptides produced by enzymatic hydrolysis of crude egg white with pepsin.
Four peptides included in the protein sequence of ovalbumin possessed radical scavenging activity higher than that of Trolox.
The hydrolysate of egg white with pepsin for 3 h was previously found to exhibit a strong angiotensin Iconverting enzyme
(ACE) inhibitory activity in vitro. The combined antioxidant and ACE inhibition properties make it a very useful multifunc-
tional preparation for the control of cardiovascular diseases, particularly hypertension. No correlation was found between
antioxidant and ACE inhibitory activities. However, the peptide Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu, which was a strong
ACE inhibitor (50% inhibitory concentration, 4.7 mM) also exhibited a high radical scavenging activity (oxygen radical
absorbance capacity-fluorescein value, 3.8 mmol of Trolox equivalent per mmol of peptide) and delayed the low-density
lipoprotein lipid oxidation induced by Cu21 at a concentration of ;0.16 mg/mg of low-density lipoprotein. Present results
support that antioxidant peptides and amino acids not only act individually, but also cooperatively and synergistically.

Reactive oxygen species and other free radicals pro- mechanism for human blood pressure reduction in these
duce oxidative damage to DNA, proteins, and other mac- studies remains undetermined. The in vitro angiotensin I
romolecules such as lipids. Reactive oxygen species are converting enzyme (ACE) inhibitory activities of intracel-
generated in the mitochondria as part of the cell metabo- lular antioxidant peptides (glutathione, oxidized glutathi-
lism, but they can be specifically produced by phagocytes one, carnosine, homocarnosine, and anserine) have been
and other types of cells as part of a response against ex- demonstrated (15). On the other hand, the octapeptide cap-
ogenous agents. Inasmuch as reactive oxygen species are topril, a well-known antihypertensive drug and a potent
implicated in the etiology of many multifactorial degener- ACE inhibitor, also exhibits antioxidant properties (14).
ative diseases such as diabetes, cancer, cardiovascular dis- ACE is an important drug target in the treatment of cardio-
ease, neurodegenerative disorders, and, in general, aging, vascular diseases, especially hypertension. ACE, a dipep-
interest in the research and use of antioxidants for their tidyl-carboxypeptidase (EC 3.4.15.1) located in different
prevention and treatment has increased (2). In addition to tissues, hydrolyzes angiotensin I into angiotensin II, which
antioxidants from fruits and vegetables, peptides from an- has a vasoconstrictor effect. ACE also inactivates the va-
imal sources such as those enzymatically obtained from sodilator bradykinin that can be involved in the control of
porcine myofibrillar proteins (28), casein (26, 35), egg (16, blood pressure (36). ACE inhibitors have notable effects on
38), whey (37), and fish proteins (31) exhibit in vitro an- oxidative stress and are an essential step in managing es-
tioxidant activity. To elucidate the antioxidative mechanism sential hypertension by improving endothelial dysfunction
of protein hydrolysates, the antioxidative properties of ami-
(4).
no acids, synthetic peptides, or both have also been inves-
It has been reported that certain egg whitederived
tigated (6, 18, 29).
peptides and synthetic analogs can play a role in controlling
Antioxidant deficiency has been implicated in the oc-
the development of hypertension by exerting vasorelaxing
currence of hypertension. Antioxidant-rich diets signifi-
effects (12, 13, 19, 20). In addition, we have shown that
cantly reduced blood pressure in spontaneously hyperten-
the proteolysis of crude egg white with pepsin produces
sive rats (1, 32), probably because of a combination of va-
peptides with in vitro ACE inhibitory properties that derive
sodilator and antioxidant actions (27). In humans, diets rich
in fruits, vegetables, and low-fat dairy foods (3) as well as from the hydrolysis of ovalbumin (21). Reversed-phase
diets supplemented with ascorbic acid (10) were shown to high-performance liquid chromatography, in conjunction
lower blood pressure in hypertensive patients. Although it with tandem mass spectrometry (RP-HPLC-MS/MS) and
was postulated that nitric oxide is implicated and that as- followed by peptide synthesis and confirmation of ACE in-
corbic acid enhances the production of prostaglandin E1, hibitory activity, allowed the identification of very active
which is a platelet antiaggregator and vasodilator (7), the sequences (21). We evaluated the antioxidant activity of the
hydrolysates of egg white proteins, peptide fractions, and
* Author for correspondence. Tel: 1 34 91 5622900; Fax: 1 34 91 synthetic peptide sequences that presented strong ACE in-
5644853; E-mail: rosina@ifi.csic.es. hibitory activity in vitro, with the objective of finding mul-
1940 VALOS ET AL.
DA J. Food Prot., Vol. 67, No. 9

tifunctional ingredients with improved biological activities. TABLE 1. ORAC-FL values of crude egg white protein, a pep-
For that purpose, two methodologies were selected: (i) the sin hydrolysate, its fraction with a molecular mass lower than
oxygen radical absorbance capacity (ORAC) method, and 3,000 Da, and semipreparative RP-HPLC subfraction F1
(ii) the in vitro ion-dependent oxidation of human low-den- through F9; results are the mean of at least three experiments
6 standard deviation
sity lipoprotein (LDL) method. The ORAC method mea-
sures the scavenging activity of a compound against per- ORAC-FL
oxyl radicals, the most abundant radicals in biological sys- valuea
tems, while the LDL method evaluates several mechanisms Egg white protein 0.131 6 0.008
of action of an antioxidant: binding of copper ions, scav- Pepsin hydrolysate 0.381 6 0.017
enging activity, and nonradical decomposition of hydroper- Fraction of ,3,000 Da 2.78 6 0.11
oxides. F8 1.81 6 0.10
F9 1.87 6 0.04
MATERIALS AND METHODS F6 2.18 6 0.05
Chemicals and apparatus. Fluorescein (FL) disodium salt F7 2.33 6 0.18
and cupric sulfate were purchased from Sigma Chemical Co. (St. F5 2.59 6 0.03
Louis, Mo.). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic F4 3.32 6 0.07
acid (Trolox), 2,29-azobis (2-methylpropionamidine) dihydrochlo- F2 3.66 6 0.13
ride (AAPH), amino acid standards (Tyr [Y], Met [M], His [H], F1 3.70 6 0.27
Ser [S], and Leu [L]) were obtained from Aldrich (Milwaukee, F3 3.73 6 0.33
Wis.). Human LDL was purchased from Calbiochem (La Jolla, a
Expressed as micromoles of Trolox equivalent per milligram
Calif.) and the Econo-Pac 10 DG desalting column was obtained
of protein.
from Bio-Rad (Hercules, Calif.). Samples and standards were dis-
solved in 75 mM phosphate buffer (pH 7.4). An FL stock solution
(1.17 mM) was made in the same buffer and stored in dark con-
calculated by subtracting the AUC of the blank. Regression equa-
ditions at 48C for 4 weeks. AAPH and Trolox solutions in 75 mM
tions between net AUC and antioxidant concentration were cal-
phosphate buffer (pH 7.4) were prepared daily.
culated for all of the samples. ORAC-FL values were expressed
A Polarstar Galaxy plate reader (BMG Labtechnologies
as Trolox equivalents by using the standard curve calculated for
GmbH, Offenburg, Germany) with 485-P excitation and 520-P
each assay. Final ORAC-FL values were expressed as micromoles
emission filters was used. The fluorescent plate reader was con-
of Trolox equivalent per milligram of protein for egg white pro-
trolled by Fluostar Galaxy software (Version 4.11-0). Black 96-
tein, egg white protein hydrolysates, and fractions, and as micro-
well microplates (96F untreated microwell, Nunc, Roskilde, Den-
moles of Trolox equivalent per micromole of compound for model
mark) were used. Fluorescence measurement was carried out at
peptides.
378C.
LDL oxidation. For each set of LDL oxidation experiments,
Hydrolysates from egg white proteins, peptide fractions,
EDTA was removed from commercial human LDL using an
and synthetic peptides. The pepsin hydrolysate of crude egg
Econo-Pac 10 DG desalting column. LDL was eluted with 0.01
white, the hydrolysate fraction with a molecular mass lower than
M phosphate-buffered saline (PBS) (0.15 M NaCl), pH 7.4, and
3,000 Da, and nine peptide fractions separated by semipreparative
protein concentration was determined according to the Bradford
RP-HPLC (F1 through F9) were prepared as explained by Miguel
method (5). LDL oxidation was carried out following the method
et al. (21). Synthetic peptides were obtained by conventional
of Esterbauer et al. (11), slightly modified. Salt-free LDL, diluted
Fmoc solid-phase synthesis with a 431 peptide synthesizer (Ap-
berlingen, Germany), and their purity was with 0.01 M PBS (0.15 M NaCl), pH 7.4, to give a final concen-
plied Biosystem Inc., U
tration of 50 mg of protein per liter, was mixed with the aqueous
verified by RP-HPLC-MS/MS. Protein concentration was deter-
hydrolysate or peptide solution (10 ml) in a 1-cm capped cuvette.
mined by the bicinchoninic acid assay (Pierce, Rockford, Ill.) us-
Oxidation was initiated by the addition of 10 ml of freshly pre-
ing bovine serum albumin as the standard.
pared cupric sulfate at a final concentration of 20 mM. The total
ORAC-FL assay. The ORAC-FL assay was based on the volume of the mixture was 600 ml. The kinetics of lipid oxidation
method proposed by Ou et al. (23) and was modified as previously were determined by monitoring the absorbance at 234 nm at 378C,
described (8). Briefly, the final assay mixture (200 ml) contained measured every 5 min for 15 h. From this curve, the lag time,
FL (70 nM) as an oxidizable substrate, AAPH (12 mM) as oxygen the maximum rate of oxidation (Vmax), the maximum quantity of
radical generator, and antioxidant (Trolox [1 to 8 mM] as a stan- dienes formed (CDmax), and the maximum time (tmax) (in minutes)
dard or sample [at different concentrations]). Fluorescence was required to reach CDmax were determined as indicated in Sanchez-
recorded every minute for 80 min. All reaction mixtures were Moreno et al. (30). At least two independent experiments were
prepared in duplicate, and at least three independent runs were carried out for each sample.
performed for each sample. Measurements of fluorescence were
normalized to the curve of the blank (no antioxidant). From the
RESULTS AND DISCUSSION
normalized curves, the area under the fluorescence decay curve Radical scavenging activity. Table 1 shows the
(AUC) was calculated as follows: ORAC-FL values of the hydrolysate of crude egg white

O
i580 with pepsin as well as those of its fractions. A 3-h prote-
AUC 5 1 1 f i/ f 0 olysis of crude egg white with pepsin increased the radical
i51
scavenging activity of crude egg white by approximately
where f0 is the initial fluorescence reading at 0 min, and fi is the threefold. Filtration of the hydrolysate through a 3,000-Da
fluorescence reading at time i. The net AUC of the sample was cut-off membrane further improved the activity (by ;20-
J. Food Prot., Vol. 67, No. 9 ANTIOXIDANT PROPERTIES OF EGG-DERIVED PEPTIDES 1941

TABLE 2. ORAC-FL values of synthetic peptides and amino Arg-Ala-Asp-His-Pro-Phe-Leu and Arg-Ala-Asp-His-Pro-
acids; results are the mean of at least three experiments 6 stan- Phe-Leu) antioxidant activity, as well as peptides devoid of
dard deviation radical scavenging activity at the highest concentration test-
Sample ORAC-FL valuea ed (0.1 mg/ml). Compared to known antioxidants of plant
origin measured under the same conditions, the ORAC-FL
Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu 3.809 6 0.155 value of Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu (3.81 mmol
Ser-Ala-Leu-Ala-Met 2.659 6 0.018 of Trolox equivalent per mmol of peptide) is almost sixfold
Tyr-Gln-Ile-Gly-Leu 1.718 6 0.073
higher than that of a-tocopherol (0.64 6 0.048) (measured
Tyr-Arg-Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe 1.184 6 0.069
Phe-Arg-Ala-Asp-His-Pro-Phe-Leu 0.132 6 0.003
by using methylated b-cyclodextrin as a water-solubility
Arg-Ala-Asp-His-Pro-Phe-Leu 0.128 6 0.005 enhancer) (unpublished data) and is approximately two- and
Val-Leu-Leu-Pro-Asp-Glu-Val-Ser-Gly-Leu ,0.022 threefold lower than those of caffeic acid and the flavonoid
Arg-Asp-Ile-Leu-Asn-Gln ,0.022 quercetin, respectively (8). Therefore, this peptide could be
Glu-Ser-Ile-Ile-Asn-Phe ,0.022 considered a strong antioxidant. Note that Tyr-Ala-Glu-Glu-
Val-Ala-Leu-Asp-Gly-Gly-Leu ,0.022 Arg-Tyr-Pro-Ile-Leu was also a very active ACE inhibitor,
Glu-Leu-Ile-Asn ,0.022 with an IC50 value of only 4.6 mM (21).
Ile-Val-Phe ,0.022 We evaluated the ORAC-FL values of some of the ami-
Phe-Ser-Leu ,0.022 no acids present in the peptides exhibiting the highest ac-
Pro-Pro ,0.022 tivity, as well as those corresponding to some peptide frag-
Tyr-Pro-Ile 1.570 6 0.032
ments, with a view toward estimating their contribution to
Tyr-Pro-Ile-Leu 0.943 6 0.039
Arg-Ala-Asp-His-Pro-Phe 0.137 6 0.005
radical scavenging activity (Table 2). The radical scaveng-
Arg-Ala-Asp-His-Pro 0.105 6 0.006 ing activity of protein hydrolysates from edible meat were
Ala-Asp-His-Pro 0.081 6 0.002 attributed to the presence of His, Tyr, and Met (28). The
Tyr 1.574 6 0.023 antioxidant activity of Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-
Met 1.126 6 0.067 Leu, Tyr-Gln-Ile-Gly-Leu, and Tyr-Arg-Gly-Gly-Leu-Glu-
His 0.073 6 0.001 Pro-Ile-Asn-Phe could be due to the presence of Tyr at the
Ser ,0.003 N terminus, since Tyr showed the highest ORAC-FL value
Leu ,0.003 of all the amino acids tested (Table 2). This has also been
observed for certain secretory peptide hormones that lost
a
Expressed as micromoles of Trolox equivalent per micromole
of compound. their antioxidant activity when Tyr residues were removed
(22). The presence of a hydroxyl group in the aromatic
structure of Tyr may make it act, as is the case for other
fold). This may reflect the enhanced accessibility of small phenolic compounds, as a chain-breaking antioxidant fol-
peptides to the redox reaction system or the degree of free- lowing a hydrogen atom transfer mechanism (23, 24). Thus,
dom of the possible critical amino acid residues responsible it is highly likely that the oxygen radical quenches the Tyr
for antioxidant action (22). phenolic hydrogen (H1), resulting in the formation of a
The permeate (molecular weight, ,3,000 Da) obtained more stable Tyr-like radical. To confirm the role of Tyr in
from the 3-h pepsin hydrolysate of chicken egg white was the antioxidant activity of Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-
separated by RP-HPLC on a semipreparative scale into nine Ile-Leu, two shorter derived fragments, Tyr-Pro-Ile-Leu and
different subfractions (F1 through F9) exhibiting different Tyr-Pro-Ile, were synthesized, and their activity was tested
polarity (21) whose radical scavenging activities were mea- (Table 2). Both showed lower activities than Tyr-Ala-Glu-
sured (Table 1). The ORAC-FL values of F1, F2, F3, and Glu-Arg-Tyr-Pro-Ile-Leu; however, Tyr-Pro-Ile presented a
F4 were higher than that of the parent fraction of the hy- higher radical scavenging activity than Tyr-Pro-Ile-Leu. As
drolysate with a molecular mass lower than 3,000 Da. F5, shown in Table 2, Leu was not active using this method.
F6, and F7 presented a similar activity, while F8 and F9 This could point to the importance of a second Tyr residue
exhibited a lower activity. in the Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu sequence, al-
No apparent relationship was found between the anti- though the presence of other amino acids and their position
oxidant and the ACE inhibition properties of these subfrac- in the peptide structure are, perhaps, important for antiox-
tions (21). Nevertheless, to further test the antioxidant ac- idant activity (35). Suetsuna et al. (35) found a high su-
tivity of the peptides responsible for the ACE inhibitory peroxide anion scavenging activity in the peptide Tyr-Phe-
activity, we selected 14 peptides, which were identified by Tyr-Pro-Glu-Leu, which strongly depended on the dipeptide
RP-HPLC-MS/MS analysis of the RP-HPLC subfractions Glu-Leu, although deletion of N terminus Tyr, Tyr-Phe, and
that presented the highest ACE inhibition (i.e., F6, F7, and Tyr-Phe-Tyr caused a loss of activity. In addition, Tyr-Ala-
F8) (50% inhibitory concentration [IC50], ,40 mg/ml) (21). Glu-Glu-Arg-Tyr-Pro-Ile-Leu has two acidic amino acids in
These were chemically synthesized, and their ORAC-FL the sequence that, according to Saiga et al. (28), might con-
values were determined, as shown in Table 2. The peptides tribute with their metal chelating properties to the radical
studied could be divided into four groups: peptides showing scavenging activity of the sequences.
very high (Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu and Ser- In addition to Leu, Ser did not exhibit radical scaveng-
Ala-Leu-Ala-Met), intermediate (Tyr-Gln-Ile-Gly-Leu and ing activity with this method at the concentration tested.
Tyr-Arg-Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe), and low (Phe- However, Met exhibited an ORAC-FL value of 1.13 (Table
1942 VALOS ET AL.
DA J. Food Prot., Vol. 67, No. 9

measurement of the radical scavenging activity of these


three peptides showed that they had very similar ORAC-
FL values, close to 0.13 mmol of Trolox equivalent per
mmol of peptide (Table 2). This activity was reduced in the
shorter derivatives Arg-Ala-Asp-His-Pro and Ala-Asp-His-
Pro. His (ORAC-FL value, 0.073) could be responsible for
the radical scavenging activity of these peptides. It has been
reported that His-containing peptides display antioxidant
properties, since they can act as metal ion chelators, active-
oxygen quenchers, and hydroxy radical scavengers (6). His
FIGURE 1. Oxidation of LDL with Cu21 in the absence (a) and was considered responsible for the antioxidant activity of
presence of 0.167 mg (b) and 1.67 mg (c) of Ser-Ala-Leu-Ala-Met the peptides Ala-His, Val-His-His, and Val-His-His-Ala-
per mg of LDL and 0.167 mg (d) and 0.667 mg (e) of Tyr-Ala- Asn-Glu-Asn obtained from an egg white albumen hydro-
Glu-Glu-Arg-Tyr-Pro-Ile-Leu per mg of LDL.
lysate (38).

Inhibition of the LDL-induced oxidation. The kinet-


2). This amino acid could be the main, but not the only,
one responsible for the radical scavenging activity of Ser- ics of LDL oxidation induced by Cu21 in the absence and
Ala-Leu-Ala-Met, which again points to the importance of in the presence of peptides from egg white is depicted in
amino acid position and synergism with other amino acids Figure 1. In the absence of any antioxidants, the average
for the scavenging activity of peptides. Met is a sulfur- values for the commercial human LDL oxidation were as
containing amino acid very prone to oxidation to Met sulf- follows: lag time 5 127 6 20 min, Vmax 5 5.59 6 0.74,
oxide (33, 39). Met could act as an endogenous antioxidant CDmax 5 560 6 91, and tmax 5 270 6 29. Table 3 shows
for proteins that react readily with oxidizing agents at a the kinetic parameters of the fraction of the peptic hydro-
physiological pH (17, 34). lysate with a molecular mass lower than 3,000 Da and the
The octapeptide Phe-Arg-Ala-Asp-His-Pro-Phe-Leu, synthetic peptides Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu
named Ovokinin, was reported to exhibit a vasorelaxing and Ser-Ala-Leu-Ala-Met. The variation between experi-
activity in canine mesenteric arteries (13) and to lower sys- ments was as high as 17%. In all cases, the lag time in-
tolic blood pressure in spontaneously hypertensive rats if creased with the protein concentration. At the same con-
administered orally at a dose of 100 mg/kg (12). Phe-Arg- centration level (0.167 mg of LDL per mg), Tyr-Ala-Glu-
Ala-Asp-His-Pro-Phe-Leu and Arg-Ala-Asp-His-Pro-Phe- Glu-Arg-Tyr-Pro-Ile-Leu exhibited a higher antioxidant ac-
Leu, a derivative lacking the N terminus Phe residue, were tivity than Ser-Ala-Leu-Ala-Met, measured as the delay in
found in the pepsin hydrolysate of the crude egg white and lag time of LDL oxidation. Moreover, Tyr-Ala-Glu-Glu-
possessed very potent ACE inhibitory activities, with IC50 Arg-Tyr-Pro-Ile-Leu and Ser-Ala-Leu-Ala-Met showed dif-
values of 3.2 and 6.2 mM, respectively (21). Similarly, the ferent antioxidant kinetics, since Vmax and CDmax decreased
hexapeptide Ovokinin(2-7) (Arg-Ala-Asp-His-Pro-Phe) as the concentration of Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-
was found to cause endothelium-dependent vasorelaxation Leu increased, which was not the case for Ser-Ala-Leu-Ala-
and to lower blood pressure in spontaneously hypertensive Met. The trend in the variation of the LDL oxidation pa-
rates when administered at a dose of 10 mg/kg (20). The rameters is indicative of the type of mechanism that is re-

TABLE 3. LDL oxidation inhibitory capacity of the most active radical scavenging egg white peptides; results are presented as the
mean (n 5 2)
Lag time Vmax
Concn increase (mmol/ming21 CDmax tmax increase
Sample (mg/mg of LDL)a (min)b of LDL)c (mmol/g of LDL)d (min)e

Fraction of ,3,000 Da 3.72 22 4.34 607 55


9.31 16 3.59 597 120
Ser-Ala-Leu-Ala-Met 0.167 22 5.70 564 15
0.667 14 5.76 645 40
1.667 25 5.70 572 45
Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu 0.040 23 5.15 663 20
0.167 27 2.58 594 193
0.667 32 0.81 NDf ND
a
Concentration is expressed as milligrams of sample per milligram of protein.
b
Lag time delay with respect to the control.
c
Maximum rate of oxidation.
d
Maximum quantity of dienes formed.
e
Maximum time delay with respect to the control.
f
ND, not determined.
J. Food Prot., Vol. 67, No. 9 ANTIOXIDANT PROPERTIES OF EGG-DERIVED PEPTIDES 1943

sponsible for antioxidant action (25). Accordingly, Ser-Ala- 8. Davalos, A., C. Gomez-Cordoves, and B. Bartolome. 2004. Extend-
Leu-Ala-Met either would act as a scavenger of free ing applicability of the oxygen radical absorbance capacity (ORAC-
fluorescein) assay. J. Agric. Food Chem. 52:4854.
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the mechanism involved may include blocking of the cop- Food Chem. 49:511516.
per-binding site of the LDL, binding of copper ions, or 10. Duffy, S. J., N. Gokce, M. Holbrook, A. Huang, B. Frei, J. F. Kea-
catalyzing a nonradical decomposition of hydroperoxides. ney, and J. A. Vita. 1999. Treatment of hypertension with ascorbic
acid. Lancet 354:20482049.
The trend in the variation of Vmax and CDmax in the case
11. Esterbauer, H., G. Striegl, H. Puhl, and M. Rotheneder. 1989. Con-
of the fraction of the hydrolysate with a molecular mass tinuous monitoring of in vitro oxidation of human low-density li-
lower than 3,000 Da was intermediate between both pep- poprotein. Free Radical Res. Commun. 6:6775.
tides. 12. Fujita, H., R. Sasaki, and M. Yoshikawa. 1995. Potentiation of the
Despite the recent interest in the antioxidant activities antihypertensive activity of orally administered Ovokinin, a vasore-
of protein hydrolysates, to date, there is not much infor- laxing peptide derived from ovalbumin, by emulsification in egg
phosphatidyl-choline. Biosci. Biotechnol. Biochem. 59:23442345.
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13. Fujita, H., H. Usui, K. Kurahashi, and M. Yoshikawa. 1995. Isolation
the antioxidant activity of peptides. The antioxidant activ- and characterization of Ovokinin, a bradykinin B1 agonist peptide
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dipeptide carnosine and of certain human secretory peptide 14. Gurer, H., R. Neal, P. Yang, S. Oztezcan, and N. Ercal. 1999. Cap-
hormones have been reported (9, 22). Our results suggest topril as an antioxidant in lead-exposed Fisher 344 rats. Hum. Exp.
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15. Hou, W.-C., H.-J. Chen, and Y.-H. Lin. 2003. Antioxidant peptides
LDL oxidation are in the same range as those reported for with angiotensin converting enzyme inhibitory activities and appli-
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Compared to antioxidants from plant origin, the flavonoid Food Chem. 51:17061709.
quercetin had a higher antioxidant activity than the peptides 16. Jung, W.-K., K.-S. Nam, F. Shaidi, and S.-K. Kim. 2001. Purification
analyzed, since it increased the lag time by approximately and characterization of antioxidative peptides from protein hydro-
100 min at a concentration of 0.012 mg of LDL per mg lysate of lecithin-free egg. JAOCS 78:651656.
17. Levine, R. L., L. Mosoni, B. S. Berlett, and R. Stadtman. 1996.
(data not shown). The combined antioxidant and ACE in- Methionine residues as endogenous antioxidants in proteins. Proc.
hibitory properties of the peptic hydrolysate of egg white, Natl. Acad. Sci. USA 93:1503615040.
its fractions, or the isolated peptides suggest that they are 18. Marcuse, R. 1960. Antioxidant effect of amino-acids. Nature 186:
useful in the prevention or treatment of cardiovascular dis- 886887.
eases. However, to exert their bioactive effects in vivo, 19. Matoba, N., H. Usui, H. Fujita, and M. Yoshikawa. 1999. A novel
these substances must get into the bloodstream in an active anti-hypertensive peptide derived from ovalbumin induces nitric ox-
ide-mediated vasorelaxation in an isolated SHR mesenteric artery.
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20. Matoba, N., Y. Yamada, H. Usui, R. Nakagiri, and M. Yoshikawa.
ACKNOWLEDGMENTS 2001. Designing potent derivatives of Ovokinin(2-7), an anti-hyper-
This work was supported by the Comision Interministerial de Cien- tensive peptide derived from ovalbumin. Biosci. Biotechnol. Bio-
cia y Tecnologa (CICYT) projects AGL2001-1261 and ALI2003-01088 chem. 65:636739.
and by the Comunidad de Madrid, Project 07G/0036/2003 1. 21. Miguel, M., I. Recio, J. A. Gomez-Ruiz, M. Ramos, and R. Lopez-
Fandino. 2004. Angiotensin Iconverting enzyme inhibitory activity
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