Angiotensin I-converting enzyme inhibitory peptides in a hydrolyzed chicken breast muscle extract

Article abstract of DOI:10.1021/jf020604h

The blood pressure of spontaneously hypertensive rats (SHRs) decreased after oral administration of an extract prepared from chicken breast muscle, falling maximally to 50 mmHg lower than before. This effect continued for at least 4 h after administration. The peptides possessing hypotensive activity in the chicken extract were examined by measuring the inhibitory activity (IC50) against angiotensin I-converting enzyme (ACE). The inhibitory activity of the chicken extract was 1060 mg%, whereas the activity of the extract treated with an Aspergillus protease and gastric proteases (trypsin, chymotrypsin, and intestinal juice) became stronger, reaching 1.1 mg%. Peptides in this hydrolysate of the extract were isolated by HPLC on a reversed-phase column, and their N-terminal sequences were analyzed. Three peptides possessed a common sequence, Gly-X-X-Gly-X-X-Gly-X-X, which was homologous with that of collagen. The peptide Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe showed the strongest inhibitory activity (IC₅₀ = 42 μM).

Full text of DOI:10.1021/jf020604h

J. Agric. Food Chem. 2003, 51, 1741−1745 1741
Angiotensin I-Converting Enzyme Inhibitory Peptides in a
Hydrolyzed Chicken Breast Muscle Extract
AI SAIGA,† TOMOYUKI OKUMURA,‡ TOSHIKAZU MAKIHARA,‡
SHINICHIRO KATSUTA,§ TSUYOSHI SHIMIZU,§ RYOJI YAMADA,‡ AND
TOSHIHIDE NISHIMURA*,†
Graduate School of Biosphere, Hiroshima University, Higashi-hiroshima 739-8528, Japan;
Japan Research and Development Center, Nippon Meat Packers, Inc., Tsukuba,
Ibaraki 300-2646, Japan; and School of Medicine, Fukushima Medical University,
Fukushima 960-1295, Japan
The blood pressure of spontaneously hypertensive rats (SHRs) decreased after oral administration
of an extract prepared from chicken breast muscle, falling maximally to 50 mmHg lower than before.
This effect continued for at least 4 h after administration. The peptides possessing hypotensive activity
in the chicken extract were examined by measuring the inhibitory activity (IC50) against angiotensin
I-converting enzyme (ACE). The inhibitory activity of the chicken extract was 1060 mg%, whereas
the activity of the extract treated with an Aspergillus protease and gastric proteases (trypsin,
chymotrypsin, and intestinal juice) became stronger, reaching 1.1 mg%. Peptides in this hydrolysate
of the extract were isolated by HPLC on a reversed-phase column, and their N-terminal sequences
were analyzed. Three peptides possessed a common sequence, Gly-X-X-Gly-X-X-Gly-X-X, which
was homologous with that of collagen. The peptide Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe
showed the strongest inhibitory activity (IC50 ) 42 µM).
KEYWORDS: Angiotensin I-converting enzyme inhibitor; spontaneously hypertensive rats; hypotensive
activity; chicken breast muscle extract; peptide
INTRODUCTION
discovered in hydrolysates of gelatin (8), casein (9-14), zein
(a maize endosperm protein) (15-17), and fish muscle proteins
(18-21).
It is well-known that chicken soup is an extremely nutritious
and palatable food, because it contains a lot of nitrogen
compounds including free amino acids and possesses a stronger
umami and brothy taste than beef and pork soups. Furthermore,
it is thought in China that chicken soup has physical functions
such as improvement of the blood circulation or a weak
constitution (1).
Recently, peptides derived from proteins in foods have been
shown to regulate physical functions in the alimentary, neural,
and circulatory systems. For example, gluten exorphin isolated
from wheat gluten hydrolysate releases insulin like an opioid
hormone (2, 3). â-Casomorphin derived from casein has been
shown to regulate peristalsis of the small intestine (4). Peptides
derived from soybean and pork proteins can suppress the
increase in cholesterol in the serum after a meal (5, 6). A peptide
in a casein hydrolysate treated with pepsin promotes calcium
absorption, being utilized as a functional material (7). Further-
more, peptides possessing hypotensive activity have been
Among many functional peptides, ones possessing hypoten-
sive activity are thought to be useful as functional food materials
for high blood pressure patients. This activity is mainly due to
the inhibition of the angiotensin I-converting enzyme (ACE)
(EC 3.4.15.1), which plays an important role in the regulation
of blood pressure in two ways. One is the conversion of the
inactive decapeptide angiotensin I into a strong vasoconstrictor
and salt-retaining octapeptide, angiotensin II. The other is the
inactivation of the vasodilator and natriuretic nanopeptide
bradykinin. Cheung et al. (22) systematically synthesized many
peptides and showed that the carboxyl-terminal sequences
(-HL, -FR, and -AP) of their peptides are involved in the
inhibition of ACE. A tripeptide, Leu-Arg-Pro, in a zein
hydrolysate has also been reported to exhibit hypotensive activity
in spontaneously hypertensive rats (SHR), decreasing their blood
pressure by 15 mmHg after a 30 mg/kg intravenous injection
(17). An octapeptide, Pro-Thr-His-Ile-Lys-Trp-Gly-Asp, from
a tuna muscle hydrolysate has been shown to exhibit inhibitory
activity toward ACE and hypotensive activity in SHR (18). A
lacto-tripeptide, Val-Pro-Pro, from casein has hypotensive
activity and is utilized for functional foods as an antihypertensive
agent (23). Although many peptides possessing hypotensive or
* Author to whom correspondence should be addressed (telephone +
81-824-24-7984; fax +81-824-24-7984; e-mail toshixy@hiroshima-u.ac.jp).
† Hiroshima University.
‡ Nippon Meat Packers Inc.
§ Fukushima Medical University.
10.1021/jf020604h CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/12/2003

1742 J. Agric. Food Chem., Vol. 51, No. 6, 2003
Saiga et al.
ACE inhibitory activity have been discovered in hydrolysates
of food proteins, as described above, there is little information
on the functions of ACE inhibitory peptides derived from animal
meat proteins.
In the present study, we investigated the hypotensive effect
of a chicken muscle extract in SHR. ACE inhibitory peptides
were isolated from the hydrolyzed chicken extract, and their
structures were clarified. Furthermore, the peptide exhibiting
the highest inhibitory activity was synthesized and characterized.
MATERIALS AND METHODS
Materials. Chicken breast muscle and fresh porcine small intestines
were obtained from Nippon Meat Packers Inc. (Osaka, Japan).
Aspergillus protease was bought from Sankyo Co. (Tokyo, Japan). ACE
from rabbit lung, trypsin, and chymotrypsin were purchased from Sigma
Chemical Co. (St. Louis, MO). Amino acid derivatives for peptide
syntheses were purchased from Shimadzu Co. (Kyoto, Japan). Hippuryl-
L-histidyl-L-leucine (Hip-HL) was obtained from Peptide Institute Inc.
(Osaka, Japan). Other chemical reagents were of reagent grade or better.
Preparation of an Extract of Chicken Breast Muscle. A chicken
extract was made from chicken breast muscle according to the following
method. One kilogram of chicken breast muscle blocks was boiled in
a 1.5 L aqueous solution (pH 4-4.5) for 3.5 h to extract proteins from
muscle blocks adequately. After boiling, the extract was filtered and
centrifuged to remove the precipitate. Furthermore, the supernatant was
concentrated with Brix 25 and used as the chicken extract.
Preparation of Small Intestine Juice. Immediately after slaughter,
fresh porcine small intestines were removed from the carcasses and
then washed with cold 0.85% NaCl. Small intestinal epithelia and
mucosae were collected using cover glasses and then homogenized with
7 volumes (w/v) of 0.1 M Tris-HCl buffer (pH 7.2) containing 12%
glycerol (1000 rpm, 3 min). After centrifugation (30000g), the
supernatant was filtered and dialyzed against 10 mM Tris-HCl buffer
(pH 7.2). Protein concentrations were determined with a protein assay
kit (Bio-Rad, Hercules, CA) according to the colorimetric method.
Digestion of the Chicken Extract with Proteases. The chicken
extract was first hydrolyzed with 0.06% Aspergillus protease at 50 °C
and pH 7.0 for 1 h. After this reaction had been stopped by boiling for
10 min, the supernatant was further hydrolyzed by successive treatment
with 1% trypsin/chymotrypsin and small intestinal juice at 37 °C and
pH 7.0 for 1 h and 30 min, respectively. Each digestion was also stopped
by boiling for 10 min to inactivate proteases. After treatment, all
hydrolysates were centrifuged to remove the precipitates.
Measurement of Blood Pressure and Heartbeat in SHRs. Eight-
week-old male SHRs were fed a commercial nonpurified diet (AIN-
76; Oriental Yeast, Tokyo, Japan) and water for 2 weeks ad libitum in
an environment-controlled room (23 °C, 55% humidity), and then either
saline or a protein hydrolysate (1 g/kg weight) dissolved in saline was
administered orally. Their tail systolic blood pressure and heartbeat
were determined at 0, 1, 2, 3, and 4 h after the oral administration by
tail-cuff method using a plethysmographic tail apparatus (Softron 98A;
Softron Co., Tokyo, Japan).
Assay of Inhibitory Activity toward ACE. The inhibitory activity
of a chicken extract or peptides toward ACE was assayed according to
the method reported by Cheung (22). The following assay components,
in a final volume of 0.25 mL, were incubated at 37 °C for 30 min:
100 mM sodium borate buffer (pH 8.3), 5 mM Hip-HL, 500 mM NaCl,
20 milliunits of rabbit lung ACE, and a chicken extract or peptides.
The enzyme reaction was stopped by the addition of a 1 N HCl solution.
The rate of hydrolysis of Hip-HL was determined by measuring the
absorbance of the released hippuric acid at 228 nm after successive
extraction with ethyl acetate, evaporation of the ethyl acetate, and
dissolution of the residue in water. The ACE inhibitor concentration
required to inhibit 50% of the ACE activity under the conditions
described above was expressed as IC50, which was calculated using
the net weight of the protein hydrolysate.
Figure 1. Effect of chicken extract administration on the blood pressure
of spontaneously hypertensive rats: [, control (saline); 2, protease-
treated extract; 9, untreated extract. The blood pressure of 8-week-old
SHRs was measured by the tail-cuff method after oral administration of
samples (1.0 g/kg of rat weight). Average blood pressure values in five
SHRs are shown. Each bar shows the standard error. *, significantly
different from the control (*, p < 0.05) with Dunnett’s test.
into two groups, with molecular masses of less and more than 1000
Da, respectively, using an ultrafiltration membrane (Millipore Co.,
Bedford, MA). Peptides in the former group possessing high ACE
inhibitory activity were purified by HPLC on an ODS column (22 ×
250 mm and 4.6 × 250 mm; Senshu, Tokyo, Japan) using a linear
gradient of CH3CN (8-40% in 80 min or 8-40% in 64 min,
respectively) containing 0.1% trifluoroacetic acid at the flow rate of
1.0 mL/min. The elution peaks were monitored at 220 nm.
Analysis of the N-Terminal Amino Acid Sequences of Peptides.
The N-terminal amino acid sequences of the isolated hypotensive
peptides were determined with a protein sequencer G1005A (Hewlett-
Packard Co., Wilmington, DE).
Peptide Syntheses. Peptides were synthesized by the fluorenyl-
methoxycarbonyl (Fmoc) strategy using a simultaneous multiple peptide
synthesizer (model PSSM-8, Shimadzu, Kyoto, Japan) according to the
method reported by Nokihara et al. (24). After being synthesized, a
peptide was purified by HPLC on an ODS column (Pegasil-300, 20 ×
250 mm; Senshu, Tokyo, Japan) with a linear gradient of 0-50% CH3-
CN containing 0.1% trifluoroacetic acid in 100 min (flow rate ) 5.0
mL/min; monitoring at 220 nm). Furthermore, the molecular mass of
an isolated peptide was determined by mass spectrometry with an ESI
mass spectrometer LC-Q (Thermo Finnigan, San Jose, CA).
Statistical Evaluations. Data on blood pressure and heartbeat count
were evaluated by one-way ANOVA, followed by Dunnett’s test to
compare the mean of each dose group with that of the control group.
The probability level used to determine statistical significance was p
< 0.05.
RESULTS
Hypotensive Activity of Protein Hydrolysates in SHR. The
blood pressure of SHRs was measured at 0, 1, 2, 3, and 4 h
after oral administration of the Aspergillus protease-treated or
untreated chicken extract. The administration of a treated or an
untreated chicken extract significantly lowered the blood
pressure of SHRs 1 h later, whereas no decrease in blood
pressure was observed in control SHRs fed no chicken extract.
This effect of both chicken extracts on blood pressure continued
for at least 4 h. The SHRs fed the untreated chicken extract
exhibited the greatest decrease, 50 mmHg, at 3 h after
administration. The administration of the chicken extract treated
with the Aspergillus protease also caused a greatest decrease,
45 mmHg, at 2 h after administration (Figure 1). There is a
little difference in continuity between the Aspergillus protease-
treated and untreated chicken extracts. This difference may be
due to the difference in the speeds of hydrolysis of the two
chicken extracts by gastric proteases in SHRs.
Separation and Purification of Hypotensive Peptides from a
Hydrolyzed Chicken Extract. Peptides in a chicken extract digested
with trypsin, chymotrypsin, and small intestine juice were first divided

Angiotensin I-Converting Enyzme Inhibitory Peptides
J. Agric. Food Chem., Vol. 51, No. 6, 2003 1743
Figure 2. Effect of chicken extract administration on the heartbeat of
spontaneously hypertensive rats: [, control (saline); 2, protease-treated
extract; 9, untreated extract. The heartbeat of 8-week-old SHRs was
measured by the tail-cuff method after oral administration of sample (1.0
g/kg of rat weight). Average heartbeat counts in five SHRs are shown.
Each bar shows the standard error.
Table 1. Inhibitory Activity of Each Chicken Extract against
Angiotensin I-Converting Enzyme
Figure 3. Purification of ACE inhibitory peptides obtained from chicken
extract by HPLC on an ODS column: (A) first HPLC; (B) second HPLC.
F6 on the first HPLC was applied to the second HPLC. Gradient conditions
for elution on both HPLCs are given under Materials and Methods.
ultrafiltration after
untreated
chicken protease-
protease treamentb
lacto-tripeptide
extract
IC a (mg%) 1060 ± 13.0 1.1 ± 0.4 0.8 ± 0.2
treatedb MW < 1000 MW > 1000
V-P-P
14 ± 2.5
13
50
Table 2. Angiotensin I-Converting Enzyme Inhibitory Activity of the
Peptide Fractions Obtained on First HPLC
a Concentration of peptide needed to inhibit 50% of the ACE activity. b Chicken
extract treated with Aspergillus protease and gastric enzymes.
fraction
IC (mg%)
50
fraction
IC (mg%)
50
The heartbeat in SHRs was also measured after administration
in order to check the effect of the administration of chicken
extracts on the physical condition. As shown in Figure 2, there
was no change in the heartbeat of SHRs at 1, 2, 3, and 4 h after
oral administration, suggesting that the administration of chicken
extracts did not have a bad effect on the circulatory system of
SHRs.
F0
F1
F2
F3
1.417 ± 0.667
0.374 ± 0.042
0.176 ± 0.027
0.159 ± 0.016
F4
F5
F6
0.173 ± 0.015
0.110 ± 0.016
0.084 ± 0.011
a Fraction numbers correspond to F0−F6 in Figure 3A.
Table 3. Angiotensin I-converting Enzyme Inhibitory Activity of the
Inhibition of ACE Activity by Chicken Extract Hydroly-
sates. Some peptides in protein hydrolysates have been shown
to suppress blood pressure in SHRs through the inhibition of
ACE. To examine the peptides in chicken extracts involved in
the lowering of the blood pressure, ACE inhibitory activities
of chicken extracts were first examined. Because peptides in
chicken extracts were further hydrolyzed by gastric systems after
administration, the ACE inhibitory activity of the chicken
extracts treated with trypsin, chymotrypsin, and small intestine
juice was also measured.
The untreated chicken extract exhibited very low inhibitory
activity (IC50 ) 1060 mg%). This extract treated with an
Aspergillus protease, trypsin/chymotrypsin, and small intestine
juice, however, exhibited high inhibitory activity (IC50 ) 1.1
mg%). Small peptides (<1000 Da) in this extract separated with
an ultrafilter membrane showed higher activity (IC50 ) 0.8
mg%) than those with molecular masses of >1000 Da (Table
1). These results suggested that proteins and peptides would
exhibit higher inhibitory activity on hydrolysis by gastric
proteases.
Peptide Peaks Obtained on Second HPLC
peak
IC50 (mg%)
peak
IC50 (mg%)
P1
P2
P3
0.204 ± 0.016
0.169 ± 0.019
0.070 ± 0.004
P4
P5
P6
0.068 ± 0.003
0.192 ± 0.017
0.081 ± 0.003
a Peak numbers correspond to P1−P6 in Figure 3B.
inhibitory activity and fraction F5 exhibited the second highest
(Table 2). Therefore, peptides in F6 were further separated by
HPLC on the same column (Figure 3B). The ACE inhibitory
activities of the six isolated peptides (P1-P6) were measured.
Three peptides, designated P3, P4, and P6, exhibited strong
inhibitory activities against ACE. On the other hand, the
inhibitory activities of P1, P2, and P5 were weak (Table 3).
Analyses of the Structures of ACE Inhibitory Peptides.
The structures of the six peptides isolated by HPLC on an ODS
column were analyzed with a protein sequencer. The sequences
of four peptides were clarified, as shown in Table 4. Among
them, P3, P4, and P6 possessed a unique and repeated sequence,
Gly-X-X-Gly-X-X-Gly-X-X-Gly (X, amino acid). Homology
analysis with known proteins showed that these peptides were
derived from collagen.
Purification of ACE Inhibitory Peptides. The ACE inhibi-
tory peptides in a chicken extract treated with the Aspergillus
protease and gastric proteases were separated by HPLC on a
reversed-phase column (Figure 3A). The separated peptides
were divided into seven groups (F0-F6) according to the
retention time. The ACE inhibitory activity of each group was
measured. Among all groups, fraction F6 showed the highest
ACE Inhibitory Activity of Synthesized Peptides. To
confirm the ACE inhibitory activity of the isolated peptide (P4),
Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe was synthe-
sized with a peptide synthesizer, and its inhibitory activity was

1744 J. Agric. Food Chem., Vol. 51, No. 6, 2003
Saiga et al.
Many peptides possessing ACE inhibitory activity have been
isolated from various hydrolysates of food proteins. Phe-Phe-
Val-Ala-Pro (11) and Ile-Pro-Pro (14) derived from casein
exhibited ACE inhibitory activity (IC50 ) 2.0 and 5.0 µM,
respectively). Val-His-Leu-Pro-Pro (15) and Leu-Gln-Pro (16),
having IC50 values of 18 and 9.6 µM, respectively, were also
found in a zein hydrolysate. Ile-Trp-His-His-Thr (IC50 ) 5.1
µM), Ile-Val-Gly-Arg-Pro-Arg-His-Gln-Glu (IC50 ) 6.2 µM),
Ala-Leu-Pro-His-Ala (IC50 ) 10 µM), Phe-Gln-Pro (IC50 ) 12
Table 4. Sequences of ACE Inhibitory Peptides Isolated on Second
HPLC
peak
sequence
P1
P3
P4
P6
Leu-Phe
Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-X
Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe
Gly-Val-Asn-Gly-Glu-Glu-Gly-Val-Pro-Gly
a Peak numbers correspond to P1−P6 in Figure 3B.
µM), Ile-Tyr (IC50 ) 3.7 µM), Leu-Lys-Pro-Asn-Met (IC50
)
Table 5. ACE Inhibitory Activities of Synthetic Peptides
17 µM), Asp-Tyr-Gly-Leu-Tyr-Pro (IC50 ) 62 µM), and Ile-
Lys-Pro-Leu-Asn-Tyr (IC50 ) 43 µM) were isolated from a
thermolysin digest of dried bonito (17). An ACE inhibitory
peptide was found in a proteolytic hydrolysate of tuna muscle
sequence
IC50 (µM)
Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe (P4)
42.4 ± 24.0
436.0 ± 160.8
26
41
Gly-Phe-Pro-Gly-Thr-Pro-Gly-Leu-Pro-Gly-Phe
proteins. Met-Asn-Pro (IC50 ) 66.6 µM), Asn-Pro-Pro (IC50
)
Val-Tyra
Val-Pro-Prob
290.5 µM), and Thr-Asn-Pro (IC50 ) 207.4 µM) were discov-
ered in a porcine myosin hydrolysate (26). An ACE inhibitory
peptide, Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe, iso-
lated in our study exhibited an IC50 of 42 µM. This activity
was not so high compared with those of peptides derived from
casein and zein hydrolysates. However, it exhibited almost the
same activity as some peptides derived from dried bonito and
a higher activity than peptides derived from porcine myosin.
Judging from these results, the chicken extract seems to be useful
as a food material possessing antihypertensive activity.
a Dipeptide from bonito muscle hydrolysate. b Tripeptide from milk protein.
measured (Table 5). This peptide exhibited an IC50 of 42 µM.
A peptide, Gly-Phe-Pro-Gly-Thr-Pro-Gly-Leu-Pro-Gly-Phe, was
also synthesized by replacing Hyp in P4 with Pro, exhibiting
an IC50 of 436 µM. This result suggested that Hyp in these
peptides played an important role in the ACE inhibitory activity.
The inhibitory activity of P4 was compared with that of the
known hypotensive peptides. Peptide P4 showed almost the
same activity as Val-Tyr and Val-Pro-Pro, which were derived
from sardine muscle and milk protein, respectively.
The relationship between the structure and ACE inhibitory
activity of peptides remains ambiguous. Cheung et al. (22)
reported that N-terminal glycyl dipeptides vary 300-fold in
inhibitory potency, those with Trp, Tyr, Pro, or Phe being the
most inhibitory. Although many of the inhibitory peptides
described above certainly possessed Pro, Tyr, or Phe at their C
terminus, peptides without them at the C terminus also exhibited
high activity. The rule proposed by Cheung et al. may apply to
evaluation of the activities of short peptides such as di- and
tripeptides. However, ACE exhibits relatively broad substrate
specificity like peptidyldipeptidase, which hydrolyzes brady-
kinin, a nanopeptide, and angiotensin I, a decapeptide. Further-
more, in our work, the substitution of Pro for Hyp in Gly-Phe-
Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe decreased the inhibi-
tory activity 10-fold, indicating that the hydroxyl group of Hyp
in peptides plays an important role in the binding of peptides
to the catalytic site of ACE. The amino acid sequences at the
N termini of oligopeptides also seem to significantly influence
their ACE inhibitory activities. The relationship between the
N-terminal structure and ACE inhibitory activity is the next
problem to be resolved.
DISCUSSION
There have been many studies on the ACE inhibitory
activities of peptides derived from proteins in food. Some of
these peptides have been shown to possess hypotensive activity
in SHRs. However, there is little information on the ACE
inhibitory and hypotensive activities of peptides derived from
animal muscle proteins. Therefore, in this study, we examined
the hypotensive activity of a chicken extract, because chicken
muscle is a popular food throughout the world. We clarified
that an extract prepared from chicken breast muscle showed
hypotensive activity in SHRs. Furthermore, peptides possessing
ACE inhibitory activity were isolated from this extract and their
structures were determined.
Thus far, a zein hydrolysate treated with thermolysin has been
shown to possess hypotensive activity on intraperitoneal or oral
administration to SHRs. The blood pressure was decreased at
1 h after intraperitoneal administration of zein hydrolysate, and
this activity continued until 7 h after administration. On the
other hand, oral administration of zein hydrolysate exhibited
hypotensive activity in SHRs only 6 h later. A hexapeptide
derived from casein was reported to have antihypertensive
activity and to cause a 10 mmHg reduction in blood pressure
at 100 min after an intravenous administration. Although a
myofibrillar protein hydrolysate also exhibited hypotensive
activity in SHRs on intraperitoneal administration, its activity
appeared at 2 h after administration and had disappeared by 4
h (25). In our study, the hypotensive activity of a chicken extract
in SHRs appeared at 1 h after oral administration and continued
for at least 4 h. This is the first observation that hypotensive
activity appeared upon oral administration of an animal food
material. Furthermore, the hypotensive activity of the chicken
extract upon oral administration was also shown to be higher
than those of the casein hexapeptide and myofibrillar protein
hydrolysate.
Homology analysis of three isolated peptides, Gly-Phe-Hyp-
Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe, Gly-Phe-Hyp-Gly-Thr-
Hyp-Gly-Leu-Hyp-Gly-X, and Gly-Val-Asn-Gly-Glu-Glu-Gly-
Val-Pro-Gly, has shown that these peptides are derived from
collagen in chicken muscle. Oshima et al. (8) reported that ACE
inhibitory peptides were found in digests of gelatin with bacterial
collagenase, possessing the structures of Gly-Pro-Hyp-Gly-Thr-
Asp-Gly-Ala-Hyp (IC50 ) 10.5 µM), Gly-Pro-Ala-Gly-Ala-Hyp
(IC50 ) 8.3 µM), Gly-Pro-Pro-Gly-Ala-Hyp (IC50 ) 8.6 µM),
Gly-Pro-Ile-Gly-Ser-Val-Gly-Ala-Hyp (IC50 ) 31.8 µM), and
Gly-Pro-Ala-Gly-Ala-Pro-Gly-Ala-Ala (IC50 ) 37.0 µM). The
structures of the peptides in our study were different from those
of peptides in gelatin digests. These differences in structure
seemed to be caused by the differences in the origin of collagen,
because it is well-known that there are many types of collagens,
which have heterogeneous structures.

Angiotensin I-Converting Enyzme Inhibitory Peptides
J. Agric. Food Chem., Vol. 51, No. 6, 2003 1745
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Received for review May 28, 2002. Revised manuscript received
November 5, 2002. Accepted December 16, 2002.
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