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Peptides 24 (2003) 791–798
New antihypertensive peptides isolated from rapeseed
Ewa D. Marczaka,b, Hachiro Usuia, Hiroyuki Fujitac, Yanjun Yanga,d, Megumi Yokooa,
Andrzej W. Lipkowskib,e, Masaaki Yoshikawaa,∗
aDivision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan
bDepartment of Biotechnology, Industrial Chemistry Research Institute, 01-793 Warsaw, Poland
cThe Nippon Supplement Inc., Osaka 531-0076, Japan
dSchool of Food Science and Technology, Southern Yangtze University, Wuxi, China
eMedical Research Center Polish Academy of Sciences, 02-106 Warsaw, Poland
Received 9 December 2002; accepted 15 April 2003
Abstract
Four potent angiotensin converting enzyme (ACE) inhibitory peptides, IY, RIY, VW and VWIS, were isolated from subtilisin digest of
rapeseed protein. Among them RIY and VWIS are new peptides with IC50 28 and 30 M, respectively. All isolated peptides lowered blood
pressure of spontaneously hypertensive rats (SHR) following oral administration. The maximum effect in the case of RIY was observed
4h after administration, while maximum effect of other peptides on blood pressure occurred 2h after administration. Furthermore, the
antihypertensive effect of RIY was observed even in old rats, in which ACE inhibitors become less effective, suggesting that a different
mechanism other than ACE inhibition is also involved in lowering blood pressure by this peptide. Subtilisin digest of rapeseed protein also
significantly lowered blood pressure of SHR after oral administration of a single dosage 0.15g/kg, exerting maximum antihypertensive
effect 4h after administration. This digest appears promising as a functional food, which may be useful in the prevention and treatment of
hypertension.
© 2003 Elsevier Inc. All rights reserved.
Keywords: Angiotensin-converting enzyme inhibitory peptides; Protein digest; Rapeseed; Antihypertensive effect; Spontaneously hypertensive rat
1. Introduction
Angiotensin I-converting enzyme (ACE) is a dipeptidyl
carboxypeptidase, which participates in regulation of blood
pressure by virtue of two different reactions, i.e. conversion
of inactive peptide Angiotensin I into powerful vasoconstric-
tor Angiotensin II and inactivation of the vasodilator peptide
Bradykinin [2,12,18]. Specific inhibitors of ACE have been
shownto be useful asantihypertensivedrugs. Many synthetic
ACE inhibitors including Captopril, Enalapril, Lisinopril and
others are available for clinical use [4,17]. ACE inhibitors
are well tolerated by most patients, however some unde-
sirable side effects may occur such as cough, lost of taste,
renal impairment and angioneurotic oedema [1]. Recently,
there has been a trend toward development of natural ACE
inhibitory peptides generated during hydrolysis of various
proteins [7–11,13–17,19,22,23]. Naturally occurring ACE
inhibitors are much less potent than synthetic ones, however,
they have an advantage of having no known side effects.
∗Corresponding author. Tel.: +81-774-38-3725; fax: +81-774-38-3774.
E-mail address: yosikawa@kais.kyoto-u.ac.jp (M. Yoshikawa).
In this study, rapeseed was selected as a source of protein
since it has never been tested for biologically active peptides.
Rapeseed is an oilseed plant cultivated all over the world.
Rapeseed meal is a byproduct of the oil removal process, and
is comprised of approximately 40% of protein. The aim of
this study was to investigate rapeseed protein as a source of
new peptides having ACE inhibitory activity and possessing
the ability to lower blood pressure after oral administration.
2. Methods
2.1. Materials
2.1.1. Enzymes
Pepsin from porcine stomach mucosa, trypsin from bovine
pancreas, chymotrypsin from bovine pancreas, pancreatin
from porcine pancreas, subtilisin Carlsberg—type VIII from
Bacillus licheniformis, thermolysin—type X from Bacillus
thermolyticus, angiotensin-converting enzyme (ACE) from
rabbit lung were obtained from Sigma. Hippuryl-histydyl-
leucine (HHL) was obtained from Peptide Institute Inc.
0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0196-9781(03)00174-8
792 E.D. Marczak et al./Peptides 24 (2003) 791–798
Fmoc-amino acids were obtained from Watanabe Chemical
Industries Ltd. HPLC solvents and other chemicals were
obtained from Nacalai Tesque Inc.
2.2. Isolation of protein from rapeseed meal
Forty grams of rapeseed meal was mixed with 400ml
of 1M solution of sodium chloride and homogenized at
4◦C (20,000U/min for 5min) with the use of Ultraturrax
homogenizer type 18-10. Homogenate was centrifuged at
15,000rpm through 30min. After extensive dialysis against
water, the supernatant was freeze-dried and kept in sealed
pack at 4◦C.
2.3. Hydrolysis of protein with the use of different
enzymes
Rapeseed protein was dissolved in water (20mg/ml)
and digested by various proteases. Enzymatic digestion
by trypsin, chymotrypsin, pancreatin, subtilisin and ther-
molysin was performed in the presence of 200g/ml of
enzyme at 37◦C for 5h (pH 7.5). Digests were subse-
quently boiled for 10min to inactivate the enzyme. In case
of pepsin, digestion was performed at pH 2 (adjusted with
hydrochloric acid). After 5h of digestion pH was adjusted
to 7 with sodium hydroxide solution before boiling. When
additional digestion with other proteases was made fol-
lowing pepsin digestion, pH was adjusted to 7.5, protease
was added, and further digestion was carried out for 5h at
37◦C. Finally, digest was boiled for 10min to inactivate
the enzyme.
2.4. Determination of ACE inhibitory activity of digests
and peptides
ACE inhibitory activity was measured using HHL as a
substrate and ACE from rabbit lung using the method of
Cushman and Cheung [5] with modification by Yamamoto
et al. [20] and presented as an IC50 value. Each assay mix-
ture comprised the following components at final concentra-
tion: borate buffer (pH 8.3)—100mM, sodium chloride—
400mM, HHL—5mM, enzyme—3 U per 200 l of assay
volume.
2.5. Isolation and purification of active peptides
The digest was injected on octadecyl silica (ODS) column
(Cosmosil 5C18-ARII 20 mm ×250 mm, Nacalai Tesque
Inc.). The column was developed at a flow rate of 10ml/min
using a linear gradient of acetonitrile containing 0.1% triflu-
oroacetic acid (from 0 to 40% in 40min). Individual frac-
tions were dried using a centrifugal concentrator and their
activity was tested. Active fractions were further purified
on a cyanopropyl silica (CN) column (Cosmosil 5 CN-R
10mm ×250 mm, Nacalai Tesque Inc.), which was deve-
loped using the same gradient at a flow rate of 5ml/min. Ac-
tive fractions from cyanopropyl column were further purified
on a phenetyl silica (5PE-MS 4.6mm×250mm, Nacalai
Tesque Inc.) and a nitrophenetyl (5NPE 4.6mm×150mm,
Nacalai Tesque Inc.) column with the same gradient at a
flow rate of 1ml/min.
The amino acid sequence of purified peptides was ana-
lyzed with the use of the 492 protein sequencer (Applied
Biosystems Inc.).
2.6. Synthesis of peptides
Peptides were synthesized with the peptide synthesizer
PS3 (Rainin Instrument Co. Inc.) using the solid phase
method. Fmoc-amino acids were successively coupled in
the presence of HBTU. Peptides, after deprotection, were
purified using HPLC (ODS column).
2.7. Determination of stability of peptides for ACE
ACE inhibitory peptides were incubated with 32mU of
ACE at 37 ◦C for 3 h. IC50 values were compared before and
after pre-incubation. Stability of the inhibitory peptides for
ACE was also tested using HPLC equipped with an ODS
column.
2.8. Determination of antihypertensive activity of peptides
Peptides were dissolved in saline and injected orally using
a metal gastric zonde in 16–30-week-old SHR. Following
oral administration of sample, blood pressure was measured
by the tail cuff method using a MK-2000 blood pressure
meter (Muromachi Kikai).
2.9. Statistical analysis
All results are expressed as means ±S.E.M. Statistical
comparisons of the results between two groups were per-
formed using the Student’s t-test. Values of P<0.05 were
considered significant.
3. Results
3.1. ACE inhibitory activity of rapeseed protein digests
Protein extracted from rapeseed meal was digested with
the use of several different proteolytic enzymes such as
pepsin, pancreatin, trypsin, chymotrypsin, thermolysin, sub-
tilisin and combinations of pepsin with other enzymes. ACE
inhibitory activity of crude digests was determined (Table 1).
Pepsin and subtilisin digests had the most potent ACE in-
hibitory activity (IC50 0.16mg/ml). Subtilisin digest, which
lowered blood pressure in preliminary tests in SHR (data
not shown), was selected for further study.
E.D. Marczak et al./Peptides 24 (2003) 791–798 793
Table 1
Angiotensin-converting enzyme inhibitory activity of rapeseed protein
digests
Enzyme used for hydrolysis IC50 (mg/ml)
Trypsin 0.95
Chymotrypsin 0.42
Pancreatin 1.30
Thermolysin 0.32
Subtilisin 0.16
Pepsin 0.16
Pepsin/trypsin 0.28
Pepsin/chymotrypsin 0.24
Pepsin/pancreatin 0.70
3.2. Isolation of ACE inhibitory peptides
Three active fractions were isolated (Fig. 1) following the
HPLC separation of the subtilisin digest of rapeseed protein
on reversed phase octadecyl (ODS) column. After further
HPLC purification of active fractions with subsequent use
of cyanopropyl (CN-R), phenethyl (PE) and nitrophenethyl
(NPE) columns five active fractions were isolated. Sequence
analysis identified the following structures: IY, VW, RIY,
VWIS and AMQS. Separation schema with retention times
of peptides on subsequent columns is shown in Table 2.
ACE inhibitory activities of isolated peptides are presented
in Table 3. IY and VW are sequences that have previously
been identified, with IC50 3.7 and 1.6M, respectively, and
RIY and VWIS are new sequences with IC50 28 and 30M,
respectively. AMQS showed very low activity for ACE in-
hibition with IC50 greater than 1mM, and was therefore not
subject to further study. Sequences of IY, RIY and AMQS
can be found in the primary structure of napin [6] and se-
quences of VW and VWIS exist in the primary structure
of cruciferin [3] and ribosomal protein [21], respectively
(Table 2).
Fig. 1. Separation of rapeseed subtilisin digest on preparative ODS column. Active fractions are shown with arrows.
Table 2
Purification of peptides from subtilisin digest of rapeseed protein
Peptide Retention time (min) during purification
on HPLC columns Origin
ODS CN-R PE-MS NPE ODS
AMQS 14.8 7.5 14.5 3.2 – Napin
IY 24.5 19.5 22.8 – – Napin
RIY 24.5 21.6 24.0 – – Napin
VW 31.0 27.5 30.8 27.3 27.3 Cruciferin
VWIS 31.0 27.5 30.8 27.3 28.2 Actin,
ribosomal
protein
ACE inhibitory activity of all isolated peptides was much
lower than activity of synthetic ACE inhibitor captopril
(Table 3). In the case of VWIS and RIY they were less ac-
tive in vitro than captopril by a factor higher than 1000. In
the case of VW and IY, they were less active than captopril
in vitro by a factor of 72 and 168, respectively.
3.3. Determination of stability of peptides for ACE
ACE inhibitory peptides can be classified into three
groups depending on their interaction with ACE [11]. The
first group comprises true inhibitors; the second group
comprises substrates for ACE, which converts them to in-
active peptides, and the third group comprises the so called
pro-drug peptides. Pro-drugs are also substrates for ACE,
but they are converted by this enzyme to true inhibitors.
In our experience only true inhibitors and pro-drugs have
the ability to lower blood pressure [9–11,22]. In order to
check the inhibitory character of isolated peptides, the IC50
values of these peptides were determined before and after
pre-incubation with ACE. It was found that the inhibitory
activity of VWIS was intensified by six times from IC50
794 E.D. Marczak et al./Peptides 24 (2003) 791–798
Table 3
ACE inhibitory activities of peptides isolated from subtilisin digest of
rapeseed protein and captopril
Peptide IC50 (M)
Before incubation
with ACE After incubation
with ACE
VW 1.6 1.6
VWIS 30 5
IY 3.7 3.7
RIY 28 20
AMQS >1mM Not tested
Captopril 0.022 Not tested
Comparison of IC50 values before and after pre-incubation with ACE.
30M prior to pre-incubation with ACE to IC50 5M after
pre-incubation. The inhibitory activity of RIY was also in-
creased by a factor of 1.4 from IC50 28MtoIC
50 20M
following pre-incubation (Table 3). Pre-incubation did not
change the activity of VW and IY. A HPLC analysis of the
reaction mixtures after the incubation showed, that VWIS
Fig. 2. Stability of VWIS (A) and RIY (B) during incubation with ACE. HPLC analysis of reaction mixtures before and after incubation with ACE on
ODS column. (A) (1) VWIS before incubation with ACE; (2) VWIS after incubation with ACE; (3) VW standard; (B) (1) RIY before incubation with
ACE; (2) IY standard; (3) RIY after incubation with ACE.
was hydrolyzed with the release of VW (Fig. 2A), which
is a known true inhibitor of ACE. In the case of RIY only
small amount of this peptide was converted by ACE to IY
(Fig. 2B).
3.4. Antihypertensive activity of peptides in vivo
The antihypertensive activities of VW, VWIS, IY and RIY
were tested following oral administration to SHR. The ac-
tivities of peptides were compared in pairs, VWIS with VW
and RIY with IY, respectively. The results of oral adminis-
tration of peptides to SHR are presented on Fig. 3 for VW
and VWIS and on Fig. 4 for IY and RIY, respectively. Oral
administration of VW and VWIS at the dosage of 25 M/kg
(7.5mg/kg for VW and 12.5mg/kg for VWIS) to SHR ex-
erted a significant hypotensive effect 2 and 4h after adminis-
tration (Fig. 3). A hypotensive effect of −10.8±2.7mmHg
(P<0.01) and −6.8±1.8mmHg (P<0.05) occurred for
VW, 2 and 4 h after oral administration, respectively. In the
case of VWIS, it was −12.5±2.9mmHg (P≤0.01) and
E.D. Marczak et al./Peptides 24 (2003) 791–798 795
Fig. 3. Antihypertensive effects of VW and VWIS after oral administration
to 19-week-old SHR at the dosage of 25mol/kg (7.5mg/kg for VW
and 12.5mg/kg for VWIS). Changes of systolic blood pressure from time
zero were expressed as mean ±S.E.M. ∗P<0.05, ∗∗ P<0.01 indicate
significant difference against control.
−9.5±2.5mmHg (P<0.01), at 2 and 4 h after administra-
tion, respectively. The time course of antihypertensive effect
of VW and VWIS shows that maximum hypotensive activ-
ity for both peptides occurred 2h after oral administration.
RIY and IY administered orally at a dosage of 7.5mg/kg
to SHR exerted significant hypotensive effect in comparison
to the control group (Fig. 4). In the case of IY, a significant
effect of −9.8±2.1 mmHg (P<0.01) was observed 2h after
administration. For RIY, the hypotensive effect was −10.8±
3.2 mmHg (P<0.001) 2h after administration and −11.3±
1.8mmHg (P<0.01) 4h after administration. IY, similar
to VW, exerted maximum activity 2h after administration,
whereas RIY had maximum effect 4h after administration.
Fig. 4. Antihypertensive effects of IY and RIY after oral administration to
20-week-old SHR at the dosage of 7.5mg/kg. Changes of systolic blood
pressure from time zero were expressed as mean ±S.E.M. ∗∗ P<0.01
indicate significant difference against control.
Fig. 5. Dose-dependent antihypertensive activity of peptides 2 and 4h
after oral administration to 18–22-week-old SHR: (A) VW and VWIS;
(B) IY and RIY. Changes of systolic blood pressure from time zero were
expressed as mean ±S.E.M. ∗P<0.05, ∗∗ P<0.01 indicate significant
difference against control.
All peptides displayed dose-dependent antihypertensive
effect (Fig. 5A and B). The minimum doses necessary to
lower blood pressure by 10mmHg, 2 and 4h after admin-
istration are shown in Table 4. These dosages are 6.0 and
16.0mg/kg, respectively for VW, and 9.0 and 15.0 mg/kg,
Table 4
Doses of peptides necessary to lower blood pressure by 10mmHg, 2 and
4h after oral administration to SHR in comparison to captopril [11]
Peptide Dosage (mg/kg)a
2h after administration 4h after administration
VW 6.0 (19.8) 16.0 (52.7)
VWIS 9.0 (17.9) 15.0 (29.8)
IY 7.0 (23.8) 14.8 (50.3)
RIY 5.8 (12.9) 5.8 (12.9)
Captopril 2.5 (11.5) 2.5 (11.5)
aValues in parentheses: dosage in mol/kg.
796 E.D. Marczak et al./Peptides 24 (2003) 791–798
Fig. 6. Comparison of blood pressure lowering activity of VW and VWIS
2h after oral administration at the dosage of 25mol/kg (7.5mg/kg for
VW and 12.5mg/kg for VWIS) to 19- and 28-week-old SHR. Decreases
of systolic blood pressure in relation to time zero are expressed as
mean ±S.E.M.
respectively for VWIS. In the case of IY, the minimum dose
necessary to lower blood pressure by 10mmHg 2 and 4h
after administration are 7.0 and 14.8mg/kg, respectively. In
case of RIY these dosages are 5.8mg/kg for both 2 and
4h after administration. Comparing the activity of pairs of
peptides, it can be seen that in case of VW and VWIS, the
latter is the more active. It is noticeable 4h after adminis-
tration especially when dosage is calculated on a molar ba-
sis. Also, in the case of IY and RIY the latter peptide is
the more active, by a factor of almost 2, 2h after admin-
istration, and by a factor of almost 4 (3.87), 4h after oral
administration.
Usually the hypotensive effect of ACE inhibitors in old
SHR is lower than in young SHR. We compared hypoten-
sive activity of VW, VWIS, IY and RIY after oral admin-
istration to young and old SHR (Figs. 6 and 7). In the case
of VW and VWIS they significantly lowered blood pressure
both in young and old rats, however, their hypotensive ef-
fect in old rats was lower than in young ones (Fig. 6). The
effect in 19 and 28 week old rats of a dosage of 25mol/kg
was −11.8±2.7 and −8.5±1.9mmHg, respectively for
VW and −12.5±2.9 and −9.3±0.6mmHg, respectively
for VWIS. In the case of RIY, the effect was almost the
same in 20 week old rats (−15.6±1.8mmHg) and 30 week
old rats (−15.8±3.7mmHg), whereas administration of
IY to older rats exerted lower hypotensive effect than in
young rats (−15.5±2.1mmHg in 20-week-old rats and
−10.5±2.0mmHg in 30-week-old rats) (Fig. 7). These re-
sults suggest that in the case of RIY, which had the same
hypotensive effect in both young and old rats, another mech-
anism besides ACE inhibition may also be involved in low-
ering blood pressure.
Fig. 7. Comparison of blood pressure lowering activity of IY and RIY 2h
after oral administration at the dosage of 10mg/kg to 20- and 30-week-old
SHR. Decreases of systolic blood pressure in relation to time zero are
expressed as mean ±S.E.M.
ACE inhibitory activity of all isolated peptides was much
lower than activity of synthetic ACE inhibitor captopril.
However, when the antihypertensive effects are compared,
VWIS is less potent than captopril 4h after oral administra-
tion only by a factor of 6 on weight base and by a factor of
2.6 on molar base. In the case of RIY it is less active than
captopril 4h after oral administration only by a factor 2.3
on weight base and by a factor of about 1.1 on molar base,
what means that RIY shows almost the same antihyperten-
sive effect to captopril after oral administration to SHR on
Fig. 8. Comparison of antihypertensive effect of pepsin- and subtilisin di-
gest of rapeseed protein (0.5g/kg) after oral administration to 20-week-old
SHR. Changes of systolic blood pressure from time zero were expressed
as mean ±S.E.M. ∗P<0.05, ∗∗P<0.01 indicate significant difference
against control.
E.D. Marczak et al./Peptides 24 (2003) 791–798 797
Fig. 9. Antihypertensive effect of different dosages of subtilisin digest of
rapeseed protein after oral administration to 20-week-old SHR. Changes
of systolic blood pressure from time zero were expressed as mean±S.E.M.
∗P<0.05, ∗∗P<0.01 indicate significant difference against control.
molar base (Table 4). RIY exerted also long-lasting antihy-
pertensive activity comparable to captopril [11].
3.5. Antihypertensive activity of rapeseed protein digests
It has been observed that oral administration of subtilisin
and pepsin digests of rapeseed protein lowered blood pres-
sure in SHR. A comparison of their antihypertensive effect
at the dosage 0.5g/kg (Fig. 8) shows that both digests have
maximum antihypertensive effect 4h after oral administra-
tion. Subtilisin digest exerted not only more than twice the
antihypertensive effect (−15.5±2.6mmHg) of the pepsin di-
gest(−6.8±1.7 mmHg) 4 hafter administration, butalsosig-
nificantly lowered blood pressure 2 and 6h after administra-
tion, when pepsin digest was not effective. Subtilisin digest
of rapeseed protein displayed dose-dependent antihyperten-
sive effect after oral administration to SHR (Fig. 9) and its
effect was significant even at a single dosage of 0.15g/kg
(−5.5±1.7mmHg; P<0.05). ACE inhibitory activity of
the subtilisin digest of rapeseed protein was changed only
Table 5
Effect of hydrolysis of rapeseed subtilisin digest with different proteases
on its ACE inhibitory activity (time of hydrolysis: 5h; amount of enzyme:
1%)
Enzyme used for digestion IC50 (mg/ml)
Subtilisin 0.16
Subtilisin–pepsin 0.18
Subtilisin–pepsin–pancreatin 0.27
Subtilisin–pepsin–trypsin 0.14
Subtilisin–pepsin–chymotrypsin 0.18
Subtilisin–pancreatin 0.22
Subtilisin–trypsin 0.14
Subtilisin–chymotrypsin 0.16
slightly after treatment with different enzymes (Table 5),
demonstrating that ACE inhibitory peptides present in sub-
tilisin digest of rapeseed protein are relatively resistant to
digestion by enzymes present in digestive tract.
4. Discussion
VW, VWIS, IY and RIY are potent ACE inhibitory pep-
tides isolated from subtilisin digest of rapeseed protein.
Among them RIY and VWIS are new sequences with IC50
28 and 30M, respectively, and IY and VW are previ-
ously known ACE inhibitors with IC50 3.7 and 1.6M,
respectively.
ACE inhibitory peptides can be classified into three
groups, depending on their interaction with ACE [11].
The first group comprises true inhibitors, which activity
is not changed by pre-incubation with ACE. The second
group comprises substrates for ACE, which converts these
substrates to inactive peptides, resulting in considerably
reduced activity of such peptides following pre-incubation
with ACE. The third group consists of so called pro-drug
inhibitory peptides. They are also substrates for ACE, but
they are converted by this enzyme to true inhibitors, result-
ing in increased activity after pre-incubation with ACE. In
our experience only true inhibitors and pro-drugs have abil-
ity to lower blood pressure [9–11,22]. Among the peptides
isolated from rapeseed subtilisin digest, IY and VW can
be considered true ACE inhibitors because IC50 values for
these peptides before and after pre-incubation with ACE
were found to be the same. VWIS is pro-drug type ACE
inhibitor, as pre-incubation with ACE of VWIS intensified
inhibitory activity of this peptide by a factor of 6. This is
also confirmed by HPLC analysis of reaction mixture after
pre-incubation (Fig. 2A) showing that ACE quickly con-
verts VWIS releasing VW, which is a true ACE inhibitor.
RIY may also be regarded as a pro-drug ACE inhibitor, be-
cause activity of RIY was increased by ACE by a factor of
1.4, although RIY is very slowly converted by ACE since
only a small amount of RIY was converted to IY (Fig. 2B).
VW, VWIS, IY and RIY caused an antihypertensive ef-
fect after oral administration to SHR. VW, VWIS and IY
had a maximum antihypertensive effect 2h after admin-
istration. In the case of VWIS, which is a pro-drug type
ACE inhibitory peptide, the peak of activity is expected
to occur later due to a delay caused by true inhibitor re-
lease. A similar time-course of the antihypertensive effect
of VW and VWIS can be explained by quick conversion
of VWIS to VW in vivo. Nevertheless, when dosage is
calculated on a molar basis, VWIS had almost two times
more potent antihypertensive effect than VW 4h after ad-
ministration. RIY is found to be a unique peptide, because
unlike other reported di- and tri-peptidic ACE inhibitors
[10,11], it exerted a long-lasting antihypertensive effect with
a maximum occurring 4h after oral administration. More-
over, in opposite to the action of ACE inhibitors, it was
798 E.D. Marczak et al./Peptides 24 (2003) 791–798
unaffected by the age of the SHR, which suggests that in
the case of RIY, some additional mechanism besides that of
ACE inhibition, may also be responsible for its hypotensive
action.
All peptides had much lower ACE inhibitory activity
in vitro than synthetic ACE inhibitor captopril. However,
when the antihypertensive effect in vivo is compared,
ACE-inhibitory peptides isolated from subtilisin digest
of rapeseed protein had only slightly lower activity than
captopril. This indicates that ACE inhibitors derived from
protein digests possess higher in vivo activities than the
efficacy levels extrapolated from in vitro activities. Similar
effects could be find in the case of ACE-inhibitory peptides
derived from other protein digests [11]. This phenomenon
may be attributed to higher affinity of these peptides to tis-
sue and slower elimination than did a synthetic compound
captopril.
Subtilisin digest of rapeseed protein exerted a long-lasting
antihypertensive effect when administered orally to SHR.
Properties of RIY might contribute to such long-lasting an-
tihypertensive effect of this digest. At a single dosage of
0.5g/kg rapeseed subtilisin digest caused significant hy-
potensive effect 2, 4 and 6h following oral administration
with a maximum (−15.5±2.6 mmHg) occurring 4 h after ad-
ministration. It significantly lowered blood pressure 4h after
oral administration even at a single dosage of 0.15g/kg. It
was found that ACE inhibitory peptides present in subtilisin
digest of rapeseed protein are relatively stable to digestion
by enzymes present in the digestive tract, as ACE inhibitory
activity of the subtilisin digest after treatment with different
enzymes was changed only slightly (Table 5). Subtilisin di-
gest of rapeseed protein, due to its digestion-resisting prop-
erties and long-lasting antihypertensive activity, may poten-
tially be used as a functional food useful in prevention and/or
treatment of hypertension.
Acknowledgments
This work was supported by Postdoctoral Fellowship of
the Japan Society for the Promotion of Science for Foreign
Researchers (for E. Marczak).
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