Microencapsulation and modification of synthetic peptides of food proteins reduces the blood pressure of spontaneously hypertensive rats

Article abstract of DOI:10.1021/jf020900u

Synthetic peptides were microencapsulated into liposomes, cycled with a disulfide bond or modified with D-phenylglycine (D-phg) at the N-terminal, and their antihypertensive effects as orally administered (0.18 mM/kg body weight) to spontaneously hypertensive rats (SHR) were measured. The microencapsulated Leu-Lys-Pro reduced significantly the systolic blood pressures of SHR by 45 mmHg and showed a prolonged duration, revealing the significant protective effect of encapsulation. D-phg-Leu-Arg-Pro showed a duration about 2 h shorter than that of the peptide without modification. In addition, cyclic Leu-Arg-Pro peptide with a disulfide bond between the N- and C-terminal amino acids reduced the systolic blood pressure of SHR by 35 mmHg and displayed a lengthy duration.

Full text of DOI:10.1021/jf020900u

J. Agric. Food Chem. 2003, 51, 1671−1675 1671
Microencapsulation and Modification of Synthetic Peptides of
Food Proteins Reduces the Blood Pressure of Spontaneously
Hypertensive Rats
TZY-LI CHEN,† YUNG-CHIEH LO,† WEI-TZE HU,† MING-CHANG WU,‡
SHUI-TEN CHEN,§ AND HUNG-MIN CHANG*,†
Graduate Institute of Food Science & Technology, National Taiwan University,
Taipei 106-17, Taiwan, Department of Food Science and Technology, National Pingtung University of
Science and Technology, Pingtung 912-01, Taiwan, and Institute of Biochemistry, Academia Sinica,
Taipei 115, Taiwan
Synthetic peptides were microencapsulated into liposomes, cycled with a disulfide bond or modified
with D-phenylglycine (D-phg) at the N-terminal, and their antihypertensive effects as orally administered
(0.18 mM/kg body weight) to spontaneously hypertensive rats (SHR) were measured. The microen-
capsulated Leu-Lys-Pro reduced significantly the systolic blood pressures of SHR by 45 mmHg and
showed a prolonged duration, revealing the significant protective effect of encapsulation. D-phg-Leu-
Arg-Pro showed a duration about 2 h shorter than that of the peptide without modification. In addition,
cyclic Leu-Arg-Pro peptide with a disulfide bond between the N- and C-terminal amino acids reduced
the systolic blood pressure of SHR by 35 mmHg and displayed a lengthy duration.
KEYWORDS: Angiotensin-converting enzyme inhibitor; oligopeptide; liposome; modification; spontane-
ously hypertensive rats (SHR)
INTRODUCTION
nine, or proline on the C-terminal and valine or isoleucine on
the N-terminal exhibited potent ACE inhibitory activity. Ko-
hmura et al. (17) synthesized some peptide fragments of human
â-casein and found that the length of those peptides had an
influence on the ACE inhibitory activity. Namely, peptides
composed of 3-10 amino acids with proline on the C-terminal
were necessary for ACE inhibitors (17, 18). Blackburn et al.
(19) tested the synthetic peptides, Ser-Lys-Pro, Ser-Gln-Pro, and
Gln-Ser-Pro, and pointed out the importance of the peptide
sequence to the ACE inhibitory activity. Thus far, the peptide
Leu-Arg-Pro from food protein hydrolysates has been reported
to be the most potent natural ACE inhibitor, with an IC50 value
of 0.27 (17) or 1.0 µM (18).
However, peptides orally administered to reduce blood
pressure are liable to be digested and lose their activities. Micro-
encapsulation using a spray-drying method is an industrially
important process for physically coating solids, gases, and
liquids with a thin, protective layer or wall of material to inhibit
their loss by volatilization and to protect them against chemical
deterioration (22, 23). Other encapsulations, such as formation
of liposomes, using phospholipids such as lecithin as lipid
bilayers have been reported to be effective in encapsulating
drugs and other bioactive substances (24, 25). Since liposome
is biodegradable (26), it is potentially useful as a drug delivery
system that would transport desired drugs to a specific site
(27, 28).
Enzymatic hydrolysis of food proteins has produced various
biologically active peptides with immunostimulating (1, 2),
opioid (3, 4), antithrombotic (5), caseino-phosphopeptic (6, 7),
bactericidal (8), or angiotensin-converting enzyme (ACE)
(dipeptidyl carboxypeptidase, EC 3.4.15.1) inhibitory (9, 10)
functions and has been the focus of recent research. ACE cleaves
angiotensin I to the potent vasodepressor angiotensin II and
releases C-terminal dipeptide (11). As a consequence, the
angiotensin II causes the constriction of blood vessels in the
renin-angiotensin system and the release of aldosterone from
the adrenal cortex, which leads to an accumulation of sodium
ions in the kidneys and increases the systolic blood pressure
(12). In addition, ACE also hydrolyzes and inactivates brady-
kinin, a vasodilator, and increases the blood pressure (13).
Therefore, development of substances that inhibit ACE activity
has been a main interest in recent research (14, 15). ACE
inhibitors have been synthesized for clinical treatment of
hypertension, such as Captopril, which exhibits potent competi-
tive inhibition on ACE (14).
Cheung et al. (16) and Ariyoshi (10) pointed out that peptides
with aromatic amino acids, such as tryptophan and phenylala-
* To whom correspondence should be addressed (E-mail changhm@
ccms.ntu.edu.tw, Telephone +886-2-2363-0231 ext 2776, Fax +886-2-2362-
0849).
† National Taiwan University.
Peptide modification is also one of the available approaches
to retain peptide activity. Terada et al. (29) compared the
‡ National Pingtung University of Science and Technology.
§ Academia Sinica.
10.1021/jf020900u CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/04/2003

1672 J. Agric. Food Chem., Vol. 51, No. 6, 2003
Chen et al.
tometer. Sample with hydrochloric acid added before mixing with ACE
was used as a blank, while the peptide sample replaced by the same
volume of distilled water was treated as a control. The inhibitory ratio
(%) of ACE was calculated as (C - A)/(C - B) × 100%, where A is
A228 of the inhibitor, B is A228 of the blank, and C is A228 of the control.
The concentration of the peptide sample in the reaction mixture with
substrate was controlled to exhibit ACE inhibitory activity between
40 and 60% (12, 32). Each peptide sample was tested at a minimum
of three concentrations to construct the standard cure for the determi-
nation of the IC50 value (concentration of inhibitor required to inhibit
50% of the ACE activity). Each peptide sample was tested in triplicate.
Preparation of Liposome. Liposomes were prepared according to
the method described by Shimizu et al. (33) and Chang et al. (25) with
dehydration-rehydration processes. Initially, egg lecithin and choles-
terol (Sigma) in a ratio of 1:0.25 (total lipid, 300 µmol) were dissolved
in 5 mL of chloroform, and the mixtures were dried (50 ( 2 °C) under
reduced pressure in round-bottom flasks with a rotary evaporator (model
Re-111, Buchi, Flawil, Switzerland) to form a thin film on the flasks.
Subsequently, 2 mL of distilled water was added to the flask, and the
mixture was agitated with glass beads (0.5 cm in diameter) at 60 ( 2
°C for 5 min, followed by incubation at ambient temperature for 10
min. Next, 2 mL of Leu-Lys-Pro [5.0 mg/mL in 0.01 M phosphate
buffer saline (PBS), pH 7.2] was thoroughly mixed with the above
suspension and dehydrated in a freeze-dryer (Eyela FD-5N, Rikakikai
Co., Tokyo, Japan). The freeze-dried material was rehydraded by adding
2 mL of distilled water, and the mixture was centrifuged (30000g, 30
min, 5 °C) after incubation at ambient temperature (25-28 °C) for 10
min. The supernatants thus obtained were sampled to determine the
encapsulation efficiency (EE, %) (33) of the peptide by the TNBS
method described above: EE (%) of peptide ) [total peptide (mg) in
solution - peptide (mg) in supernatant of liposomal suspension/total
peptide (mg) in solution] × 100%. Each sample was tested in triplicate.
Antihypertensive Activity of Peptides. The effect of orally
administered peptides on the systolic blood pressure was determined
according to the method described by Maruyama and Suzuki (32) with
minor modification. Twenty male spontaneously hypertensive rats (6
week of age) (Experimental Animal Center, National Taiwan Univer-
sity) were raised in an air-conditioned room (24 °C). All rats had free
access to Lab Diet (5001 Rodent Diet, PMI, St. Louis, MO) and water.
After being raised for eight more weeks, SHR were randomly divided
into four groups (five rats/group) and warmed for 10 min in a 39 (
1 °C thermostated box before each measurement of blood pressure.
Tail systolic blood pressure was measured four times at each the desired
time using a indirect blood pressure meter (BP-98-A meter, Softron
Co. Ltd., Tokyo, Japan) for each treatment.
proteolytic coefficients of linear and cyclic hexapeptides against
pepsin and trypsin and concluded that cyclic formation of the
peptide was beneficial to its stability. In addition, Wang et al.
(30) pointed out the advantages of coupling D-phenylglycine
(D-phg) to R-methyldopa in an investigation of the concentration
dependence of the intestinal bruss-border membrane vesicle
(BBMV) uptake of R-methyldopa. They found that the level of
D-phg bound to R-methyldopa was linearly related (r 2 ) 0.99)
to the R-methyldopa uptake through the BBMV. In addition,
the oral bioavailability of D-phg-R-methyldopa in rabbit blood
was 83% when it was orally administered.
In the present research, to retain the activities of peptides in
the gastrointestinal tract, peptides were encapsulated in lipo-
somes or modified into cyclic form by a disulfide bond linked
between N- and C-terminal amino acids. Subsequently, the
changes in systolic blood pressure of spontaneously hypertensive
rats (SHR) induced by different types of peptides were measured
and compared with the change induced by a potent clinical ACE
inhibitor, Captopril.
MATERIALS AND METHODS
Synthesis, Purification, and Mass Analysis of Oligopeptide. All
the amino acids referred to in this paper were of L-configuration. The
protected N-Fmoc-L-amino acids (Fmoc ) fluoren-9-ylmethoxycarbo-
nyl) and D-phg for the solid-phase synthesis (Instruction to Cleavage
Techniques, Rainin Instrument Co., Inc., Mack Woburn, MA) were
the products of ABI Co. (Foster City, CA). Two oligopeptides, Leu-
Lys-Pro and Leu-Arg-Pro, cyclic Leu-Arg-Pro with a disulfide bond
between Leu and Pro, and D-phg-Leu-Arg-Pro were synthesized with
a PS-3 automated solid-phase peptide synthesizer (Rainin Instrument
Co., Inc.) using preloaded proline as the resin (17).
After being cleaved from the solid support, the peptides were purified
to homogeneity by reversed-phase high-performance liquid chroma-
tography (HPLC) (S-3702 variable-wavelength UV-vis detector, Soma,
Tokyo, Japan, with pump PU-980, Jasco, Tokyo, Japan) on a C18
column (250 mm in length and 4.6 mm in diameter) (Protein and
Peptide C18, Vydac, Hesperia, CA) under the following experimental
conditions: eluent A, 5.0% acetonitrile (ACN)/0.1% trifluroroacetic
acid (TFA) aqueous solution; eluent B, 95% ACN/0.1% TFA aqueous
solution. Purification conditions: 0-100% eluent B; flow rate, 1.0 mL/
min; separation time, 20 min. All the solutions were filtered through a
0.45-µm membrane and then deaerated by sonication prior to use.
The purity of each synthetic peptide was further confirmed by mass
spectrometric analysis (Finnigan MAT, LCQ, San Jose, CA) to reach
a level higher than 95% (data not shown).
The tested peptides (Leu-Lys-Pro, Leu-Arg-Pro, cyclic Leu-Arg-
Pro, and D-phg-Leu-Arg-Pro) were dissolved in 1.0 mM normal saline
before administration. After being warmed up, rats were orally
administered (0.18 mmol/kg bw) with peptides or normal saline
(control). Tail systolic blood pressures were measured at 2-h intervals
(0, 2, 4, 6, 8, 10, and 12 h) after the administration. Captopril [(2S)-
N-(3-mercapto-2-methylpropionyl)-L-proline] (Sigma), a potent ACE
inhibitor for clinical hypertension treatment, was used as a positive
control (0.18 m mol/kg bw) in vivo for comparison with the synthetic
peptides.
Statistical Analysis. Analysis of variances of results was carried
out using the General Linear Model Procedure of SAS Statistical
Software, version 6.11 (SAS Institute, Cary, NC, 1995) (31). Multiple
comparisons of means were carried out by Duncan’s multiple range
tests at p < 0.05 (*) and p < 0.01 (**).
Determination of Peptide. The peptide concentration in the sample
was determined according to the TNBS (2,4,6-trinitrobenzenesulfonic
acid) method described by Adler-Nissen (31). Various levels (0.025,
0.0625, 0.125, and 0.25 mM) of L-leucine solutions were used to
construct the standard curve (r 2 ) 0.98) for the calibration of peptide
concentration.
Assay of ACE Inhibitory Activity. The synthetic peptides were
assayed in vitro for their capacity to inhibit the ACE activity with the
method described by Cushman and Cheung (12) and Maruyama and
Suzuki (32). To 300 µL of 5.0 mM hippuryl-L-histidyl-L-leucine (Sigma,
St. Louis, MO)/300 mM NaCl/100 mM borate buffer solution (pH 8.3)
was added 30 µL of peptide, and the mixture was then preincubated in
a water bath at 37 ( 1 °C for 30 min. Next, 50 µL (8.0 mU) of ACE
(Sigma)/300 mM NaCl/100 mM borate buffer solution (pH 8.3) was
mixed with the above substrate solution to initiate the enzyme reaction.
After incubation of the enzyme-substrate mixture in a water bath at
37 ( 1 °C for 30 min, 1 N hydrochloric acid was added to terminate
the reaction. The liberated hippuric acid was extracted with 1.5 mL of
ethyl acetate by vortex mixing for 15 s. After a brief centrifugation
(3600g, 5 min), a 1.0-mL aliquot of each acetyl acetate layer was
transferred to a test tube and then dried in a boiling water bath. Distilled
water (1.0 mL) was added to dissolve the hippuric acid, and the
absorbance of the solution at 228 nm was monitored by a spectropho-
RESULTS AND DISCUSSION
IC50 Values of Synthetic Peptides. The potent ACE inhibitor
Leu-Arg-Pro, with IC50 values of 0.27 and 1.0 µM, has
previously been isolated from protein hydrolysates of R-zein
(20) and fish (21), respectively. However, the synthetic Leu-
Arg-Pro (Table 1) showed an IC50 value of about 1.17 µM,
higher than the reported values. Variations in determining the
IC50 value could be responsible for the difference. On the other

Microencapsulation and Modification of ACEI on SHR
J. Agric. Food Chem., Vol. 51, No. 6, 2003 1673
Table 1. IC50 Valuesa of Modified Oligopeptidesb and Encapsulation
Efficiency (EE)c of Leu-Lys-Pro in Liposomed
oligopeptideb
IC50 (µM)a
EE (%)c
Leu-Lys-Pro
Leu-Arg-Pro
D-phg-Leu-Arg-Pro
0.87 ± 0.13
1.17 ± 0.24
4.55 ± 0.22
61.8 ± 2.7
e
e
a Concentration of an inhibitor required to inhibit 50% of ACE activity. b See
the methods in the text. c (Weight of added peptide − weight of uncapsulated
peptide)/weight of added peptide × 100%. d Lecithin/cholesterol ) 1:0.25; total
lipid content in liposome was 300 µmol/mL; rehydration temperature 60 ± 2 °C.
e Not determined.
Figure 1. Antihypertensive effect of synthetic oligopeptides on spontane-
ously hypertensive rats (p < 0.01, **). Fourteen-week old rats were
randomly divided into groups (five rats/group) and orally administered (0.18
mmol/kg bw). The control group received the same volume of normal
saline.
Figure 2. Changes in systolic blood pressure of spontaneously hyper-
tensive rats orally administered (0.18 mmol/kg bw) with synthetic peptide
(p < 0.05, *; p < 0.01, **). Captopril was used as the positive control. The
control group received the same volume of normal saline.
hand, the synthetic Leu-Lye-Pro showed an IC50 value of 0.87
µM, similar to that for the protein separated from food proteins
(35), revealing no diversity of bioactivity in synthetic and
hydrolyzed proteins (36).
Antihypertensive Activity of Encapsulated Peptides. Leu-
Lys-Pro was encapsulated in liposomes with an EE of about
62%. The EE of immunoglobulin in yolk in liposomes was
reported to be 30% (33) and 70% (25), depending upon the
level and composition of lipids as well as the methods used
(33, 37). An increase in freeze-drying-rehydration cycles was
favorable for increasing EE (25, 38).
Figure 1 represents the changes in tail systolic blood
pressures of SHR orally administered (0.18 mmol/kg bw) Leu-
Lys-Pro. It was observed that the blood pressure remarkably
declined at the fourth hour after administration. The maximal
reduction of blood pressure of SHR administered with this
peptide without encapsulation was about 30 mmHg. However,
the encapsulated peptide in liposomes exhibited more significant
(p < 0.01, **) reduction effects on blood pressure at the fourth
and sixth hours and, furthermore, showed a maximal reduction
effect of 45 mmHg between the sixth and tenth hours after the
administration. Therefore, it was clear that the cholesterol-
lecithin bilayers of liposomal structure facilitated the peptide
stability against proteases in vivo. Some similar results showed
the efficiency of liposomal structures in protecting IgY specific
against certain antigens or amylase against the pepsin hydrolysis
in strong acidic pH conditions (25, 33, 38). In addition, emulsion
formation of sardine hydrolysates by 30% egg yolk were also
reported to be effective in enhancing the antihypertensive effect
on SHR (39).
the fourth to the tenth hours after oral administration (Figure
2A). However, Leu-Arg-Pro peptide reduced the blood pressure
by about 15 mmHg at the fourth hour and showed a maximal
reduction effect of about 35 mmHg at the eighth hour after oral
administration. In contrast, the D-phg-Leu-Arg-Pro peptide
showed a significant (p < 0.05, *) reduction effect (20 mmHg)
of blood pressure at the fourth hour and reached the maximal
reduction effect of about 35 mmHg (p < 0.01, **) at the sixth
hour after oral administration. It was observed that the duration
of this modified peptide was about 2 h shorter than that of the
unmodified peptide (Figure 2B). Therefore, the D-phg group
appeared to facilitate the absorption of the modified peptide,
as it did in increasing the uptake and bioactivity of R-methyldopa
through the BBMV of rabbits (30). D-phg has been used as a
“seeing-eye dog” and, coupled with drugs with amino acid
structure, as a type of prodrug, which is hydrolyzed to release
drug after absorption in small intestine through the oligopeptide
transporter system (30).
Figure 3 shows the reduction profile of blood pressure of
SHR induced by cyclic Leu-Arg-Pro peptide. It was obvious
that the maximal reduction effect of blood pressure induced by
this cyclic peptide was about 35 mmHg, similar to that induced
by linear peptide; however, a prolonged duration of modified
peptide was observed. Terada et al. (29) indicated that the cyclic
hexapeptide (-Gly2-Phe2-Gly-Lys-) was not hydrolyzed at all
by pepsin at 37 °C and at pH values between 1.5 and 4.0.
However, it showed a proteolytic coefficient as low as 0.015-
0.025 when incubated with 2.0 mg of trypsin/mL at 30 °C and
pH values between 7.0 and 9.0. In the present study, Leu-Arg-
Pro peptide was cycled with a disulfide bond between N- and
C-terminal amino acids and displayed a marked improve-
ment in duration, revealing the stability of the cyclic peptide
(Figure 3).
Antihypertensive Activity of Modified Peptides. The clini-
cal ACE inhibitor, Captopril, exhibited a fast and potent
antihypertensive effect, reducing the systolic blood pressure of
SHR from about 200 to 160 mmHg at the second hour, and
then maintained the blood pressure at about 170 mmHg from

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SHR, spontaneously hypertensive rat; ACEI, angiotensin-
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Received for review August 21, 2002. Revised manuscript received
December 6, 2002. Accepted December 6, 2002.
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