U.S. patent application number 13/604969 was filed with the patent office on 2014-03-06 for peptides and use thereof in the inhibition of angiotensin converting enzyme.
This patent application is currently assigned to FOOD INDUSTRY RESEARCH AND DEVELOPMENT INSTITUTE. The applicant listed for this patent is CHIEN-TI CHANG, CHU-CHIN CHEN, YI-HONG CHEN, FU-NING CHIEN, SHIAO-CHENG CHUANG, MING-YU HUNG, HSIANG-LING LAI, SU-ER LIOU, YU-HUI LIU. Invention is credited to CHIEN-TI CHANG, CHU-CHIN CHEN, YI-HONG CHEN, FU-NING CHIEN, SHIAO-CHENG CHUANG, MING-YU HUNG, HSIANG-LING LAI, SU-ER LIOU, YU-HUI LIU.
Application Number | 20140066361 13/604969 |
Document ID | / |
Family ID | 50188357 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140066361 |
Kind Code |
A1 |
CHEN; YI-HONG ; et
al. |
March 6, 2014 |
PEPTIDES AND USE THEREOF IN THE INHIBITION OF ANGIOTENSIN
CONVERTING ENZYME
Abstract
Peptides useful as angiotensin converting enzyme inhibitors are
provided. Also provided are compositions comprising one or more of
the peptides and methods for preventing, treating and/or
diminishing one or more syndromes associated with angiotensin
converting enzyme by using the peptides.
Inventors: |
CHEN; YI-HONG; (Hsinchu
City, TW) ; LAI; HSIANG-LING; (Hsinchu City, TW)
; CHUANG; SHIAO-CHENG; (Hsinchu City, TW) ; CHANG;
CHIEN-TI; (Hsinchu City, TW) ; HUNG; MING-YU;
(Hsinchu City, TW) ; LIU; YU-HUI; (Hsinchu City,
TW) ; LIOU; SU-ER; (Hsinchu City, TW) ; CHIEN;
FU-NING; (Hsinchu City, TW) ; CHEN; CHU-CHIN;
(Hsinchu City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHEN; YI-HONG
LAI; HSIANG-LING
CHUANG; SHIAO-CHENG
CHANG; CHIEN-TI
HUNG; MING-YU
LIU; YU-HUI
LIOU; SU-ER
CHIEN; FU-NING
CHEN; CHU-CHIN |
Hsinchu City
Hsinchu City
Hsinchu City
Hsinchu City
Hsinchu City
Hsinchu City
Hsinchu City
Hsinchu City
Hsinchu City |
|
TW
TW
TW
TW
TW
TW
TW
TW
TW |
|
|
Assignee: |
FOOD INDUSTRY RESEARCH AND
DEVELOPMENT INSTITUTE
Hsinchu City
TW
|
Family ID: |
50188357 |
Appl. No.: |
13/604969 |
Filed: |
September 6, 2012 |
Current U.S.
Class: |
514/1.9 ;
514/14.9; 514/16.2; 514/16.3; 514/21.8; 514/21.9; 514/21.91;
530/330; 530/331; 548/496; 562/445; 562/450; 562/561 |
Current CPC
Class: |
A61P 9/12 20180101; A61P
9/04 20180101; C07K 7/06 20130101; C07K 17/06 20130101; A61P 9/00
20180101; A61P 3/00 20180101; A61K 38/556 20130101; A61P 7/02
20180101; A61P 9/10 20180101; A61K 38/08 20130101 |
Class at
Publication: |
514/1.9 ;
562/445; 530/331; 530/330; 562/450; 562/561; 548/496; 514/21.91;
514/21.9; 514/21.8; 514/16.3; 514/16.2; 514/14.9 |
International
Class: |
C07K 5/083 20060101
C07K005/083; C07K 5/113 20060101 C07K005/113; C07K 5/087 20060101
C07K005/087; C07K 7/06 20060101 C07K007/06; C07K 5/093 20060101
C07K005/093; C07K 5/062 20060101 C07K005/062; C07K 5/068 20060101
C07K005/068; C07K 5/103 20060101 C07K005/103; C07K 5/078 20060101
C07K005/078; A61K 38/05 20060101 A61K038/05; A61K 38/06 20060101
A61K038/06; A61K 38/07 20060101 A61K038/07; A61K 38/08 20060101
A61K038/08; A61P 9/12 20060101 A61P009/12; A61P 9/10 20060101
A61P009/10; A61P 7/02 20060101 A61P007/02; A61P 9/04 20060101
A61P009/04; A61P 9/00 20060101 A61P009/00; A61P 3/00 20060101
A61P003/00; C07K 5/065 20060101 C07K005/065 |
Claims
1. An isolated peptide of Phe-Val-Asn-His-Phe (SEQ ID NO: 2).
2. A composition comprising an isolated peptide of
Phe-Val-Asn-His-Phe (SEQ ID NO: 2) and one or more carriers,
diluents, solvents, colorants, anti-oxidants, inert materials
and/or additives.
3. The composition of claim 2, which is a pharmaceutical
composition.
4. The composition of claim 2, which is a food composition.
5. The composition of claim 4, which is a nutraceutical or a food
additive.
6. A method for inhibiting angiotensin converting enzyme in a
subject, comprising administering to the subject a composition of
claim 2.
7. The method of claim 6 for treating cardiovascular complications
in the subject, wherein the cardiovascular complications are
selected from arterial hypertension, systolic hypertension,
peripheral vascular disease, atherosclerosis, restenosis, heart
failure, cardiac insufficiency, thrombosis and any thromboembolic
events, angina pectoris, cerebral vascular accidents, coronary
artery disease, myocardial infarction, vascular remodeling, and the
combination thereof.
8. (canceled)
9. The method of claim 6 for reducing hypertrophy and/or
hyperplasia of adipocytes in the subject.
10. The method of claim 9 for reducing intraperitoneal fat and/or
subcutaneous fat in the subject.
11. A method for inhibiting angiotensin converting enzyme in a
subject, comprising administering to the subject an effective
amount of an isolated peptide of Phe-Val-Asn-His-Phe (SEQ ID NO:
2).
12. The method of claim 11 for treating cardiovascular
complications in the subject, wherein the cardiovascular
complications are selected from arterial hypertension, systolic
hypertension, peripheral vascular disease, atherosclerosis,
restenosis, heart failure, cardiac insufficiency, thrombosis and
any thromboembolic events, angina pectoris, cerebral vascular
accidents, coronary artery disease, myocardial infarction, vascular
remodeling, and the combination thereof.
13. (canceled)
14. The method of claim 11 for reducing hypertrophy and/or
hyperplasia of adipocytes in the subject.
15. The method of claim 14 for reducing intraperitoneal fat and/or
subcutaneous fat in the subject.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel peptides which are
useful in the inhibition of angiotensin converting enzyme.
BACKGROUND OF THE INVENTION
[0002] Angiotensin converting enzyme (ACE) is present mainly in
human vascular endothelial cells, lungs, kidneys and brains. This
enzyme may remove two amino acid residues (His-Leu) from the
C-terminus of the inactived angiotensin I to form an activated
angiotensin II so as to cause contraction of blood vessels and
increase blood pressure (Maruyama, S. and Suzuki, H., A peptide
inhibitor of angiotensin I converting enzyme in the tryptic
hydrolysate of casein, Agric. Biol. Chem., 1982, 46: 1393-1394).
Maruyama, S. et al. (Angiotensin I-converting enzyme inhibitor
derived from an enzymatic hydrolysate of casein. II. Isolation and
bradykinin-potentiating activity on the uterus and the ileum of
rats, Agric. Biol. Chem., 1985; 49: 1405-1409) found that ACE may
inactivate bradykinin (having blood vessel dilating activity) so as
to increase blood pressure. Therefore, the binding of an
angiotensin converting enzyme inhibitor (ACEI) with ACE may reduce
the formation of angiotensin II and the inactivation of bradykinin,
so application of an ACEI may ameliorate hypertensive symptoms.
[0003] Krysiak, R. et al, (The effect of angiotensin-converting
enzyme inhibitors on plasma adipokine levels in normotensive
patients with coronary artery disease, Polish Journal of
Endocrinology, 2010, 61: 280-286) discloses that ACE inhibitors are
effective agents in patients with coronary artery disease (CAD),
even if their blood pressure is within normal limits. U.S. Pat. No.
8,021,697 B2 discloses that ACE inhibitors can alter the
distribution of body mass by decreasing overall percentage of fat
and/or increasing the ratio of lean mass to fat mass. In other
words, ACE inhibitors can reduce the amount of fat that is produced
from consumed food.
[0004] In addition to chemically synthesized drugs, it has been
found that many peptides having different lengths and amino acid
residues can effectively inhibit ACE. The peptides having ACE
inhibitory activity can be isolated from foods including, e.g.,
hydrolysates of animal or plant proteins, such as casein (Maruyama
and Suzuki, 1982; Maruyama, S. and Suzuki, H., 1985; and Yamamoto,
N. et al., Antihypertensive effect of peptides derived from casein
by an extracellular proteinase from Lactobacillus helveticus CP790,
J. Dairy Sci., 1994, 77: 917-922), corn protein (e.g.,
.alpha.-zein) (Miyoshi et al., Structures and activity of
angiotensin-converting enzyme inhibitors in an .alpha.-zein
hydrolysate, Agric. Biol. Chem., 1991, 55: 1313-1318 and Yano, S.
et al., Isolation from .alpha.-zein of thermolysin peptides with
angiotensin I-converting enzyme inhibitory activity, Biosci.
Biotech. Biochem., 1996, 60: 661-663), sardine (Matsui, T. et al.,
Inhibition of angiotensin I-converting enzyme by Bacillus
licheniformis alkaline protease hydrolyzates derived from sardine
muscle, Biosci. Biotech. Biochem., 1993, 57: 922-925 and Matsufuji,
H. Et al., Angiotensin I-converting enzyme inhibitory peptides in
an alkaline protease hydrolyzate derived from sardine muscle,
Biosci. Biotech. Biochem., 1994, 58: 2244-2245) and bonito
(Matsumura, N. et al., Isolation and characterization of
angiotensin I-converting enzyme inhibitory peptides derived from
bonito bowels, Biosci. Biotech. Biochem., 1993, 57: 1743-1744 and
Fujita, H. et al., Antihypertensive effect of thermolysin digest of
dried bonito in spontaneously hypertensive rat, Clin. Exp.
Pharmacol. Physiol. Suppl., 1995, 22: S304-S305), and fermented
foods, such as sake and wine residue (Saito, Y. et al., Structure
and activity of angiotensin I converting enzyme inhibitory peptides
from sake and sake lees, Biosci. Biotechnol. Biochem., 1994, 58:
1767-1771), soy sauce (Kinoshita, E. et al., Purification and
identification of an angiotensin I-converting enzyme inhibitor from
soy sauce, Biosci. Biotechnol. Biochem., 1993, 57: 1107-1110),
cheese (Okamoto, A. et al., Angiotensin I converting enzyme
inhibitory activities of various fermented foods, Biosci.
Biotechnol. Biochem., 1995, 59: 1147-1149) and sour milk (Masuda,
O. et al., Antihypertensive peptides are present in aorta after
oral administration of sour milk containing these peptides to
spontaneously hypertensive rats, J. Nutr., 1996, 126:
3063-3068).
[0005] JP7289281 (A) discloses that the fermented product of soy
bean with Aspergillus niger has ACE inhibitory activity. JP4299991
(A) discloses that a peptide product obtained by hydrolyzing soy
bean with bromelain (a proteinase) has ACE inhibitory activity.
JP2002053595 (A) discloses that the peptides from a soy bean
hydrolysate can inhibit ACE activity. JP6298794 (A) discloses a
process for the preparation of ACEI from the proteins of animal and
plant sources, such as fish meats, pork and chicken. The
hydrolysate is subjected to centrifugation, filtration,
concentration and resin absorption to obtain the peptides with ACE
inhibitory activity. JP5331192 (A) discloses that dried katsuobushi
can be hydrolyzed by thermolysin to produce a peptide with ACE
inhibitory activity. JP4264098 (A) discloses the preparation of a
peptide with ACE inhibitory activity from chicken meat containing
no fat. U.S. Pat. No. 5,854,029 discloses a process for the
preparation of a dipeptide exhibiting ACE inhibitory activity. U.S.
Pat. No. 6,767,990 B1 discloses peptides isolated from the
hydrolysate of chicken residue. US 20120107409 A1 discloses a
method for preparing a fish skin fermentation product which can
inhibit the activity of tyrosinase, inhibit the activity of
angiotensin-converting enzyme and/or improve the survival of
fibroblasts.
[0006] A need still exists in the art for ACE inhibitors,
particularly peptides from natural sources, which are more safe
than the chemically synthesized compounds.
SUMMARY OF THE INVENTION
[0007] The present invention relates to novel peptides of Tyr-Tyr,
Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1), Phe-Asp-Thr,
Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe, Val-Val-Asn,
Thr-Tyr-Ala, Asn-Ser-Leu, Leu-Phe, Gly-Asn-Phe, Lys-Lys,
Val-Gly-Gly-Ser (SEQ ID NO: 3), and Trp-Asn that inhibit ACE.
[0008] In another embodiment, the present invention relates to a
composition comprising an effective amount of one or more peptides
selected from Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ
ID NO: 1), Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2),
Gly-Leu-Phe, Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser-Leu, Leu-Phe,
Gly-Asn-Phe, Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn,
Phe-Val, and Leu-Leu and one or more carriers, diluents, solvents,
colorants, anti-oxidants, inert materials and/or additives.
[0009] In a further embodiment, the present invention provides a
method for preventing and treating one or more syndromes associated
with angiotensin converting enzyme in a subject in need thereof,
comprising administering to the subject the composition of the
invention or an effective amount of one or more peptides selected
from Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1),
Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe,
Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser-Leu, Leu-Phe, Gly-Asn-Phe,
Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn, Phe-Val, and
Leu-Leu.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The invention provides isolated novel peptides having the
following sequences:
[0011] Tyr-Tyr;
[0012] Thr-Phe;
[0013] Thr-Ser-Phe;
[0014] Asn-Asp-Glu-Gly (SEQ ID NO: 1);
[0015] Phe-Asp-Thr;
[0016] Phe-Val-Asn-His-Phe (SEQ ID NO: 2);
[0017] Gly-Leu-Phe;
[0018] Val-Val-Asn;
[0019] Thr-Tyr-Ala;
[0020] Asn-Ser-Leu;
[0021] Leu-Phe;
[0022] Gly-Asn-Phe;
[0023] Lys-Lys;
[0024] Val-Gly-Gly-Ser (SEQ ID NO: 3); and
[0025] Trp-Asn.
[0026] These peptides can be isolated and purified from the
hydrolysates of natural substances (such as animal (e.g., fish or
chicken) and plant (e.g., soy bean) proteins). For example, the
proteins can be obtained by the methods disclosed in U.S. Pat. No.
6,767,990 B1, U.S. Pat. No. 8,063,017 B2 and US 20120107409 A1, the
contents of which are incorporated by reference.
[0027] The peptides of the invention with ACE inhibitory activity
can also be prepared by known chemical synthesis. For example, the
azide method, the acid chloride method, the acid anhydride method,
the mixed acid anhydride method, the DCC method, the active ester
method, the carboimidazol method, the oxidation-reduction method,
the DCC-active method (see, for example, Schroder & Luhke, The
Peptide, Vol. 1 (1996), Academic Press, New York, USA; or Izumiya
et al., Peptide Synthesis, Maruzen Co., Ltd. (1975), the contents
of which are incorporated by reference). These peptide synthesis
methods can be performed in either solid phase or liquid phase
synthesis. The amino acids having a side chain functional group
such as tyrosine and threonine are preferably protected in their
side chain functional groups with known protective groups such as a
benzyloxycarbonyl group, t-butoxycarbonyl group or benzyl group,
etc. The protective groups can be removed by any known methods.
[0028] Alternatively, according to the amino acid sequences, they
can also be prepared by cloning the nucleotide sequences
corresponding to the amino acid sequences of the peptides into
suitable vectors and expressed in suitable host cells, plants or
animals.
[0029] According to the invention, the term "isolated" or
"isolation" means that the material is removed from its original
environment (e.g., the natural environment if it is naturally
existing). The term "isolated" does not necessarily reflect the
extent to which the material has been purified by removing all
other substances (e.g., impurities).
[0030] In a further embodiment of the invention, a composition
comprising an effective amount of one or more peptides selected
from Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1),
Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe,
Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser-Leu, Leu-Phe, Gly-Asn-Phe,
Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn, Phe-Val, and
Leu-Leu is provided. In an another embodiment of the invention, the
composition comprises an effective amount of one or more peptides
selected from Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ
ID NO: 1), Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2),
Gly-Leu-Phe, Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser-Leu, Leu-Phe,
Gly-Asn-Phe, Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn,
Phe-Val, and Leu-Leu, provided that Phe-Val and Leu-Leu are not
simultaneously presented. The composition of the invention is a
pharmaceutical composition or a food composition. The
pharmaceutical composition or food composition can be prepared in a
conventional manner by mixing the peptide product with one or more
conventional carriers, diluents, solvents, colorants,
anti-oxidants, inert materials and/or additives to formulate the
composition in the form of tablets, capsules, powder, pellets,
concentrates, beverages, nutraceuticals, food additives or
feeds.
[0031] According to the invention, the term "effective amount" of
an active ingredient as provided herein means a sufficient amount
of the ingredient to provide the desired inhibition of the activity
of ACE. The exact amount required may vary from subject to subject,
depending on the disease state, physical conditions, age, sex,
species and weight of the subject, the specific identity and
formulation of the composition, etc. Thus, it is not possible to
specify an exact "effective amount." However, an appropriate
effective amount can be determined by one of ordinary skill in the
art using only routine experimentation.
[0032] In a further embodiment of the invention, a method for
preventing or treating one or more syndromes associated with
angiotensin converting enzyme in a subject in need thereof, which
comprises administering to the subject the composition or an
effective amount of one or more peptides selected from Tyr-Tyr,
Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1), Phe-Asp-Thr,
Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe, Val-Val-Asn,
Thr-Tyr-Ala, Asn-Ser-Leu, Leu-Phe, Gly-Asn-Phe, Lys-Lys,
Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn, Phe-Val, and Leu-Leu.
[0033] According to the invention, the term "preventing" or
"prevention" represents that use in relation to a condition
includes administering, prior to onset of the condition, an agent
to reduce the frequency or severity of or delay the onset of
symptoms of a medical condition in a subject relative to a subject
which does not receive the agent.
[0034] According to the invention, the term "treating" or
"treatment" represents reversing, alleviating, inhibiting the
progress of, or improving the disorder or condition to which such
term applies, or one or more symptoms of such disorder or
condition.
[0035] According to the invention, the term "subject" represents
any animal, preferably a mammal, and more preferably a human.
Examples of subjects include humans, non-human primates, rodents,
guinea pigs, rabbits, sheep, pigs, goats, cows, horses, dogs and
cats.
[0036] Artisans skilled in this field know that ACE is associated
with syndromes such as cardiovascular complications and hypertrophy
and/or hyperplasia of adipocytes. Therefore, the peptides and
composition of the invention have the ability to treat or prevent
arterial hypertension, systolic hypertension, peripheral vascular
disease, atherosclerosis, restenosis, heart failure, cardiac
insufficiency, thrombosis and any thromboembolic events, angina
pectoris, cerebral vascular accidents, coronary artery disease,
myocardial infarction, vascular remodeling, and the combination
thereof, and/or to reduce intraperitoneal fat and/or subcutaneous
fat.
[0037] The following examples are for explication of the
feasibility of the invention to substantiate the technical contents
of the invention but not to limit the scope of the invention. Any
variations and modifications to the invention by persons skilled in
the art on the basis of the teaching of the prior art are within
the scope of the invention.
EXAMPLES
Example 1
Preparation of Soybean Residues
[0038] 25 kg of defatted soybean powder (purchased from Central
Union Oil Corp.) was mixed with an amount of water half the weight
of the defatted soybean powder; the mixture was steamed at
100.degree. C. for 45 minutes and then cooled to 45.degree. C. 4.2
g of koji (purchased from Higuchi Matsunosuke Shouten Co Ltd.
Japan) containing Aspergillus sojae was added to and mixed with the
mixture, and the mixture was fermented at 27.degree. C. with 95%
relative humidity for 48 hours. After fermentation, an amount water
3 times the weight of the fermented mixture was added to the
mixture and the mixture was hydrolyzed at 45.degree. C. for 8
hours. The hydrolysate was pumped through a filter cloth to
separate the fluid portion from the solid soybean residue.
Example 2
Preparation of Peptides with ACE Inhibition Activity
[0039] Soybean residue prepared by the process of Example 1 was
mixed with 1 L of water and the pH of the mixture was adjusted to
2.0. 0.5 g of pepsin was added to the mixture, which was allowed to
be hydrolyzed at 37.degree. C. for 2 hours to obtain a hydrolysate.
The pH of the hydrolysate was adjusted to 7.2 by using NaHCO.sub.3.
0.5 g of trypsin and 0.5 g of chymotrypsin were then added to the
hydrolysate, which was allowed to be further hydrolyzed at
37.degree. C. for 2.5 hours to obtain a further hydrolysate. The
further hydrolysate was heated in a boiling water bath for 15
minutes to inactive the enzymes. After cooling, the further
hydrolysate was centrifuged at 10,000 rpm for 10 minutes to
separate the supernatant from the solid pellet. The supernatant was
then lyophilized to obtain a dried powder of 43 g, in which the
amount of protein was 24.5%.
[0040] The dried powder was dissolved in water to prepare a 1%
(w/v) water solution. The solution was filtered through a film with
a pore size of 0.45 .mu.m. The filtrate was subjected to high
performance liquid chromatography (HPLC). The conditions used in
the size exclusion column were as follows:
[0041] System: AKTA purifier
[0042] Elution column: Superdex Peptide HR 10/30
[0043] Sample amount: 500 .mu.L
[0044] Elution fluid: 5% alcohol in pure water
[0045] Flow rate: 0.25 mL/min
[0046] Detector: 214 nm.
[0047] Every 20 minutes, a 5-mL sample eluted from the column was
collected, and a total of 7 eluted fractions were collected. The
above HPLC process was repeated 60 times, and all the collected
corresponding fractions were respectively mixed and lyophilized to
obtain 7 dried powder samples. Each dried powder sample was
dissolved in water to prepare a 1% (w/v) water solution. The ACE
inhibition activities of all the water solutions were respectively
determined by the protocol disclosed by Vermeirssen, V. et al.
(Optimisation and validation of an angiotensin-converting enzyme
inhibition assay for the screening of bioactive peptides, J.
Biochem. Biophys. Methods, 2002, 51: 75-87). As shown in Table 1,
among all the solutions, the ACE inhibition activities of the
solutions of Fractions 3, 4 and 5 were stronger, and the solution
of Fraction 3 had the strongest activity.
TABLE-US-00001 TABLE 1 Protein concentration ACE IC.sub.50 (mg/mL)
(mg/mL) Fraction 1 6.61 -- Fraction 2 21.33 0.871 Fraction 3 17.10
0.014 Fraction 4 3.80 0.018 Fraction 5 0.75 0.025 Fraction 6 0.69
0.102 Fraction 7 0.53 0.593
[0048] The solution of the 3.sup.rd fraction was further eluted by
Sephasil Peptide RP-18 column under the following conditions:
[0049] System: AKTA purifier
[0050] Elution column: Sephasil Peptide RP-18 (5 .mu.m) ST
4.6/250
[0051] Sample amount: 100 .mu.L
[0052] Elution fluid A: H.sub.2O containing 0.1% trifluoroacetic
acid
[0053] Elution fluid B: MeOH containing 0.1% trifluoroacetic
acid
[0054] Gradient: 0% B for 5 min [0055] 0-100% B for 30 min [0056]
100% B for 10 min
[0057] Flow rate: 1 mL/min
[0058] Detector: 214 nm.
[0059] Every 5 minutes, a sample eluted from the column was
collected, and a total of 6 eluted fractions were collected. The
above elution was repeated 250 times. All the 6 eluted fractions
were respectively mixed and lyophilized to obtain 6 dried powder
samples. Each dried powder sample was dissolved in 1 mL water to
prepare a water solution. The ACE inhibition activity of the
solution of each fraction was measured. The results show that
Fractions 3-2, 3-3 and 3-4 have a better inhibition activity and
that Fraction 3-4 has the best inhibition activity (see Table 2
below).
TABLE-US-00002 TABLE 2 Protein concentration ACE IC.sub.50 (mg/mL)
(mg/mL) Fraction 3-1 1.86 1.269 Fraction 3-2 8.88 0.072 Fraction
3-3 5.77 0.069 Fraction 3-4 4.97 0.051 Fraction 3-5 1.32 0.112
Fraction 3-6 0.04 --
[0060] The materials at m/z 100-800 in Fraction 3-4 were determined
by mass spectrometer after eluted by Luna C18(2) column under the
following conditions:
[0061] Pump: Waters 600
[0062] Elution column: Luna C18(2) 150*2 mm, 3.mu.
[0063] Sample amount: 20 .mu.L
[0064] Elution fluid A: H.sub.2O containing 0.1% formic acid
[0065] Elution fluid B: acetonitrile containing 0.1% formic
acid
[0066] Gradient: 5% B for 1 min [0067] 5-95% B for 24 min [0068]
95% B for 10 min
[0069] Flow rate: 0.2 mL/min
[0070] Detector: Quattro LC MS/MS.
[0071] 17 peptides were identified. The ACE inhibition activities
of the 17 peptides were measured by the method disclosed in Pripp,
A. H. et al. (Quantitative structure-activity relationship modeling
of ACE-inhibitory peptides derived from milk proteins, Eur. Food
Res. Technol., 2004, 219: 579-583). According to the results shown
in Table 3 below, all the 17 peptides effectively inhibit the
activity of ACE, and thus can be used as ACE inhibitors.
TABLE-US-00003 TABLE 3 Retention SEQ ID time IC.sub.50 IC.sub.50
Peptides NOs MW (min) log IC.sub.50 (.mu.mol/L) (mg/mL) YY 344
24.61 2.41 256.92 0.088 TF 266 5.46 1.66 46.23 0.012 TSF 353 8.61
1.19 15.64 0.006 NDEG 1 433 24.88 2.89 768.42 0.333 PDT 331 2.86
2.44 272.33 0.090 FVNHF 2 663 4.04 1.71 51.10 0.034 GLF 335 25.2
1.92 83.04 0.028 VVN 330 7.24 1.89 77.05 0.025 TYA 353 6.88 2.72
526.14 0.186 NSL 332 17.86 1.81 64.86 0.022 LF 278 7.12 1.92 83.04
0.023 GNF 336 25.39 1.43 26.85 0.009 KK 274 3.78 2.96 921.30 0.252
VGGS 3 318 4.82 1.55 35.46 0.011 WN 318 25.56 2.57 367.71 0.117 FV
264 5.33 2.99 978.59 0.258 LL 244 5.58 2.54 344.51 0.084
Sequence CWU 1
1
314PRTArtificial SequenceSynthetic peptide 1Asn Asp Glu Gly 1
25PRTArtificial SequenceSynthetic peptide 2Phe Val Asn His Phe 1 5
34PRTArtificial SequenceSynthetic peptide 3Val Gly Gly Ser 1
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