Download (número 1) 2012 Angiotensin converting enzyme inhibitors from cas

Revista Computadorizada de Producción Porcina
Volumen 19 (número 1) 2012
Angiotensin converting enzyme inhibitors from casein/Inhibidores de la enzima convertidora de angiotensina a partir de caseína
A JOURNEY FROM PORCINE PANCREATIN TO PRODUCTION OF ANGIOTENSIN CONVERTING ENZYME (ACE)
INHIBITORS FROM CASEIN
1
12
1
L. Ozimek , Sirinda Kusump , Takuo Nakano and E.R. Silva
3
1
Department of Agricultural, Food and Nutritional Science, University of Alberta. Edmonton, Alberta T6G 2P5, Canada
email: [email protected]
2
Department of Food Science and Technology.Thammasat University. Tha Phra Cha Campus, Bangkok, Thailand
3
University of Veracruz, Xalapa, Veracruz, Mexico
SUMMARY
Angiotensin converting enzyme (E.C. 3.4.15.1) plays an important role in the regulation of blood pressure and pathophysiology of
hypertension, which causes various diseases of the circulatory system including stroke, arteriosclerosis, and coronary heart
disease. Angiotensin converting enzyme (ACE) can increase blood pressure by converting angiotensin I to a potent vasoconstrictor,
angiotensin II, and by degrading an arteriolar vasodilator, bradykinin, into inactive peptides. Inhibition of ACE may exert an
antihypertensive effect as a consequence of a decrease in angiotensin II activity and a concomitant increase in bradykinin activity.
The ACE inhibiting activity determined in the pancreatin hydrolysate of bovine casein was considerably high with its concentration of
50% inhibition (IC50) being 600 μg/mL. These results suggest that the pancreatin hydrolysate of bovine casein is a potential source
of hydrophobic peptides with ACE inhibiting activity, and that chromatography on phenyl-agarose in 5 M NaCl is a useful technique
to purify such peptides. Further studies to separate and characterize individual hydrophobic peptides are under progress in our
laboratory.
Key words: ACE inhibitor, casein hydrolysate, hydrophobic peptide, hydrophobic interaction chromatography, phenyl-agarose
Short title: Angiotensin converting enzyme inhibitors from casein
UNA VIA A PARTIR DE LA PANCREATINA PORCINA PARA PRODUCIR INHIBIDORES DE LA ENZIMA CONVERTIDORA DE
ANGIOTENSINA (ECA) PRESENTES EN LA CASEINA
RESUMEN
La enzima convertidora de angiotensina (E.C. 3.4.15.1) desempeña un papel importante en la regulación de la presión sanguínea y
en la patofisiología de la hypertensión, que causa varias enfermedades del sistema circulatorio, incluyendo infacto,
arterioesclerosis y enfermedades de las arterias coronarias. La enzima convertidora de angiotensina (ACE, acrónimo en inglés)
puede incrementar la presión sanguínea mediante la conversión de angiotensina I en un potente vasoconstrictor, la angiotensina II,
y por la degradación de un vasodilatador arterial, la bradiquinina, a péptidos inactivos. La Inhibición de la ACE puede ejercer un
efecto antihipertensor como consecuencia de una disminución en la actividad de la angiotensina II y el aumento concomitante de la
actividad de la bradiquinina.
La actividad inhibidora de la ACE en hidrolizados pancreáticos de caseína bovina fue considerablemente alta para su concentración
con un 50% de inhibición (IC50) de 600 μg/mL. Estos resultados sugieren que el hidrolizado de pancreatina de caseína bovina es
una fuente potencial de péptidos hidrofóbicos con actividad inhibidora de ACE, y que la cromatografía en fenil-agarosa en NaCl 5 M
es una técnica útil para purificar tales péptidos. En nuestro laboratorio están en progreso más estudios para separar y caracterizar
los péptidos hidrofóbicos individuales.
Palabras claves: inhibidor de ACE, hidrolizado de caseína, péptido hidrofóbico, cromatografía de interacción hidrofóbica, fenilagarosa
Título corto: Inhibidores de la enzima convertidora de angiotensina a partir de caseína
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Revista Computadorizada de Producción Porcina
Volumen 19 (número 1) 2012
Angiotensin converting enzyme inhibitors from casein/Inhibidores de la enzima convertidora de angiotensina a partir de caseína
to the prevention and treatment of hypertension. These ACE
inhibitory peptides can be derived from many food proteins.
INTRODUCTION
Food classified as functional are generally associated with
and individual primary health concerns, with include
cardiovascular disease, high blood pressure, stroke, high
cholesterol and cancer. Hypertension or high blood pressure
is a significant health problem worldwide (Ozimek 2010a,b).
Angiotensin converting enzyme, the peptidyl dipeptide
hydrolase (E.C. 3.4.15.1), commonly named ACE (see for
example, Coates 2003), is an exopeptidase which cleaves
dipeptides from the C-terminal of various peptide substrates.
This enzyme plays an important role in the regulation of
blood pressure and pathophysiology of hypertension, which
causes various diseases of the circulatory system including
stroke, arteriosclerosis, and coronary heart disease.
Angiotensin converting enzyme can increase blood pressure
by converting the inactive decapeptide angiotensin I to a
potent vasoconstrictor, angiotensin II (Ondetti and Cushman
1982; Matsui et al 1999), and by degrading an arteriolar
vasodilator, bradykinin (Ferreira et al 1970), into inactive
peptides. A schematic diagram describing the mode of
action of ACE in presented in figure 1. Inhibition of ACE
may exert an antihypertensive effect as a consequence of a
decrease in angiotensin II activity and a concomitant
increase in bradykinin activity. A relevant review concerning
this subject was published by Murray and FitzGerald (2007).
Renin.angiotensin
system
Angiotensinogen
<Angiotensin I
<Angiotensin II
vasoconstrictor
Renin
Kallikrein
Angiotensin
converting
enzyme, ACE
Kallikreinkinin system
Kininogen
->
Bradykinin
vasodilator
->
Inactive
fragments
Figure 1. ACE regulation of blood pressure. Angiotensin
converting enzyme, the enzyme found in reninangiotensin system, converts angiotensin I into
angiotensin II, which is a strong vasoconstrictor. Also the
enzyme catalyses bradykinin, which is a strong
vasodilator into inactive peptides. The formation of
angiotensin II and the degradation of bradykinin is a
strong potential factor for a rise in blood pressure
Biologically active peptides are of particular interest in food
science and nutrition because they have been shown to play
physiological roles. Hidden or inactive in the amino acid
sequence of native dairy proteins, they can be released or
activated in vivo during gastrointestinal digestion, or
upstream during food processing through enzymatic
proteolysis, for example during cheese ripening or yogurt
manufacturing. Bioactive peptides derived from milk proteins
that inhibit ÁCE in the cardiovascular system can contribute
Recently, numerous researchers have fconfirmed that food
proteins are precursors of many different biologically
peptides such as opiate, antithrombotic, antihypertensive,
immunomodulating and antibacterial (Kitts and Weiler 2003;
Korhonen and Pihlanto 2003, 2006; Hartmann and Meisel
2007), as it was previously claimed (Ariyoshi 1993)..
It is well known that milk protein derived peptides do have
several physiological activities (Meisel 1998, 2005). In this
connection, several studies have demonstrated ACE
inhibiting activities in peptides released after enzymatic
hydrolysis of food proteins including milk proteins (Kusump
2006; Kusump and Ozimek 2007; Kusump et al 2007).
These peptides are known to contain between two and 25
amino acid residues, and the majority are hydrophobic in
nature (Bouhallab et al 1992, 1993). In this connection, there
is limited information available concerning ACE inhibitors
from caseins treated with pancreatin, a mixture of pancreatic
enzymes.
The objective of this study was, therefore, to determine the
ACE inhibiting activity in a pancreatin hydrolysate of casein
by
fractionation
using
hydrophobic
interaction
chromatography on phenyl-agarose. This medium was used
because most ACE inhibitors are known to be hydrophobic
peptides. A preliminary report concerning this subject was
already done (Ozimek et al 2010).
MATERIALS AND METHODS
A sample of bovine casein hydrolysate was prepared by
hydrolysis with pig pancreatin. Pancreatin:substrate ratio
was 1:100. Pancreatin was added to an aqueous solution of
6% casein adjusting the medium to pH 7.6 by adding 1 N
NaOH. Hydrolysis was undertaken at 37ºC for 8 hours
(Ozimek et al 1993; Kusump 2006). The mixture containing
the casein hydrolysate was adjusted to pH 4.6 with 1 N HCl
and was centrifuged at 12 000 g during 15 minutes, at 20ºC.
The resulting supernatant from each sample was
fractionated using an ultrafiltration system at 20 psi of
pressure with 2000 Da molecular weight cut-off membrane
to give permeate and retentate. The obtained impregnate
was liophilyzed and stored at -20ºC for subsequent
purifications.
A portion of hydrolysate was dissolved in 0.01M sodium
phosphate, pH 6.8 containing 5 M NaCl (solution A). This
preparation was applied to a 1.5 x 6.5 cm column of phenylagarose (Sigma Chemical Co) equilibrated with solution A,
and materials retained in the column were eluted with water.
Fractions (2 mL) collected at a flow rate of 7.5 mL/hour were
monitored for peptide contents by measuring the absorbance
at 210 nm (Stoschek 1990) using serum bovine albumin as
protein standard, and for ACE inhibiting activities.
The described fractionating method is a consequence of
employing techniques of column chromatography separation
developed in our laboratory (Nakano and Ozimek 2001;
Nakano et al 2009).
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Revista Computadorizada de Producción Porcina
Volumen 19 (número 1) 2012
Angiotensin converting enzyme inhibitors from casein/Inhibidores de la enzima convertidora de angiotensina a partir de caseína
Assays of ACE inhibiting activity were carried out using
hippuryl-L-histidyl-L-leucine as substrate, following Cushman
and Cheung (1971) recommendations, as modified by
Nakamura et al (1995). ACE was obtainied from rabbit lungs.
Briefly, 80 μL of each sample were added to 200 μL of 0.1
mol/L potassium phosphate containing 0.3 mol/L NaCl and 5
mmol/L hippuryl-L-histidyl-L-leucine, pH 8.3. The reaction
was stopped by adding 250 μL of 1 N HCl. The content of
hippuric acid was released from the substrate by ACE
hydrolysis, by extraction with ethyl acetate, heat evaporated
o
at 95 C for 10 min, dissolved in distilled water. The
absorbance of the extract was spectrophotometrically
measured at the wavelength of 228 nm.
The inhibition activity was calculated using the following
equation:
Inhibition activity, % = 100 x ((A – B) – (C – D))/(A - B)
where A is the absorbance of the solution containing ACE,
but without the sample, B is the absorbance of a solution
with ACE previously inactivate by adding HCl and without
the sample, C is the absorbance in the presence of ACE and
sample, and D is the absorbance with ACE previously
inactivated with HCl and containing the sample.
The inhibitory activity of the hydrolysates or collected
fractions was expressed as percentage of ACE inhibition at a
given protein concentration. IC50 was defined as the
concentration of an ACE inhibitor, in µg peptide/mL needed
to inhibit 50% of ACE activity.
Table 1. Inhibitory activity of ACE in fractions
of the pancreatin hydrolysate of casein
as obtained by phenyl-agarose
chromatography
Phosphate
ACE
IC50, µg
peptide/mL
buffer
NaCl
inhibition, %
1
pH 6.0
4M
0
nd
3M
4
nd
2M
3
nd
0
nd
H2O
SD ±
1.6
pH 6.8
4M
14
>1 000
3M
11
>1 000
1M
4
>1 000
7
>1 000
H2O
SD ±
3.8
1
nd express not determined
Further studies to separate and characterize individual
hydrophobic peptides are under progress in our laboratory.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the facilitation activity of
the librarians, Swine Research Institute, Havana, and to
Professor W. Sauer for revising the final draft.
REFERENCES
Data are presented as means and standard deviation of
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The ACE inhibiting activity determined in the pancreatin
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bovine casein is a potential source of hydrophobic peptides
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