FEBS 27917 FEBS Letters 555 (2003) 597^600

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Inhibition of angiotensin converting enzyme (ACE) activity by provided by Elsevier - Publisher Connector

£avan-3-ols and procyanidins

Lucas Actis-Gorettaa , Javier I. Ottaviania , Carl L. Keenb , Cesar G. Fragaa;b;
a
Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Jun|¤n 956, 1113 Buenos Aires, Argentina
b
Department of Nutrition, University of California, Davis, CA 95616, USA

Received 2 October 2003; revised 4 November 2003; accepted 16 November 2003

First published online 26 November 2003

Edited by Judit Ova¤di

rate of DNA oxidative damage [8], reduce platelet reactivity

Abstract It was determined that £avan-3-ols and procyanidins
have an inhibitory e¡ect on angiotensin I converting enzyme [9,10], and modulate the oxidant-responsive transcription fac-
(ACE) activity, and the e¡ect was dependent on the number tor NF-UB [11].
of epicatechin units forming the procyanidin. The inhibition Angiotensin I converting enzyme (ACE) is a glycoprotein
by £avan-3-ols and procyanidins was competitive with the two peptidyldipeptide hydrolase, whose main known functions
substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and are to cleave histidyl-leucine from angiotensin I forming
N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). the potent vasoconstrictor angiotensin II, and to degrade
Tetramer and hexamer fractions were the more potent inhibi- bradykinin to inactive peptides [12]. ACE has two active
tors, showing Ki of 5.6 and 4.7 WM, respectively. As ACE is a sites, N- and C-terminal, with di¡erent a⁄nities for di¡erent
membrane protein, the interaction of £avanols and procyanidins substrates. ACE inhibition is considered to be an important
with the enzyme could be related to the number of hydroxyl
therapeutic approach in the treatment of high blood pres-
groups on the procyanidins, which determine their capacity to
be adsorbed on the membrane surface. sure, and the intake of certain synthetic ACE inhibitors pro-
4 2003 Published by Elsevier B.V. on behalf of the Federation vides de¢nitive positive health e¡ects [13]. However, as ACE
of European Biochemical Societies. inhibitors are pharmacological drugs their use in healthy or
low-risk populations is not advisable. The ¢nding that cer-
Key words: Flavonoid; Hypertension; Cocoa; tain £avonoid-rich foods can induce reductions in blood
Oxidative stress pressure and inhibit ACE activity, both in vivo and in vitro
[14^19], opens up the possibility that consumption of select
£avonoid-rich foods may mimic synthetic ACE inhibitors
and provide health bene¢ts but without adverse side e¡ects.
1. Introduction In this regard, it was observed that after the regular con-
sumption of a diet rich in £avan-3-ols and procyanidins for
Several epidemiological studies have shown associations be- 14 days, blood pressure was signi¢cantly diminished in aged
tween the regular consumption of £avonoid-rich foods and a people [20].
decreased risk for cardiovascular disease [1^3]. While the in- The aim of the present work was to determine if £avan-3-
verse association between the risk for vascular disease and ols and procyanidins have an inhibitory e¡ect on ACE activ-
dietary £avonoid intake does not prove causality, an increas- ity, and to characterize such inhibition from a kinetic point of
ing amount of experimental data pertaining to certain £avo- view. We studied ACE inhibition based on the two commonly
noids does lend support to this association [4]. employed methods for determining ACE activity [21,22]. It
Flavonoids and other polyphenols are widely distributed in was observed that £avan-3-ols and procyanidins isolated
the human diet, primarily in plant-derived foods and bever- from cocoa compete for enzyme-active sites with synthetic
ages. Procyanidins are a group of polymeric polyphenols com- ACE substrates. The e¡ects of £avan-3-ols and procyanidins
posed of the £avan-3-ol units, (3)-epicatechin (epicatechin) on ACE could contribute to the decreased risk for cardiovas-
and (+)-catechin (catechin). Procyanidins are present in foods cular and other diseases observed in populations that consume
such as nuts, cranberries, apples, red wine, tea and cocoa or high amounts of foods rich in £avonoids.
chocolate [5,6]. Flavan-3-ols and procyanidins are of great
interest in nutrition and medicine because of their potent anti-
2. Materials and methods
oxidant capacity and other protective e¡ects on human
health. In vitro, procyanidins can, among other e¡ects, delay
the rate of low density lipoprotein oxidation [7], reduce the 2.1. Materials
ACE from rabbit lung (puri¢ed ACE), hippuric acid (HA), hippur-
yl-L-histidyl-L-leucine (HHL), N-[3-(2-furyl)acryloyl]-L-phenylalanyl-
glycylglycine (FAPGG), epicatechin, and catechin were from Sigma
(St. Louis, MO, USA). High-performance liquid chromatography
*Corresponding author: Fax: (54)-11-4508 3646. (HPLC)-grade acetonitrile and acetic acid were from Merck KGaA
E-mail address: cfraga@¡yb.uba.ar (C.G. Fraga). (Darmstadt, Germany). Puri¢ed procyanidin fractions (epicatechin
dimers, trimers, tetramers, pentamers, and hexamers) were obtained
Abbreviations: ACE, angiotensin I converting enzyme; HHL, N-hip- from Mars Inc. (Hackettstown, NJ, USA). The purity of the procya-
puryl-L-histidyl-L-leucine; HA, hippuric acid; FAPGG, N-[3-(2-fur- nidin fractions was 99.0, 94.8, 95.4, 92.0, and 86.2% for dimer, trimer,
yl)acryloyl]-L-phenylalanylglycylglycine tetramer, pentamer, and hexamer fractions, respectively.

0014-5793 / 03 / $22.00 K 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
doi:10.1016/S0014-5793(03)01355-3

FEBS 27917 3-12-03

598 L. Actis-Goretta et al./FEBS Letters 555 (2003) 597^600

2.2. Inhibition of puri¢ed ACE activity 3. Results

A mixture consisting of 100 Wl of 6.3 nM puri¢ed ACE and 100 Wl
of £avan-3-ols or procyanidins (0^1500 WM) was preincubated for 30
min at 37‡C. For ACE activity determination, the 200-Wl mixture was 3.1. Kinetics of the inhibition of puri¢ed ACE activity by
added to 100 Wl of HHL (1.5^12 mM) in 50 mM HCl^Tris, 300 mM epicatechin, dimer and hexamer fractions
NaCl (pH 8.3) and incubated from 20 to 90 min at 37‡C [21]. The For the kinetics studies epicatechin, dimer, and hexamer
reaction was stopped by addition of 100 Wl of 12% (w/v) phosphoric fractions were used as ACE inhibitors. Inhibition was eval-
acid. The HA was separated and quanti¢ed by HPLC with UV de-
tection [23]. The system consisted of a Supelcosil LC-18-DB column uated by means of two di¡erent ACE activity assays, i.e.
(15 cmU4.6 mmU5 Wm) using a mobile phase composed of 5 mM HHL and FAPGG hydrolysis. Fig. 1 shows the inhibition
phosphoric acid:acetonitrile (80:20 v/v), pH 2.5 (£ow rate, 1 ml/min; on ACE activity (reduction of Vi ) by the hexamer fraction
retention time, 3.65 min). The UV detection was carried out at 228 using the HHL hydrolysis assay (Fig. 1A), and by the dimer
nm (Jasco UV 1575, Intelligent UV/VIS Detector, Japan Spectroscop-
fractions using the FAPGG hydrolysis (Fig. 1B). Similar
ic Co. Ltd.). Commercial HA was used as standard.
For FAPGG hydrolysis determination, a 50-Wl aliquot of the 200 Wl curves were obtained when epicatechin or dimer were assayed
mixture was added to 280 Wl of FAPGG (50^400 WM) dissolved in as inhibitors of FAPGG hydrolysis, and epicatechin or hex-
0.1 M potassium phosphate bu¡er, 0.5 M NaCl, 0.1 mM ZnCl2 , pH amer were assayed inhibiting HHL hydrolysis.
7.5. After incubating for 10 min at 37‡C, hydrolysis of FAPGG by Puri¢ed ACE showed a Michaelis^Menten mechanism us-
ACE was quanti¢ed by recording the decrease of absorbance at 340
nm [22]. ing HHL or FAPGG as substrates. The apparent maximum
rate of substrate hydrolysis (VmaxA ) and the apparent Michae-
lis constant (KMA ) were determined to characterize the kind of
2.3. Kinetic calculations
The kinetic parameters were calculated by adjusting curves to the inhibition exerted by £avan-3-ols and procyanidins. KMA for
Michaelis^Menten equation: Vi = (VmaxA US)/(KMA +S) where Vi = both HHL and FAPGG hydrolysis were plotted against epi-
initial rate; VmaxA = apparent maximum rate; KMA = apparent Mi- catechin, dimer, or hexamer concentrations (Fig. 1C,D). KMA
chaelis constant, and S = substrate concentration. KMA and VmaxA values were dependent on the inhibitor and its concentration.
were plotted vs. concentration of inhibitor (I). Ki values were calcu-
By contrast, the VmaxA were independent of the inhibitor con-
lated adjusting the curves to the equation: KMA = KM (1+I/Ki ).
The IC50 were calculated by adjusting inhibition curves to the equa- centration (3.0 T 0.3, 3.4 T 0.4, and 3.6 T 0.2 WM min31 for
tion for a two-parameter hyperbolic decay: f = (Vi UIC50 )/(IC50 +I), in FAPGG and 0.8 T 0.2, 1.2 T 0.3 and 1.5 T 0.3 WM min31 for
which Vi = initial rate with 1 mM HHL. The Ki values were calculated HHL for epicatechin, dimer, and hexamer, respectively).
from IC50 considering a competitive inhibition with the following These data indicate a competitive enzyme inhibition.
equation: Ki = IC50 /(1+(S/KM )). Gibbs free energy (vG) was calcu-
lated as vG = RTln Ki .
The dissociation constant for the binding of inhibitor to the
free enzyme (Ki ) was calculated for the ACE inhibition by
epicatechin, dimer, and hexamer, using both substrates,
2.4. Data analysis
Curves were adjusted using routines available in Sigma Plot 5.0.
HHL and FAPGG. Ki values showed signi¢cant di¡erences
Data were analyzed for statistical signi¢cance using ANOVA (Stat- between both substrates for epicatechin (1302 WM for HHL
View 5, Mountain View, CA, USA). and 39 WM for FAPGG) and dimer (227 WM for HHL and 35

Fig. 1. Initial velocities for the inhibition of ACE activity by epicatechin, dimer and hexamer vs. substrate concentration (A,B) and KMA vs.
epicatechin, dimer, and hexamer concentrations for HHL (C) or FAPGG (D). Puri¢ed lung ACE was preincubated at 37 ‡C for 30 min in the
absence (control), or the presence of di¡erent amounts of epicatechin, dimer or hexamer, and then incubated from 20 to 90 min in the presence
of di¡erent concentrations of HHL or FAPGG. Values are mean T S.E.M. of at least three independent experiments.

FEBS 27917 3-12-03

L. Actis-Goretta et al./FEBS Letters 555 (2003) 597^600 599

WM for FAPGG). However, hexamer presented Ki in the same

range with both substrates (4 WM for HHL and 12 WM for
FAPGG). The inhibition of HHL hydrolysis was more clearly
related to the number of monomer units than FAPGG hydro-
lysis.

3.2. Inhibition of puri¢ed ACE activity by epicatechin and

procyanidin fractions
Based on the results obtained above, procyanidins were
tested to investigate whether their inhibitory e¡ect was related
to polymer size. The preincubation of puri¢ed ACE in the
presence of £avan-3-ols or procyanidins inhibited the trans-
formation of HHL into HA in a dose-dependent manner
(Fig. 2). The inhibition was also dependent on the procyani-
din molecular weight. IC50 was 1781 T 381, 1593 T 443,
267 T 24, 126 T 11, 12 T 1, 25 T 3, and 10 T 1 WM, for epicate-
Fig. 3. E¡ect of the number of epicatechin units in procyanidins on
chin, catechin, dimer, trimer, tetramer, pentamer, and hexam- IC50 and vG values. Values of IC50 and vG were calculated from
er, respectively. the data shown in this ¢gure, and analyzed mathematically as indi-
The dissociation constant for the binding of inhibitor to the cated in Section 2. Inhibitors are epicatechin and procyanidins.
free enzyme (Ki ) was calculated taking into account that en-
zyme inhibition was competitive. The Ki were 828, 124, 59,
5.6, 11.6, and 4.7 WM for epicatechin, dimer, trimer, tetramer, plant extracts have been described as possible ACE inhibitors
pentamer and hexamer, respectively. These values were on the [14,15,26]. In this study, epicatechin, as well as procyanidins,
same order as those calculated previously by the kinetic assay inhibited the activity of rabbit lung ACE.
for epicatechin (1302 and 828 WM), dimer (227 and 124 WM), The kinetic analysis of the results suggests that £avan-3-ols
and hexamer (4 and 4.7 WM). and procyanidins inhibit the enzyme activity by competing
Using the Ki values, changes in Gibbs free energy (vG) were with the substrate for the active sites. The calculated Ki
calculated and IC50 and vG were plotted versus units of were di¡erent depending on the type of substrate used
monomers present in procyanidins (Fig. 3). The coe⁄cient (HHL or FAPGG) for epicatechin and dimer, but were sim-
for linear regression (R2 ) was 0.87 for vG, and the average ilar for the hexamer. It can be suggested that the larger mol-
energy increase per unit of monomers was 2.6 T 0.5 kJ/mol ecules can inhibit both the C- and the N-active sites to a
(P 6 0.01). similar extent; meanwhile, monomers and dimers seem to in-
hibit preferentially the N-active site.
4. Discussion From thermodynamic considerations, the linear relation-
ship between changes in Gibb’s free energy (vG) and procyan-
We postulate that the observed e¡ect of the consumption idin size, and the calculated binding stabilization enhancement
£avonoid-rich foods reducing blood pressure in humans of about 2.6 kJ/mol/monomer unit, suggest an increase in
[20,24] and in rats [25] can be related to the inhibition of hydrogen bond formation between ACE and procyanidin hy-
ACE by £avan-3-ols and procyanidins. Flavonoid-containing droxyl groups [27]. The calculated vG values are on the same
order as those estimated for the interaction between hydroxyl
groups and some macromolecules [28,29], but are higher than
those calculated for the interaction between £avonoids and
certain proline-rich proteins [27].
To characterize the relation between the size of procyani-
dins and their ability to inhibit ACE activity, we assayed the
ACE activity using the assay for the hydrolysis of HHL. We
observed that catechin and epicatechin were the weaker inhib-
itors, showing inhibitory e¡ects at concentrations higher than
100 WM. Increasing the number of epicatechin units in the
procyanidin yielded an increasing inhibitory e¡ect. Thus,
three levels of IC50 could be established : IC50 in the mM
range for monomeric £avan-3-ols, in the 100 WM range for
dimer and trimer, and in the 10 WM range for the larger
procyanidins. The relevance of these concentrations should
be interpreted with caution since they were estimated using
Fig. 2. E¡ect of di¡erent concentrations of £avan-3-ols and procya-
nidins on ACE activity. Puri¢ed lung ACE was preincubated at non-physiological in vitro conditions (isolated enzyme, syn-
37‡C for 30 min in the absence (control), or the presence of di¡er- thetic substrates, and high substrate concentration).
ent amounts of £avan-3-ols or procyanidins, and then during 60 With regard to the physiological relevance for the inhibition
min in the presence of HHL (5.2 mM). Inhibitors assayed were: of ACE by £avan-3-ols and procyanidins it is important to
catechin (black triangle up), epicatechin (empty circles), dimer (gray
square), trimer (black triangle down), tetramer (empty diamond),
note the actual concentrations of these substances in vivo. The
pentamer (black circle), and hexamer (gray triangle down). Values presence of catechin, epicatechin, and dimers has been deter-
are mean T S.E.M. of at least three independent experiments. mined in human plasma at low micromolar concentrations

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600 L. Actis-Goretta et al./FEBS Letters 555 (2003) 597^600

[30], but there are few reports on tissue concentrations of Schmitz, H.H. and Fraga, C.G. (2002) Arch. Biochem. Biophys.
monomer and dimer, and no studies addressing the presence 406, 203^208.
[9] Rein, D., Paglieroni, T.G., Wun, T., Pearson, D.A., Schmitz,
of larger procyanidins in tissues. As ACE is a membrane- H.H., Gosselin, R. and Keen, C.L. (2000) Am. J. Clin. Nutr.
bound enzyme, and £avan-3-ols and procyanidins, especially 72, 30^35.
the larger molecules, can adsorb to lipid^water interphases [10] Holt, R.R., Schramm, D.D., Keen, C.L., Lazarus, S.A. and
[31], it is feasible that a local enrichment of these compounds Schmitz, H.H. (2002) JAMA 287, 2212^2213.
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on the membrane surface could lead to an enhanced interac- Fraga, C.G. and Oteiza, P.I. (2003) FASEB J. (in press,
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the membranes of vascular endothelial cells, which are [12] Dzau, V.J. (2001) Hypertension 37, 1047^1052.
thought to be responsible for regulating blood pressure [32]. [13] Schi¡rin, E.L. (2002) Am. J. Med. 113, 409^418.
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198.
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shown that ACE inhibitors can modulate antioxidant defenses [16] Hackl, L.P., Cuttle, G., Dovichi, S.S., Lima-Landman, M.T. and
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In summary, the results presented here a¡ord a biochemical
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