Antihypertensive effect of caffeic acid and its analogs through dual renin-angiotensin-aldosterone system inhibition

Article abstract of DOI:10.1016/j.ejphar.2014.02.038

Hypertension is a crucial risk factor for cardiovascular diseases and contributes to one third of global mortality. In addition to conventional antihypertensive drugs such as captopril, naturally occurring phytochemicals and their analogs are used for red

Full text of DOI:10.1016/j.ejphar.2014.02.038

European Journal of Pharmacology 730 (2014) 125–132
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Antihypertensive effect of caffeic acid and its analogs through dual
renin–angiotensin–aldosterone system inhibition
a
b
Khushwant S. Bhullar , Grégoire Lassalle-Claux ,
Mohamed Touaibia
a
b,
n
,1
a,nn,2
, H.P. Vasantha Rupasinghe
Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS, Canada B2N 5E3
Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB, Canada E1A 3E9
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 January 2014
Received in revised form
Hypertension is a crucial risk factor for cardiovascular diseases and contributes to one third of global
mortality. In addition to conventional antihypertensive drugs such as captopril, naturally occurring
phytochemicals and their analogs are used for reducing the risk and occurrence of hypertension. Herein,
we demonstrate the possible use of caffeic acid and its derivatives in the treatment of hypertension
through multi-target modulation of renin–angiotensin–aldosterone system (RAAS). Caffeic acid along
with its nineteen novel derivatives, chlorogenic acid, quercetin and captopril were all investigated for the
inhibition of renin and angiotensin converting enzyme (ACE) activities and production of aldosterone.
9
February 2014
Accepted 23 February 2014
Available online 11 March 2014
Keywords:
Hypertension
Renin
Angiotensin converting enzyme
Aldosterone
Caffeic acid
Compound 22 with CH
2
CH(Ph)
2
moiety exhibited the strongest renin inhibition (IC50¼229 mM) among
all compounds tested (Pr0.05). Caffeic acid was the weakest renin inhibitor (IC50¼5704 mM) among all
the compounds assayed. Similar to renin inhibition, compound 22 (IC50¼9.1 mM) also exhibited about 47
times stronger ACE inhibition compared to the parent compound. Analysis of aldosterone revealed that
compound 8 with n-Pr moiety was the strongest modulator of aldosterone production among all the
derivatives (Pr0.05). Toxicity analysis using human fibroblasts (WI-38 cells) confirmed the non-toxic
manifestations of caffeic acid and its derivatives in comparison to clinically used drug captopril.
Polyphenols
&
2014 Elsevier B.V. All rights reserved.
1
. Introduction
of aldosterone (McMurray et al., 2008; Calhoun, 2006). These clinical
agents also extend other cardioprotective benefits beyond antihyper-
tensive action including antifibrotic effect by the ACE inhibitors (Peng
et al., 2005). On the other hand, renin inhibitors extend profound
beneficial effects on renal physiology (Heerspink et al., 2009). RAAS
inhibitors have been shown to cause significant decrease in cardio-
vascular mortality (7%) along with successful blood pressure reduction
(Ferrari, 2013). However, the use of these single target inhibitors is
sometimes compromised by resistant hypertension and toxicity
complications leading to the search for novel and multi-target RAAS
inhibitors.
Multi-target modulation of RAAS activity is characterized by
the interaction of antagonists with multiple drug targets such as
ACE, renin and aldosterone synthase simultaneously, thus result-
ing in stronger modulation of RAAS. Moreover, the single RAAS
blockade may lead to incomplete therapeutic intervention which
can result in persistent pathology by other mechanisms in RAAS
(Pichler and deBoer, 2010). Theoretically, the multi-target RAAS
inhibition by a drug may improve its ability to reduce the rate of
cardiovascular disorder and hypertension (Bakris and Weir, 2008).
Besides conventional drugs (McMurray et al., 2008; Calhoun,
Renin–angiotensin–aldosterone system (RAAS) system has evolved
as a principal clinical target for the treatment of hypertension,
myocardial infraction, stroke and renal diseases (Ma et al., 2010).
RAAS is now emerging as a unifying system involved in maintenance
of haemodynamic equilibrium, normal blood pressure and pathophy-
siology of hypertension. RAAS-mediated hypertension is initiated by
the cleavage of angiotensinogen to form angiotensin I (Ang I) by renin.
The Ang I is further converted to angiotensin II (Ang II) by angiotensin-
converting enzyme (ACE), while Ang II also triggers aldosterone
secretion and thereby increases blood pressure (Ma et al., 2010;
Hsueh and Wyne, 2011). Many drugs used in the treatment of hyper-
tension and cardiac disorders elicit their activity through modulation
of RAAS enzymes such as renin and ACE as well as acting as antagonist
n
Corresponding author. Tel.: þ1 506 858 4493; fax: þ1 506 858 4480.
nn
Correspondence to: Faculty of Agriculture, Dalhousie University, Rm 219-C Cox
Institute Building, 50 Pictou Road, P.O. Box 550 Truro, NS, Canada B2N 5E3.
Tel.: þ1 902 893 6623; fax: þ1 902 893 1404.
E-mail addresses: mohamed.touaibia@umoncton.ca (M. Touaibia),
vrupasinghe@dal.ca (H.P.V. Rupasinghe).
2006) such as captopril (1), many phytochemicals including plant
1
polyphenols (Fig. 1) have gained attention as antihypertensive
agents (Li et al., 2013; Suzuki et al., 2006). Principally, these plant
Contribution: Synthesis of caffeic acid derivatives.
Contribution: Biological evaluation.
2
http://dx.doi.org/10.1016/j.ejphar.2014.02.038
0
014-2999/& 2014 Elsevier B.V. All rights reserved.

126
K.S. Bhullar et al. / European Journal of Pharmacology 730 (2014) 125–132
secondary metabolites, including flavonoids such as quercetin (Perez-
Vizcaino et al., 2009) and phenolic acids, exhibit strong antihyperten-
sive activities and promise non-pharmacological dietary intervention
renin (human recombinant), sulfuric acid, quercetin and Tris buffer
were purchased from Sigma Aldrich Canada Ltd., Oakville, ON. Sterile
96 and 6 well cell culture grade plates were obtained from BD
Biosciences (Mississauga, ON).
(Zhao et al., 2011). Caffeic acid (4 Fig. 2), a phenolic acid commonly
found in fruits and vegetables, has emerged as a strong antihyperten-
sive agent in both in vitro (Actis-Goretta et al., 2006) and in vivo
models (Li et al., 2005). Although these studies exhibit antihyperten-
sive action of caffeic acid, (4) they do not assess its potential multi-
target RAAS blockade and toxicology analysis. Herein, we report the
first evidence of multi-target RAAS regulation by caffeic acid (4)
along with its 19 novel derivatives (Fig. 2), indicating the possible
examination of these drug candidates as antihypertensive agents. The
antihypertensive activities were evaluated against three principal
RAAS targets including renin, ACE and aldosterone in vitro while the
toxicological aspect was evaluated using human fibroblasts (WI-38
cells).
2.2. Chemistry and synthesis
As recently reported (Sanderson et al., 2013), alkyl esters 6–12
were prepared by esterification with selected alcohol and commer-
cially available caffeic acid (4) in the presence of sulfuric acid. Aryl
esters 14–23, cyclohexyl ester 13, and amide 24 were synthesized
from selected alcohol or 2-phenylethanamine and acetylated caffeic
acid. Base-induced de-O-acetylation resulted in the desired caffeic acid
derivatives (Fig. 2). The substituents were chosen for their electronic
and steric properties: appending progressively larger aliphatic moi-
eties (R¼Me, to R¼I-pentyl; R¼Phenyl to R¼CH
2 2
CH Naphtyl) not
2
. Materials and methods
only modifies the steric bulk of the molecules but also augments
the general lipophilicity. The addition of insaturation (R¼3-methyl-
but-3-enyl) modulates flexibility of the side chain while appending
p-substituents to the aromatic moiety should allow investigation of
the effects of additional steric bulk as well as the presence of an
electron withdrawing and donating groups. The synthesized com-
pounds were screened for antihypertensive activities by preparing the
stock solutions of 1000 mM in DMSO. This stock solution was further
diluted to achieve working solutions of different concentrations.
2.1. Chemicals and reagents
Angiotensin converting enzyme (ACE), captopril, chlorogenic acid,
caffeic acid, sodium borate buffer, NaOH, o-pthaldehyde, Arg-Glu
EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg
renin substrate), dimethylsulfoxide (DMSO), ethyl acetate, HCl, histi-
dine–leucine (His-Leu), histidine- -hippuryl- -leucine-chloride (HHL),
(
(
L
L
Fig. 1. Chemical structures of captopril (1), quercetin (2), and chlorogenic acid (3).
O
O
HO
HO
HO
HO
OH
O
Caffeic Acid (4)
CAPE (5)
O
O
R
R
O
HO
HO
HO
HO
X
6
7
8
9
1
1
1
1
: R = Me
14 : X = O, R = Ph
: R = Et
15: X = O, R = CH Ph
2
: R = n-Pr
1
1
1
1
2
2
2
2
2
6: X = O, R = CH CH PhF(4-F)
2 2
: R = I-Pr
7: X = O, R = CH CH PhMe(4-Me)
2
2
2
0: R = n-But
8: X = O, R = CH CH PhOMe(4-OMe)
2
1: R = I-pentyl
2: R = 3-methylbut-3-enyl
3: R = CH CH cyclohexyl
9: X = O, R = CH CH Ph(4-NO )
2
2
2
2
2
0: X = O, R = CH CH cycloHexyl
2
2
2
1: X = O, R = CH CH Naph
2
2: X = O, R = CH CH(Ph)
2
2
3: X = O, R = CH CH CH Ph
2
2
2
4: X = NH, R = CH CH Ph
2
2
Fig. 2. Caffeic acid derivatives evaluated as multi-target RAAS regulators.

K.S. Bhullar et al. / European Journal of Pharmacology 730 (2014) 125–132
127
2.3. Cell culture
2.8. Toxicity analysis
s
Human lung fibroblast cell line WI-38 (ATCC CCL75™) was
obtained from Cederlane labs (Burlington, ON) and cultured
according to supplier's instructions. Briefly, cells were maintained
in 75 cm2 culture flasks in Eagle's minimum essential medium
s
Cell viability assay was performed using the CellTiter 96 AQueous
non-radioactive cell proliferation assay (Promega, Madison, WI).
Assays were performed according to the manufacturer's instructions
using human fibroblasts (WI-38) as cellular model. Toxicity analysis
was conducted at active concentration of 100 mM (concentration of
test compounds was 100 mM inside the 96 well plate wells). Briefly,
20,000 cells were subjected to drug candidates (100 mM) for 6 h and
levels of cellular viability were measured spectrophotometrically at
490 nm using BMG FLUOstar OPTIMA spectrophotometer (BMG
Labtech, Durham, NC).
(
EMEM) supplemented with 10% fetal bovine serum and incubated
at a temperature of 37 1C in a humidified incubator containing 5%
CO (VWR International, Mississauga, ON). Cells were allowed to
2
grow till they were 80% confluent and then sub-cultured. Cells
were observed for normal growth and morphology every day
while the growth medium was changed every 48 h.
2.4. Animal model
2.9. Statistical analysis
The animal model setup and experiment was conducted in the
All experiments were performed in triplicates (n¼3) and statistical
animal facility of the Atlantic Veterinarian College, University of Prince
Edward Island (UPEI) Charlottetown, PE, Canada. Experimental proce-
dures were conducted in accordance with the guidelines of the
Canadian Council for Animal Care and the study was approved by
the Animal Care and Use Committee (ACUC) at UPEI prior to the
experiment. Spontaneously hypertensive rats (SHR) were obtained
from Charles River Laboratories Inc. (Quebec City, QC). The SHR rats
were subjected to 12 h light and dark cycle in individual chambers and
were fed a casein–corn starch–sucrose-based AIN-93G diet. The rats
were anesthetized by isoflurane inhalation after 30 days, and kidney
tissues were removed and immediately stored at ꢀ80 1C until further
analysis.
analysis of the data was performed by analysis of variance (one-way
ANOVA) using Minitab 16 software (State College, PA, USA). A proba-
bility value obtained from statistical analysis with Pr0.05 was
considered statistically different. The IC50 values were determined
using the regression analysis in Microsoft excel.
3. Results
Caffeic acid (4, Fig. 2) is found in relatively high concentrations in
many plants as a key intermediate in the biosynthesis of lignins.
Caffeic acid and its naturally occurring derivative, caffeic acid phe-
nethyl ester (CAPE) (5, Fig. 2), is found in high concentrations in
propolis of the honeybee hive, have anticancer properties as well
as immunomodulatory, anti-inflammatory and antioxidant effects
(Grunberger et al., 1988). Caffeic acid (4) and CAPE (5) have been
found to have antiproliferative and cytotoxic properties in a variety of
cancer cell lines without displaying significant toxicity toward healthy
cells (Gomes et al., 2003). The caffeic acid (4) has been reported as a
potent antihypertensive agent (Li et al., 2005) and an inhibitor of ACE
(Actis-Goretta et al., 2006). However, no studies to date have system-
atically evaluated synthetic derivatives of caffeic acid (4) or CAPE (5) to
identify novel molecules with improved potencies. Given the biologi-
cal significance of the caffeic acid pharmacophore, we hypothesized
that certain derivatives of caffeic acid will have stronger antihyperten-
sive activity. The current study focused on the potential of structurally
modified caffeic acid derivatives for development of new hypertensive
agents.
2
.5. ACE inhibition assay
An ACE inhibition assay was performed according to previously
described methodology (Balasuriya and Rupasinghe, 2012) using
histidine- -hippuryl- -leucine-chloride (HHL) as substrate. The histi-
L
L
dine–leucine (His-Leu) production from HHL cleavage by ACE and its
potential inhibition was measured fluorometrically using BMG FLUOs-
tar OPTIMA spectrophotometer (BMG Labtech, Durham, NC).
2
.6. Renin inhibition assay
The renin inhibitory potential of caffeic acid derivatives was
determined according to a previous report (Ajibola et al., 2011) using
fluorescent substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-
Ile-His-Leu-Val-ile-His-Thr-Lys(Dabcyl)-Arg. The fluorescent product
(
peptide-EDANS) from substrate cleavage by renin and its inhibition
Since renin, a member of the aspartic protease family, is the first
enzyme involved in the cascade of RAAS activation, we first assessed
the renin inhibitory action of these compounds in vitro. The structural
modification of caffeic acid (4) had immense impact on renin
inhibitory activity of its derivatives. The IC50 values of the compounds
ranged between 0.1 and 5704 mM (Table 1) for inhibition of renin
in vitro. Aliskiren, a direct renin inhibitor exhibited remarkably strong
renin inhibition with IC50 value of 0.1 mM while caffeic acid was the
weakest renin inhibitor (IC50¼5704 mM) among all compounds
assessed (Pr0.05). The strongest renin inhibition among caffeic acid
was measured fluorometrically using BMG FLUOstar OPTIMA spectro-
photometer (BMG Labtech, Durham, NC).
2
.7. Aldosterone production
The aldosterone concentration was determined using an Aldoster-
one Enzyme Immune Assay (EIA) kit (Cayman Chemical, Ann Arbor,
MI). The assay is based on the competitive binding of aldosterone and
aldosterone-acetylcholiesterase conjugates to aldosterone monoclonal
antibody. The aldosterone-acetylcholiesterase conjugate binding to
antibody is inversely proportional to the aldosterone present in a
sample. Kidneys were removed surgically from SHR and adrenal
tissues were cultured using complete EMEM medium as described
previously (Armato and Nussdorfer, 1972). Briefly, adrenal glands were
removed and subjected to tissue culture technique using complete
EMEM growth media, supplemented with 10% fetal bovine serum,
2 2
derivatives was shown by CH CH(Ph) moiety containing analog 22
with 25 times stronger inhibition compared to CA. Compound 22 was
closely followed by compound 21 (ꢁ23 times stronger than CA) in its
ability to inhibit renin enzyme and subsequently hypertension. The
results from Table 1 clearly indicate that addition of a phenyl group in
the CAPE (5) structure significantly (Pr0.05) improved the biological
properties when compared to CA. This could be due to better
interaction of the candidate compound with renin enzyme upon its
esterification. However, 24, an amide analog of CAPE (5), showed very
weak inhibition of renin enzyme in vitro. Interestingly, analog 12
bearing a 3-methylbut-3-enyl moiety also showed improved renin
inhibitory properties. Overall, in the first phase of our study, we
1
00 U/ml penicillin and 100 mg/ml streptomycin in a humidified
incubator (5% CO ) at 37 1C. Tissue cultures were treated with drug
2
candidates (500 mM) for 24 h and levels of steroid hormone were
measured spectrophotometrically with ELISA kit at 420 nm using
BioTek, Power wave XS2 spectrophotometer (BioTek, Winooski, VT).

128
K.S. Bhullar et al. / European Journal of Pharmacology 730 (2014) 125–132
Table 1
Renin inhibition by caffeic acid and its derivatives in comparison to aliskiren, captopril and other phytochemicals.
Concentration (μM)
In vitro percentage inhibition of renin
Aliskiren
Captopril (1)
Quercetin (2)
Chlorogenic acid (3)
CA (4)
CAPE (5)
6
7
1
0
72.50
95.74
98.47
100
ND
6.31
13.69
29.87
42.39
7.43
27.28
31.07
44.43
52.36
4.93
7.61
19.16
41.59
63.69
ND
17.30
33.11
34.82
49.20
69.35
3.75
26.93
28.72
34.09
68.80
4.56
1
00
3.11
5.99
6.62
9.81
26.33
32.18
36.77
64.72
2
5
00
00
1000
100
IC50 (μM)
0.1a
1108.04s
971.78r
730.83q
5704.12w
556.58k
679.51o
691.90p
Concentration (μM)
In vitro percentage inhibition of renin
8
9
10
11
12.98
26.59
26.68
35.24
62.58
12
13
14.10
35.01
34.60
51.94
66.77
14
18.69
26.79
28.98
35.56
73.28
15
1
0
13.27
11.67
27.02
31.29
40.22
66.01
10.74
30.49
37.36
42.52
66.15
10.17
30.05
39.64
56.44
67.77
17.35
36.19
33.60
51.41
70.13
1
00
27.44
28.44
38.03
45.57
2
5
00
00
1000
IC50 (μM)
2846.30u
655.07n
508.42i
744.47qr
331.67e
383.95g
620.32l
534.64jk
Concentration (μM)
In vitro percentage inhibition of renin
1
6
17
18
19
10.12
35.96
40.19
47.33
78.90
21
22
23
24
1
0
19.33
36.24
39.41
48.01
70.89
16.27
32.75
33.11
54.40
68.71
19.09
25.96
34.03
41.85
67.59
10.68
41.96
43.01
48.24
77.50
6.34
45.52
40.89
65.70
66.85
11.70
36.64
35.88
44.57
67.53
ND
1
00
20.10
24.30
44.84
47.67
2
5
00
00
1000
IC50 (μM)
522.08j
361.91f
627.64m
281.69d
249.31c
228.80b
433.05h
1296.81t
Data are presented as mean of three independent experiments. Mean with various superscripts (a–w) in each column represent levels of significance (Pr0.05); micro molar
μM); inhibitory concentration 50 percent (IC50). The Aliskiren as a reference compound for renin inhibition was assayed separately with IC50 value of 0.1 μM. CA, caffeic acid;
(
CAPE, caffeic acid phenethyl ester.
noticed that esterification of caffeic acid significantly improved the
renin inhibitory properties in vitro.
In the next phase to assess multi-target RAAS inhibition, novel
derivatives were screened for their ability to inhibit aldosterone
production in adrenal glands of SHR. The kidneys of SHR were
removed surgically and tissues were placed in ꢀ80 1C until used
for the assays. The adrenal cultures were then treated with caffeic
acid and its derivatives (500 mM) in comparison with selected
phenolics and captopril in six well sterile tissue culture plates.
The analysis was conducted using aldosterone EIA kit-Monoclonal
and results were obtained spectrophotometrically. The results
(Table 3) showed that treatment of adrenal glands of SHR with
caffeic acid derivatives had significant effect on the aldosterone
levels. The aldosterone concentration ranged between 530.6 and
891.8 pg/ml in sample. Parent compound, caffeic acid, emerged as
a weak inhibitor of aldosterone activity in vitro with high aldos-
terone concentration of 679.5 pg/ml. However, captopril (1)
(530.6 pg/ml aldosterone) was the strongest inhibitor (Pr0.05)
of aldosterone activity in vitro. The caffeic acid analog, 8 (574.4 pg/
ml aldosterone) with n-Pr moiety was the most potent modulator
of aldosterone activity among derivatives in vitro (Pr0.05) while
two phenolics, quercetin (2) (569.6 pg/ml aldosterone) and chloro-
genic acid (3) (598.4 pg/ml aldosterone) closely followed com-
pound 8 in their ability to inhibit aldosterone activity. Amide 24
(567.7 pg/ml aldosterone) was also an active modulator of aldos-
terone activity among assayed compounds. However, CAPE (5) was
the weakest inhibitor of steroid hormone activity (Pr0.05) among
all drug candidate compounds. Results from the third phase of
this study were consistent with renin and ACE inhibitory activity
as caffeic acid derivatives having an aryl group showed stronger
potential to modulate aldosterone activity in SHR rat adrenal
glands.
The second phase of antihypertensive studies focused on
inhibition of ACE, a peptide involved in the conversion of angio-
tensin I to angiotensin II along with degradation of bradykinin. The
involvement of ACE in two hypertension cascades makes it an
essential drug target for the treatment of hypertension. The parent
compound, caffeic acid (4) emerged as a strong ACE inhibitor
compared to its ability to inhibit renin in vitro. The IC50 value of
caffeic acid (4) was 430 mM while the IC50 value of all compounds
assayed ranged between 1.02 and 1087 mM (Table 2). The strongest
ACE inhibitor was captopril (1) (IC50¼1.02 mM), while compound
2
2 (IC50¼9.14 mM) and quercetin (2) (IC50¼9.85 mM) closely fol-
lowed the clinically used drug (Pr0.05). Among caffeic acid
derivatives, analog 22 with CH CH(Ph) moiety showed 47 times
stronger ACE inhibition compared to the parent compound.
Following this trend, compound 23 with CH CH CH Ph moiety
2
2
2
2
2
exhibited ꢁ28 times higher ACE inhibitory activity, which sug-
gests that addition of an extra carbon increases the biological
activity compared to the CAPE (5). Another phenolic acid, chloro-
genic acid (3) (IC50¼21.53 mM) also emerged as a strong antihy-
pertensive agent as shown previously (Suzuki et al., 2006).
Interestingly, CAPE (5) emerged as a stronger (IC50¼131.7 mM)
ACE inhibitor (Pr0.05) compared to its structural counterpart
amide 24 (IC50¼513.3 mM). However, most compounds with
aliphatic alkyl chain moieties including 8, 7 and 9, bearing n-
propyl, ethyl, and isopropyl, respectively, emerged as the weakest
ACE inhibitors in vitro. These compounds with aliphatic chain
exhibited ꢁ2 to 2.5 times weaker ACE inhibition compared to
caffeic acid.

K.S. Bhullar et al. / European Journal of Pharmacology 730 (2014) 125–132
129
Table 2
ACE inhibition by caffeic acid and its derivatives in comparison to captopril and other phytochemicals.
Concentration (μM)
In vitro percentage inhibition of angiotensin converting enzyme (ACE)
Captopril (1)
Quercetin (2)
Chlorogenic acid (3)
CA (4)
CAPE (5)
6
7
8
1
0
64.96
95.43
99.03
99.12
100
44.94
78.05
83.03
84.95
86.07
45.13
62.25
63.44
68.36
80.24
19.90
21.43
47.31
62.58
78.48
19.39
34.58
60.18
62.04
86.65
22.62
25.62
25.10
1
00
48.54
46.10
54.67
70.61
25.25
27.66
33.63
54.76
35.31
39.82
43.95
52.97
2
5
00
00
1000
IC50 (μM)
1.02a
9.85b
21.53d
430.01n
131.73i
185.70m
911.98t
1017.19u
Concentration (μM)
In vitro percentage inhibition of angiotensin converting enzyme (ACE)
9
10
11
20.56
35.47
48.74
63.81
83.21
12
20.24
23.78
25.45
35.15
67.97
13
14
12.71
20.95
23.11
42.62
75.74
15
16
1
0
24.04
21.07
25.23
25.66
51.39
19.31
39.44
53.69
59.41
75.22
33.81
47.71
83.10
87.27
89.54
27.41
46.61
72.40
78.74
87.10
17.25
38.51
56.44
57.27
85.01
1
00
2
5
00
00
1000
IC50 (μM)
1087.31v
166.62l
154.17k
686.31r
40.52e
600.42q
63.09f
148.29j
Concentration (μM)
In vitro percentage inhibition of angiotensin converting enzyme (ACE)
17
18
19
21
22
23
24
1
0
20.32
35.01
66.72
68.39
87.73
24.15
25.46
46.11
50.47
78.50
26.87
32.90
63.66
68.87
87.82
4.60
9.50
31.23
31.99
62.36
46.72
77.83
89.61
91.57
92.05
46.58
67.73
71.32
86.60
91.36
16.19
29.68
32.70
51.62
62.35
1
00
2
5
00
00
1000
IC50 (μM)
107.57h
456.47o
99.83g
768.49s
9.14b
15.85c
513.28p
Data are presented as mean of three independent experiments. Mean with various superscripts (a–v) in each column represent levels of significance (Pr0.05); micro molar
μM); inhibitory concentration 50% (IC50). CA, caffeic acid; CAPE, caffeic acid phenethyl ester.
(
Table 3
inhibition by active compounds. We measured the Pearson pro-
duct moment correlation (Pearson correlation) using Minitab
statistical software and results are shown in Table 4. The results
for active derivatives [compound 8 and CAPE (5)] had low and
statistically insignificant correlation among the renin and ACE
inhibitory activities. Other active compounds, 12, 23, 22 and 24
had positive and strong correlation in their ability to inhibit renin
and ACE in vitro. However, analog 21 had strong negative correla-
tion in its antihypertensive characteristics.
Modulation of aldosterone levels in SHR adrenal glands by caffeic acid and its
derivatives.
Compounds
Aldosterone concentration (pg/ml)
Captopril (1)
Quercetin (2)
Chlorogenic acid (3)
CA (4)
530.59a
bc
569.56
598.35
679.51
862.25
641.00
603.97
574.41
632.09
bcd
i
j
CAPE (5)
fghi
6
7
8
9
Finally, all the improved caffeic acid derivatives along with
captopril (1) (Fig. 1) were screened for their toxicity in a cell
culture model using human fibroblasts or WI-38 cells (Fig. 3).
Depletion of cellular viability upon incubation of drug candidates
was a toxicity biomarker in this experiment. Cellular viability was
assessed by monitoring the formation of formazan product via bio-
reduction of tetrazolium compound. The caffeic acid derivatives,
captopril (1), quercetin (2) and chlorogenic acid (3) were screened
at 100 mM (Fig. 3) and absorbance was recorded at 490 nm. As
anticipated due to their natural origin, all caffeic acid derivatives,
quercetin (2) and chlorogenic acid (3) exhibited lower toxicity
compared to captopril (1) (Pr0.05). Quercetin (2) emerged as the
least cytotoxic compound while cells treated with caffeic acid
derivatives showed 480% cellular viability. Quercetin (2) was
closely followed by chlorogenic acid (3) in line of non-toxic
antihypertensive agents (Pr0.05). Among all caffeic acid deriva-
tives, compound 13, a cyclohexyl CAPE (5) analog, showed the
lowest cellular viability (Pr0.05) compared to other derivatives.
However, the strong antihypertensive compound 22 exhibited
lower cellular toxicity compared to the parent compound, caffeic
acid (4) (Pr0.05). Captopril (1), an antihypertensive drug, exhib-
ited the highest toxicity in WI-38 cells (Pr0.05) compared to all
other assayed natural compounds and derivatives. Overall most
bcdef
b
efgh
ghi
1
0
645.50
defg
1
1
626.21
613.92
618.95
670.06
599.05
613.20
bcdefg
1
2
cdefg
1
3
hi
1
4
bcde
1
5
bcdefg
1
6
ighi
1
7
645.50
ibcd
1
8
590.72
590.03
681.92
670.84
681.10
567.74
891.81
ibcd
1
9
i
2
1
i
2
2
2
2
3
4
i
bcdef
j
SHR control
Data are presented as mean from three independent experiments. Mean with
various superscripts (a–j) in each column, are significantly different (Pr0.05). CA,
caffeic acid; CAPE, caffeic acid phenethyl ester.
As the renin and ACE activity is closely related in pathophysiol-
ogy of hypertension, we conducted correlation analysis between
these two variables to see possible correlation in renin and ACE

130
K.S. Bhullar et al. / European Journal of Pharmacology 730 (2014) 125–132
compounds with an aryl group exhibited 490% cellular viability
resulting in low toxic manifestations in WI-38 cells.
has been extensively used as dual antihypertensive therapy; however,
a recent report suggests that its long term application has not resulted
in significant improvement in terms of survival rate and clinical events
(
Makani et al., 2013). As renin is a modulator of the rate limiting step
4
. Discussion
in the RAAS, it serves as a crucial and direct target for hypertension.
A known direct renin inhibitor, aliskiren, despite its low bioavailability
serves the purpose of attenuating hypertension and makes renin
inhibition an attractive therapeutic option (Gradman and Kad, 2008).
Reports on renin inhibition by phytochemicals and their derivatives
are limited, while studies on phenolic acids remain unexplored. A few
recent reports indicate direct renin inhibition by polyphenols and
other bioactives. Baicalin, a flavone member of the flavonoid class of
polyphenols exhibits strong renin inhibition with IC50 value of 120 mM
RAAS plays an important role in the regulation and pathology of
blood pressure, thus serving as a crucial drug target for attenuation
of hypertension. In addition, multiple therapeutic targets within
RAAS, open doors for inclusive investigation of these focus points for
pharmacological intervention. Renin inhibitors directly interact with a
single endogenous substrate, angiotensinogen, and are highly specific
in their hypotensive action. Most of the recent investigations on
hypertension therapeutics have ignored renin and solely focused on
ACE inhibition and angiotensin II type I receptor blockade. This
combination of ACE inhibitors and angiotensin receptor blockade
(
Deng et al., 2012). Similarly, strong inhibitory action against renin has
been shown by catechin related compounds in vitro (Li et al., 2013).
Catechins (flavan-3-ols) including (ꢀ)-epigallocatechin gallate (IC50
¼
4
5 mM), (ꢀ)-epicatechin gallate (IC50¼619 mM) and (ꢀ)-epigallocate-
Table 4
chin (IC50¼2175 mM) have exhibited antihypertensive potential
Correlation between extent of ACE inhibition and renin inhibition.
through renin inhibition in vitro (Li et al., 2013). Similarly, saponins
(
triterpenoids), another member of plant secondary metabolites
identified for their human renin inhibitory activity (Takahashi et al.,
008, 2010). Our findings are in accordance with previous studies and
Compound
ACE inhibition
Renin inhibition Pearson
P-value
(
IC50 value)
(IC50 value)
correlation
2
Captopril (1)
Quercetin (2)
Chlorogenic
acid (3)
CA (4)
CAPE (5)
6
7
8
9
1.02
9.85
21.53
1108.04
971.78
730.83
0.201
0.806
-0.140
0.871
0.403
0.911
revealed renin inhibitors can be derived from phytochemicals. Inter-
estingly, compound 22 with IC50 value of 229 mM exerted strong and
comparable anti-renin activity to other renin inhibitors (Deng et al.,
2012; Li et al., 2013; Takahashi et al., 2008, 2010). Similar to the renin
inhibitory report of catechin compounds (Li et al., 2013), our study
showed that structural modification with respect to the parent com-
pound significantly improved the renin inhibition. Moreover, the
addition of large molecular weight moieties to the caffeic acid skeleton
may be a crucial contributory factor to improved renin inhibition,
which can be further explored through docking and molecular
modeling analysis.
430.01
131.77
185.70
911.98
1017.19
1087.31
166.62
154.17
686.31
40.52
600.42
63.09
148.29
107.57
456.47
99.83
5704.12
556.58
679.51
691.90
2846.30
655.07
508.42
744.41
331.67
383.95
620.32
534.64
522.08
361.90
627.64
281.69
249.31
288.80
433.05
1296.81
0.886
-0.119
0.686
0.111
0.306
0.924
0.519
0.929
0.920
0.254
0.661
0.787
0.047
0.829
0.182
0.048
0.318
0.762
0.142
0.816
0.336
0.620
0.264
0.402
0.125
0.921
0.507
0.329
0.997
-0.265
-0.960
-0.917
0.878
0.365
-0.975
ꢀ0.285
ꢀ0.864
0.562
0.915
1
0
1
1
1
2
1
3
ACE, a dipeptidyl carboxypeptidase is a major target for antihy-
pertensive drugs such as captopril and other similar drugs. Apart from
converting angiotensin I to angiotensin II, ACE is also involved in the
release of aldosterone from the adrenal cortex (Hsueh and Wyne,
1
4
1
5
1
6
1
7
1
8
2
011). The ACE inhibition is an important and well investigated target
1
9
2
1
768.49
9.14
15.85
in hypertension therapy and has given strong clinically viable options
like alcacepril, captopril, enalapril and lisinopril (Peng et al., 2005).
Unlike renin inhibitors, large number of polyphenols (Balasuriya and
Rupasinghe, 2011) including phenolic acids (Zhao et al., 2011) have
been assessed for their antihypertensive and ACE inhibitory potential.
An initial report (Actis-Goretta et al., 2006) demonstrated the weak
22
23
24
513.28
0.808
The correlation analysis conducted between ACE and renin inhibition using Pearson
correlation analysis at α¼0.05. CA, caffeic acid; CAPE, caffeic acid phenethyl ester.
Fig. 3. Toxicity analysis of caffeic acid and its derivatives used as multi-target RAAS regulators. All data represent the mean7S.E.M. of three independent experiments, each
with six wells per experiment and treatment conditions with appropriate controls. The concentration of drug candidates (100 μM) is an active concentration interacting with
the cells in vitro. Columns not sharing a common symbol (n, nn, nnn, #, and ##) differ significantly with each other (Po0.05). CA, caffeic acid; CAPE, caffeic acid
phenethyl ester.

K.S. Bhullar et al. / European Journal of Pharmacology 730 (2014) 125–132
131
ACE inhibitory potential of various phenolic acids including gallic acid,
chlorogenic acid and caffeic acid with IC50 41500 mM. However, a
recent report (Hidalgo et al., 2012) shows stronger ACE inhibitory
potential of phenolic acids including gallic acid (IC50¼332.4740.1
strong benchmark to evaluate the toxic effects of drug candidates. The
depleted cellular viability was established as the biomarker of toxicity
and results showed that human fibroblasts exhibited very high (80–
90%) cellular viability after exposure to most drug candidates. As
synthetic drugs are known to induce many side effects in the human
body, results showed lowest cellular viability in the captopril treated
group. This clinically approved drug is widely used in treatment of
hypertension through ACE inhibition. Our results also supported the
safety of other natural bioactives such as quercetin and chlorogenic
acid, which are potent antihypertensive agents of natural origin. Since
all of our tested compounds exhibited high cellular viability with low
toxic manifestations, we have chosen compound 22 as a potential dual
RAAS inhibitor. However, the long term efficacy and safety of
compound 22 requires mechanistic and dose–response studies to
validate its possible dual blockade of the renin–angiotensin system
in vivo.
mM), caffeic acid (IC50¼157.3 716.1 mM) and coumaric acid (IC50
¼
504.2731.5 mM). These results may be contradictory due to the
methodology adopted during the experimentation. Apart from in vitro
activity, phenolic acids like vanillic acid (Kumar et al., 2011) have also
ω
exhibited antihypertensive potential in N -nitro-
L
arginine methyl
ester (
another phenolic acid) has shown in vivo ACE inhibition potential
Ohsaki et al., 2008) as it reduced plasma ACE activity in SHR rats from
0.6 mU/ml to 16.9 mU/ml, along with the modulation of several
L-NAME) induced hypertensive rats. Similarly, ferulic acid
(
(
2
other plasma and liver biochemical parameters. Our findings are
consistent with these earlier reports (Hidalgo et al., 2012; Kumar
et al., 2011; Ohsaki et al., 2008) and with other studies which confirm
the improved biological activities of caffeic acid derivatives (Jiang et al.,
2
005). As for the most active compound 22 (IC50¼9 mM), its ACE
inhibition potential was at par with captopril (IC50¼1 mM), quercetin
5. Conclusion
(
IC50¼9 mM) and potent antihypertensive compounds such as epica-
techin–tetramer (IC50¼4 mM), epicatechin – hexamer (IC50¼8 mM)
and quercetin-3-O-glucuronic acid (IC50¼27 mM) (Balasuriya and
Rupasinghe, 2011, 2012). Overall, the ACE inhibitory activities of caffeic
acid and its derivatives were confirmed using standard methodology
and a novel ACE inhibitor compound 22 was identified. It is interesting
Most of the currently used antihypertensive drugs target RAAS for
therapeutic intervention via inhibition of enzymes triggering patho-
logical cascades. Currently, pharmacological investigations aim at the
multi-target inhibition of RAAS as an effective approach to modulate
hypertension. In this study, we present the first report of multi-target
RAAS inhibition by caffeic acid and its novel derivatives. The RAAS
inhibitor compound 22 emerged as a multi-target modulator in vitro.
2 2
to note that the addition of CH CH(Ph) moiety to caffeic acid skeleton
made the parent phytochemical a novel dual RAAS inhibitor. Further
studies using experimental animal models and human clinical models
are required to confirm the in vivo biochemical and pharmaceutical
potential of the novel compound.
2 2
The addition of CH CH(Ph) moiety at R1 domain of caffeic acid was
found to significantly improve the biological activity of the compound.
However, most of the derivatives with short aliphatic alkyl chain
remained inactive in the inhibition of RAAS enzymes. Our study
demonstrates the potential of the new caffeic acid derivatives as
antihypertensive agents, as they modulate the RAAS pathway through
enzymatic inhibition with low cytotoxic manifestations compared to a
conventional hypertension drug.
Aldosterone, a sodium retaining steroid is a downstream member
of RAAS, and an effector of angiotensin II. These biological character-
istics allow it to present itself as a target for antihypertensive therapy.
The two ways to explore the use of aldosterone as a drug target are
through its selective mineralocorticoid-receptor blockade or by aldos-
terone antagonism (aldosterone synthase inhibition). The present
study examined the ability of caffeic acid and its derivatives to modu-
late aldosterone concentration in the adrenal gland cultures of SHR,
possibly by lowering the levels of the steroid hormone. It was found
that captopril and compound 8 were the most potent inhibitors of
steroidogenesis in rat adrenal gland culture. The addition of n-Pr
moiety at R position significantly impacted the inhibition of steroid
hormone. There are a few reports indicating the aldosterone modula-
tion by phytochemicals, and mainly focus on inhibition of cortisol
production in cell line models. These findings have reported the strong
inhibition of steroid hormone synthesis in human adrenocortical
H295R cells by two isoflavones, daidzein and genistein, along with
the flavone apigenin. Similarly, treatment of the flavonoids such as
Acknowledgments
H.P.V.R. gratefully acknowledges funding from the Canada
Research Chair program and the Discovery Grant program of the
Natural Sciences and Engineering Research Council (NSERC) of
Canada. M.T. acknowledges the New Brunswick Health Research
Foundation and the New Brunswick Innovation Foundation.
Authors also thank Jason Sproule, Department of Environmental
Sciences, Faculty of Agriculture, Dalhousie University for his help
with spectrophotometric analysis.
6-hydroxyflavone, 4-hydroxyflavone, apigenin, daidzein, genistein and
formononetin significantly impacted the cortisol production in human
adrenal H295R cells stimulated with di-buthylyl cAMP (Ohno et al.,
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