Antihypertensive effects of tannins isolated from

traditional Chinese herbs as non-specific inhibitors of

angiontensin converting enzyme
Ju-Chi Liu a, Feng-Lin Hsu b, Jen-Chen Tsai c, Paul Chan a,*, Jenny Ya-Hsin Liu a,
G. Neil Thomas d, Brian Tomlinson d, Ming-Yu Lo e, Jung-Yaw Lin f
a

Division of Cardiovascular Medicine, Taipei Medical UniversityWan Fang Hospital, No 111, Hsing Lung Road,
Section 3, Wen Shan District, Taipei City 117, Taiwan
b
School of Pharmacy, Taipei Medical University, Taipei, Taiwan
c
Department of Nursing, Taipei Medical University, Taipei, Taiwan
d
Department of Medicine and Therapeutics, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong
e
Department of Chinese Medicine, China Medical College, Taichung, Taiwan
f
Department of Biochemistry, National Taiwan University College of Medicine, Taipei, Taiwan
Received 11 July 2002; accepted 14 March 2003

Abstract
The tannins are natural polyphenols, able to precipitate water-soluble alkaloids and possess an inhibitory action
on the angiotensin converting enzyme (ACE). We identified 18 polyphenolic compounds (tannins) from Chinese
herbs and examined the in vitro effects of these tannins on ACE activity, including determination of the 50%
inhibitory concentrations (IC50), specificity and mode of inhibition. We also assessed the in vivo inhibitory effect
of the tannins on angiotensin I-induced blood pressure elevation in spontaneously hypertensive rats (SHR). Nine
tannins with an IC50 < 200 AM for ACE inhibitors were identified belonging to three tannin classes:
caffeoylquinates, flavan-3-ols and gallotannins. In vitro, we found caffeoylquinates chelate the ACE zinc cofactor.
Two of the flavan-3-ols: epigallocatechin-3-O-gallate and epigallocatechin-3-O-methylgallate, and one of
gallotannin: 1, 2, 3, 4, 6-penta-O-galloyl-h-D-glucose were non-specific inhibitors because also reduced the
activity of trypsin and chymotrypsin. The ACE inhibition of 1, 2, 3, 4, 6-penta-O-galloyl-h-D-glucose was also
reduced after addition of bovine serum albumin, suggesting a non-specific mode of action. In vivo, 1,2,3,6-tetra-Ogalloyl-h-D-glucose and epigallocatechin-3-O-methylgallate had a strong dose-dependent hypotensive effect

0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0024-3205(03)00481-8

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J.-C. Liu et al. / Life Sciences 73 (2003) 15431555

reducing the blood pressure significantly in the SHR with infusion of the angiotensin I. These findings indicate that
some of the tannins isolated from herbs inhibit ACE activity non-specifically. The ACE inhibitory effect of these
tannins may explain the hypotensive effects of some traditional Chinese herbs.
D 2003 Elsevier Science Inc. All rights reserved.
Keywords: Angiotensin converting enzyme; Inhibitor; Tannins; Traditional Chinese herbs

Introduction
The angiotensin converting enzyme (ACE) is an important component of the renin-angiotensinaldosterone system, cleaving angiotensin I to the potent vasoconstrictor, angiotensin II. Randomized,
placebo-controlled trials have shown that ACE inhibitors are effective in lowering blood pressure and in
the treatment of left ventricular dysfunction and diabetic nephropathy [14].
Naturally occurring peptides, such as those isolated from snake venom, for example from Bothrops
jararaca, or microbial metabolites have ACE inhibitory activity [5,6]. These compounds have been
reported to potentiate bradykinin either by binding to the active site as a substrate for the enzyme [5] or as a
non-substrate such as the competitive inhibitor [6]. The orally active synthetic ACE inhibitors, that are
commercially available, have non-peptide structures based around sulfur, carboxyl or phosphinyl groups at
the active centre [7].
It is known that some herbal extracts, such as the tannins from Magnolia liliflora and Magnolia
officinalis lower blood pressure [8] with aqueous or alcohol extracts of these herbs. They could
reduce blood pressure in vivo after intravenous, intramuscular or intraperitoneal injection in
anaesthetized rats [9]. Moreover, some extracts of the Gentianaceae show similar effects [10].
Therefore it is of interest to identify individual components, rather than crude extracts, which act
as inhibitors of ACE activity from purified fractions of Chinese herbal medicines that are traditionally
used in the treatment of hypertension. In this study, we examined the activity of tannins isolated from
traditional Chinese herbs on the inhibition of angiotensin converting enzyme in vitro and their
hypotensive effect in vivo.

Materials and methods

Quantitative analysis on inhibition of the ACE activity
The assay method of the activity of angiotensin converting enzyme (ACE EC3.4.15.1) was based
on that described by Cushman et al. in 1970 [11] and modified by Suzuki et al. [12]. Simply, ACE
hydrolyses the angiotensin I analogue, hippuryl-L-histidyl-L-leucine forming hippuric acid, levels of
which determine ACE activity. One unit of ACE is defined as the amount of ACE required to
produce 1 Amole hippuric acid at 37 jC, pH 8.3 in one minute [13]. 2,4,6-Trichloro-s-triazine in
100% 1,4-dioxane (TT reagent, Cyanuchloride, E Merck, Germany) was used as an indicator for
screening the 18 tannins with ACE inhibiting activity isolated from over 100 Chinese herbal
medicines analyzed.

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Kinetic study of the effect of tannins on ACE activity

The nine tannins with ACE IC50 less than 200 AM were categorised into three classes: caffeoylquinates, flavan-3-ols, or gallotannins. Four representative tannins with ACE IC50 less than 200 AM were
then selected from these to elucidate the mode of inhibition. These included two caffeoylquinates, one of
the flavan-3-ol: catechin-3-O-gallate, and one of the gallotannin:1,2,3,6-tetra-O-galloyl-h-D glucose.
The tannins were dissolved in 100% DMSO to produce concentrations (S) of 500, 1000, 1500, 2000 and
2500 AM. Absorbency at 382 nm was measured at 10 and 15 min. The reaction rate (V) is defined as the
change of absorbency in unit time. Lineweaver-Burk plots (1/S against 1/V) of three curves (two
inhibition concentration curves and one control group curve) were then used to determine the MichaelisMenten equation and whether the inhibition was competitive or non-competitive [14].
Assessment of specificity of the ACE inhibition
As tannins can conjugate with protein and hence non-specifically inhibit enzyme action, we assessed
the tannin-induced ACE inhibition with increasing concentrations of bovine serum albumin (BSA, 0, 25,
50, 125, 200 and 250 Ag/ml) to assess the importance of this mode of inhibition. We also assessed the
specificity of the inhibition of ACE by investigating the effect of all nine tannins on the activity of
chymotrypsin and trypsin. In the experimental group, 6 Al of tannin in 100% DMSO was mixed with 0.5
ml 20 Ag/ml chymotrypsin under 0.1 M phosphate buffer (pH 8.0) and incubated at 37 jC for 5 min. 1
ml of 2% casein in 0.1 M phosphate buffer (pH 8.0) was then added and incubated for a further 30 min at
which time 2 ml of 10% trichloroacetate was added to quench the reaction. After 1 hour, for protein
sedimentation, the mixture was centrifuged and the absorbency of supernatant measured at 280 nm. In
the control group, no tannins were added to the DMSO and in the blank group no enzyme was added.
The experimental procedure for the measurement of the tannin inhibition of trypsin was similar to that
for chymotrypsin. In the experimental group, 6 Al of tannin solution was mixed with 0.5 ml (40 Ag/ml)
trypsin solution and incubated at 37 jC for 5 min. 1 ml of BAPNA (a-N-benzoyl-DL-arginine-Pnitroanilide HCl) solution (25 mg BAPNA in 0.5 ml DMSO made up to 50 ml in 0.1 M TrisHCl
buffer) was added and incubated at 37 jC for a further 20 min. Then, 0.5 ml of 10% acetic acid was
added to quench the reaction and the absorbency was measured at 410 nm. The Chymotrypsin/trypsin
inhibitory activity is expressed as residual Chymotrypsin/trypsin activity (%) = (absorbency of
experimental group
absorbency of experimental group blank / absorbency of control group
absorbency of control group blank)  100.
The effect of ZnCl2 on the inhibitory activity of ACE
As ACE is a Zn2 + ion containing metalloprotein, 1.5 mM ZnCl2 was added to the standard assay to
assess whether the inhibitory action of the nine tannins resulted from the chelation of the Zn2 + ions.
In vivo studies of the ACE inhibition action of tannins in spontaneously hypertensive rats
Spontaneously hypertensive rats (SHR) of body weight 250350 g and average blood pressure 150
180 mm Hg were used (n = 10). Twenty-four hours prior to the start of the experiments, polyethylene
catheters (internal 0.58 and external diameter 0.965 mm) filled with heparin solution (1000 U/ml) were

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J.-C. Liu et al. / Life Sciences 73 (2003) 15431555

inserted and secured into the exposed tail artery and vein of anaesthetized SHR. The rats were placed in
specially designed metal ring cages and allowed to recover overnight. Prior to the experiments the
arterial catheters were connected to pressure transducers and mean arterial blood pressure was recorded.

Fig. 1. The structures of three classes tannins isolated from traditional Chinese herbs: A. caffeoylquinates; B. flavan-3-ols;
C. gallotannins.

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1547

Fig. 1 (continued).

Subsequent studies were conducted in conscious rats. Single 0.2 ml volume doses of 200 ng/kg
angiotensin I were administered to each rat 5 min (control) before and at 5 min (experimental) intervals
10 min after the tannins were administered. 50 Al epigallocatechin-3-O-methylgallate, 1,2,3,6-tetra-Ogalloyl-h-D-glucose (10, 20 and 40 mg/kg dissolved in 100% DMSO) and captopril (10 mg/kg dissolved
Table 1
Summary of the IC50 (AM) and Ki (AM) for the tannins of the caffeoylquinates, flavan-3-ols and gallotannins classes extracted
from Chinese herbs
Tannin class
Caffeoylquinates
Methyl 3,5-di-O-caffeoylquinate
Methyl 3,4-di-O-caffeoylquinate
Flavan-3-ols
Gallocatechin
Catechin-3-O-gallate
Epicatechin-3-O-gallate
Epigallocatechin-3-O-gallate
Epigallaocatechin-3-O-methylgallate
Gallotannins
1,2,3,6-Tetra-O-galloyl-h-D-glucose
1,2,3,4,6-Penta-O-galloyl-h-D-glucose
All tannins shown have IC50 less than 200 AM, ND = not detectable.

IC50

Ki

82.9
169.6

72.6
287.7

195.9
113.0
18.3
37.4
26.6

218.6
168.2
20.7
37.4
35.2

101.4
73.1

86.7
ND

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Fig. 2. The inhibitory effect of 18 tannins on the angiotensin converting enzyme: A. The concentration-effect curves of
caffeoylquinates: (E-E) chlorogenic acid; (x-x) 3,4-di-O-caffeoylquinic acid; (w-w ) 3,5-di-O-caffeoylquinic acid; (D-D)
methylchlorogenate; (n-n) methyl 3,4-di-O-caffeoylquinic acid; (.-.) methyl 3,5-di-O-caffeoylquinic acid. B. The
concentration-effect curves of gallotannins: (w-w ) 1,6-di-O-galloyl-h-D-glucose; (D-D) 1,2,6-tri-O-galloyl-h-D-glucose; (nn) 1,3,6-tri-O-galloyl-h-D-glucose; (x-x) 1,4,6-tri-O-galloyl-h-D-glucose; (o-o) 3,4,6-tri-O-galloyl-h-D-glucose; (.-.)
1,2,3,6-tetra-O-galloyl-h-D-glucose; (E-E) 1,2,3,4,6-penta-O-galloyl-h-D-glucose. C. The concentration-effect curves of
flavan-3-ols (procyanidins): (5-5) gallocatechin; (D-D) catechin 3-O-gallate; (.-.) epigallocatechin 3-O-gallate; (n-n)
epigallocatechin 3-O-methylagllate; (E-E) epicatechin 3-O-gallate.

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1549

in 0.9% normal saline) were given by intravenous injection. The vasopressor action of angiotensin I was
observed for 60 minutes. The % of inhibition is calculated as the [(maximum experimental blood
pressure
maximum control blood pressure) / maximum control blood pressure]  100.

Results
Of the 18 tannins (Fig. 1) isolated from more than 100 different Chinese medicines, nine had IC50 less
than 200 AM and were classified into the three major tannin classes (Table 1). In the quantitative analysis
of the inhibitory activity, drug concentration causing 50% inhibition of the enzyme activity (IC50) can be
obtained by plotting the concentration against residual activity (Fig. 2).

Fig. 3. Lineweaver-Burk plot for the inhibition of angiotensin converting enzyme by tannins. A. Catechin 3-O-gallate: (o-o)
0.5%(v/v) DMSO, (.-.) 131.3 AM and (D-D) 262.6 AM. B. 1,2,3,6-Tetra-O-galloyl-D-glucose: (o-o) 0.5%(v/v) DMSO,
(.-.) 46.5 AM and (D-D) 93.0 AM. C. Methyl 3,4-dicaffeoylquinic acid: (o-o) 0.5%(v/v) DMSO, (.-.) 330.8 AM and
(D-D) 165.4 AM. D. Methyl 3,5-dicaffeoylquinic acid: (o-o) 0.5%(v/v) DMSO, (.-.) 132.6 AM and (D-D) 66.3 AM.

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Table 2
Comparison of inhibitory effects of various tannins on angiotensin converting enzyme in the presence or absence of bovine
serum albumin (BSA:25 Ag/ml)
Compound

Amount added
A M (Ag/ml)

Inhibition of activity (%) in the

presence or absence of BSA
None

BSA

Methyl 3,5-dicaffeoylquinic acid

Methyl 3,4-dicaffeoylquinic acid
Gallocatechin
Catechin 3-O-gallate
Epicatechin 3-O-gallate
Epigallocatechin 3-O-methylgallate
Epigallocatechin 3-O-gallate
1,2,3,6-Tetra-O-galloyl-h-D-glucose
1,2,3,4,6-Penta-O-galloyl-h-D-glucose

94.25 (50)
188.50 (100)
326.48 (100)
282.54 (125)
56.51 (25)
52.94 (25)
54.53 (25)
126.81 (100)
78.31 (75)

53.87
61.47
75.54
68.48
65.17
62.77
57.85
57.86
50.72

54.28
60.13
71.23
71.10
62.29
60.23
57.84
55.67
7.05

Kinetic study of the effect of tannins on enzyme activity

The enzyme kinetics of the ACE in the presence of the four representative tannins with IC50 less than
200 AM was determined from the Lineweaver-Burk plots from the Ki values that were calculated by
fitting the slope of linear regression in Michaelis-Menten formula (Fig. 3). Each of the four tannins from
the three classes exhibited a non-competitive mode of inhibition.
Assessment of specificity of the ACE inhibition
When the effect of the tannins on ACE inhibition was assessed in the presence of increasing
concentrations of BSA, only the activity of 78 AM 1,2,3,4,6-penta-O-galloyl-h-D-glucose was
significantly reduced, falling by 86% from an inhibition activity of 50.7% to 7.1% (Table 2). The
ACE inhibitory activity of remaining tannins was unaffected by the addition of different concentrations
of BSA. When the tannins were assessed for their ability to inhibit chymotrypsin and trypsin only three

Table 3
Inhibitory effects of various tannins on the activity of angiotensin converting enzyme, trypsin, chymotrypsin
Compound
Methyl 3,5-dicaffeoylquinic acid
Methyl 3,4-dicaffeoylquinic acid
Gallocatechin
Catechin 3-O-gallate
Epicatechin 3-O-gallate
Epigallocatechin 3-O-methylgallate
Epigallocatechin 3-O-gallate
1,2,3,6-Tetra-O-galloyl-h-D-glucose
1,2,3,4,6-Penta-O-galloyl-h-D-glucose

Amount added
A M (Ag/ml)

Activity (%)
ACE

Trypsin

Chymotrypsin

94.25 (50)
188.50 (100)
326.48 (100)
282.54 (125)
56.51 (25)
52.94 (25)
54.53 (25)
126.81 (100)
78.31 (75)

46.13
38.53
24.46
31.52
34.83
37.23
42.15
42.14
49.28

94.90
99.08
93.64
93.72
75.76
41.81
65.39
92.92
35.99

88.54
86.48
84.92
93.49
93.72
73.72
81.65
72.24
64.20

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Table 4
Effects of ZnCl2 on the inhibitory activity of various tannins on angiotensin converting enzyme
Compound

Methyl 3,5-dicaffeoylquinic acid

Methyl 3,4-dicaffeoylquinic acid
Gallocatechin
Catechin 3-O-gallate
Epicatechin 3-O-gallate
Epigallocatechin 3-O-methylgallate
Epigallocatechin 3-O-gallate
1,2,3,6-Tetra-O-galloyl-h-D-glucose
1,2,3,4,6-Penta-O-galloyl-h-D-glucose

Amount added
A M (Ag/ml)
188.50
377.00
244.86
282.54
226.04
105.88
218.12
126.81
104.41

(100)
(200)
(75)
(125)
(100)
(50)
(100)
(100)
(100)

Inhibition of activity (%) in the

presence or absence of ZnCl2
None

ZnCl2 (1.5 mM)

76.24
83.82
59.14
67.66
91.86
75.18
86.26
50.95
59.56

49.66
58.56
57.04
64.32
93.65
77.16
81.32
52.35
59.36

of the tannins, epigallocatechin-3-O-gallate, epigallocatechin-3-O-methylgallate and 1,2,3,4,6-penta-Ogalloyl-h-D-glucose, were shown to non-specifically inhibit these enzymes (Table 3). The addition of
1.5 mM ZnCl2 to the assay of the tannin-induced ACE inhibitory action showed that the methyl group

Fig. 4. The time-course of hypotensive effect of tannins on SHR after intravenous administration of angiotensin I (200 ng/kg):
A. 1,2,3,6-tetra-O-galloyl-h-D-glucose in DMSO (5: vehicle; E: 10 mg/kg; D: 20 mg/kg; .: 40 mg/kg); B. epigallocatechin
3-O-methylgallate in DMSO (D: vehicle; 5: 10 mg/kg; E: 20 mg/kg; .: 40 mg/kg) and captopril (n: 10 mg/kg).

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containing methyl 3,4-dicaffeoylquinate (377 AM) and methyl 3,5-dicaffeoylquinate (189 AM) reduced
the inhibition of ACE activity by 30.1% (83.82% to 58.56%) and 34.8% (76.2% to 49.7%), respectively.
The inhibition of ACE activity of the other tannins was insignificantly changed by the addition of ZnCl2
(Table 4).
In vivo animal study of tannins
For test the in vivo effects of the tannins, epigallocatechin-3-O-methylgallate and 1,2,3,6-tetra-Ogalloyl-h-D-glucose on the hypertensive action of exogenous angiotensin I were assessed. The results
showed that epigallocatechin-3-O-methylgallate and 1,2,3,6-tetra-O-galloyl-h-D-glucose both had
strong hypotensive effects with 10, 20 and 40 mg/kg dose-dependently decreasing blood pressure
(Fig. 4) by 27.2 F 3.6, 45.5 F 5.3 and 51.0 F 5.1 and by 37.5 F 4.7, 46.3 F 4.7 and 52.5 F 5.6
mm Hg respectively (Fig. 5). A control dose of 10 mg/kg captopril reduced the mean blood pressure of
the SHR rats following angiotensin I infusion by 9.9 F 2.3 mm Hg within 10 min post administration
(Fig. 5).

Fig. 5. The inhibitory effects of epigallocatechin 3-O-methylgallate and 1,2,3,6-tetra-O-galloyl-h-D-glucose on the maximum
systemic blood pressure (B.P.) change within 10 min after the administration of Angiotensin I (200 ng/kg) in SHR.
:
Injection of 10 mg/kg Epigallocatechin 3-O-methylgallate;
: Injection of 20 mg/kg Epigallocatechin 3-O-methylgallate;
:
Injection of 40 mg/kg Epigallocatechin 3-O-methylgallate;
: Injection of 10mg/kg 1, 2, 3, 6-Tetra-O-galloyl-h-D-glucose;
: Injection of 20 mg/kg 1, 2, 3, 6-Tetra-O-galloyl-h-D-glucose;
: Injection of 40 mg/kg 1, 2, 3, 6-Tetra-O-galloyl-h-Dglucose;
: Injection of 10 mg/kg Captopril; *P < 0.05 (vs. Captopril); **P < 0.01 (vs. Captopril).

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Discussion
We examined more than 100 different Chinese herbs, and 18 tannins were isolated. Nine of those
tannins were found to have ACE IC50 less than 200 AM and were divided into the three major tannin
classes caffeoylquinates, flavan-3-ols and gallotannins.
Comparison of the structures of caffeoylquinates extracted from Flonicera japonica Thunb and ACE
inhibitory activity suggested that both two caffeoyl groups (R1, R2, or R1, R3) and methylation at the
R4 position are the prerequisites of stronger ACE inhibition. Other tannins, for example, with one
caffeoyl (R1) and methylation at the R4 position, such as methylchlorogenate, or with two caffeoyl
groups (R1, R2, or R1, R3), but no methylation at the R4 position, such as 3,4 di-O-caffeoylquinate,
present less ACE inhibitor activity.
The ACE inhibitory activity of the three prototype flavan-3-ols, which were extracted from a Chinese
tea, gallocatechin, epicatechin and catechin were quite low, with only the activity of gallocatechin being
sufficiently high to meet the study inclusion criteria of an IC50 < 200 AM. This data suggested that the
R5V hydroxyl group might be important in determining ACE inhibitory activity. The inclusion of a
hydroxyl group at R3 position and gallate in to the flavan-3-ol structure increased the inhibitory activity
of the three flavan-3-ols. In gallotannins, we found more galloryl groups in structure, more ACE
inhibitory activity presented. According to a recent study, the tannin (penta-O-galloyl-D-glucopyranouse)/protein (bradykinin) complexes are formed by multiple weak interactions between peptide side
chains and galloyl rings. Proline and arginine are good anchoring points and the glycine gives a certain
flexibility in the peptide backbone that allows the polyphenol to approach to and interact with protein
[15].
Each of the tannin classes appeared to exhibit a non-competitive mode of inhibition, indicating that
the substrate and inhibitor bind to the ACE simultaneously in a reversible manner. Increasing
angiotensin I to maintain angiotensin II levels would not overcome this mode of inhibition of the ACE.
Tannin-induced ACE inhibition resulting from protein precipitation was only observed with 1,2,3,4,6penta-O-galloyl-h-D-glucose, with which addition of BSA reduced the inhibition by 86%. 1,2,3,4,6Penta-O-galloyl-h-D-glucose was also observed to be a non-specific inhibitor of ACE activity by
similarly reducing the activity of both chymotrypsin and trypsin. Likewise, two flavan-3-ols, epigallocatechin-3-O-gallate and epigallocatechin-3-O-methylgallate, were also found to have reduced the
activity of these enzymes, suggesting they are also non-specific inhibitors of ACE activity.
ACE is a zinc metallopeptidase, in which the zinc ion at the catalytic active site is essential for
enzyme activity. This means that non-specific metal chelaters may have apparent ACE inhibitor action.
Supplementation of the ACE activity test system with ZnCl2 is designed to reduce inhibition resulting
from tannin-induced Zn2 + ion chelation. ZnCl2 decrease the inhibitory activity of both caffeoylquinates
by approximately 30%, showing that the chelation of Zn2 + may at least in part to be responsible for the
ACE inhibitory activity of the caffeoylquinate class of tannins.
Due to the possible confounding effects of anesthesia, we conducted our in vivo studies of the effect
of tannins in conscious SHR. This approach is associated with certain disadvantages such as difficulty in
controlling the animals. Furthermore, external disturbances, surgical wound and even discomfort given
rise by injection may lead to change of blood pressure and interfere with the progress of the experiment.
For blood pressure measurement and drug administration, we selected tail artery and tail vein,
respectively, which limited the damage to the rats reducing possible indirect effects on the blood
pressure of the rats.

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Previous studies had shown that the tannins could lower the basal blood pressure within 10 min [8,9].
However, the complexity of the blood pressure regulatory mechanisms means that the lowering of the
blood pressure by the tannins cannot directly substantiate the inhibitory activity of endogenous ACE.
Therefore exogenous angiotensin I was used to elicit a temporary increase of blood pressure. Then the
blood pressures before and after drug administration were compared in order to demonstrate the
inhibitory action of the tannins on ACE activity. The rapid rise in blood pressure after infusion of
angiotensin I highlighted its rapid metabolism in the SHR. The vasopressor effect of each injection of
angiotensin I dose was consistent, eliminating the possibility of a cumulative effect of angiotensin I.
Epigallocatechin-3-O-methylgallate and 1,2,3,6-tetra-O-galloyl-h-D-glucose both reduced the vasopressor activity of exogenous angiotensin I 10 min after their administration, which was consistent with
the effect of captopril. The tannins had a stronger effect on blood pressure, epigallocatechin-3-Omethylgallate and 1,2,3,6-tetra-O-galloyl-h-D-glucose, reducing blood pressure to a significantly greater
extent than captopril in our study. This finding is consistent with the data reported by Inokuchi et al. [16]
who found the maximum antihypertensive effect of Areca II-5-C, a tannin isolated from seeds of Areca
catechu L., was 5 times as that of captopril at the same dose (15 mg/kg, i.v.). Beside angiotensin I
inhibitory effect, Areca II-5-C also produced dose-related inhibition of the pressor response of
angiotensin II [16]. Therefore, it seemed that the hypotensive effects of these tannins were not only
from ACE inhibition but also from other mechanisms. This may explain why epigallocatechin-3-Omethylgallate and 1,2,3,6-tetra-O-galloyl-h-D-glucose in our in vivo study found to be more effective
than captopril in inhibiting the hypertensive actions of angiotensin I, even the IC50 on ACE was much
more than captopril.
In vitro studies have identified a range of herbal remedies that possess ACE activity from China, India,
Africa and South America [1720]. Screening of these herbs has identified a number of natural ACE
inhibitors, which were not tannin-based [18,19]. Additionally tannin-based inhibitors have also been
identified, as in the current study. The tannins are naturally existed polyphenols and are defined based on
their main basal activity, which is the ability to precipitate water-soluble proteins and alkaloids [20].
In conclusion, a number of the tannins investigated in the current study non-specifically inhibited
ACE activity through sequestration of the enzyme metal cofactor (Zn2 +), protein precipitation or were
shown to be non-specific enzyme inhibitors, of trypsin and chymotrypsin, through other mechanisms.
However, three of the five flavan-3-ols and 1,2,3,6-tetra-O-galloyl-h-D-glucose did not appear to inhibit
ACE activity by any of the methods in this study. This suggests a possible specific inhibition of this
enzyme, although the tannins did exhibit non-competitive inhibition, which implies a mechanism that
did not involve active competition for the ACE active site. Further investigations into the blood pressurelowering efficacy and potential side effects of the tannins are required.

References
[1] Pitt B, Poole-Wilson PA, Segal R, Martinez FA, Dickstein K, Camm AJ, Konstam MA, Riegger G, Klinger GH, Neaton J,
Sharma D, Thiyagarajan B. Effect of losartan compared with captopril on mortality in patients with symptomatic heart
failure: randomized trialthe Losartan Heart Failure Survival Study ELITE II. Lancet 2000;355:1582 7.
[2] Hansson L, Lindholm LH, Niskanen L, Lanke J, Hedner T, Niklason A, Luomanmaki K, Dahlof B, de Faire U, Morlin C,
Karlberg BE, Wester PO, Bjorck JE. Effect of angiotensin-converting-enzyme inhibition compared with conventional
therapy on cardiovascular morbidity and mortality in hypertension: the Captopril Prevention Project (CAPPP) randomized
trial. Lancet 1999;353:611 6.

J.-C. Liu et al. / Life Sciences 73 (2003) 15431555

1555

[3] Maschio G, Alberti D, Locatelli F, Mann JF, Motolese M, Ponticelli C, Ritz E, Janin G, Zucchelli P. Angiotensinconverting enzyme inhibitors and kidney protection: the AIPRI trial. The ACE Inhibition in Progressive Renal Insufficiency (AIPRI) Study Group. J Cardiovasc Pharmacol 1999;33:S16 20.
[4] Swedberg K, Kjekshus J, Snapinn S. Long-term survival in severe heart failure in patients treated with enalapril. Ten year
follow-up of CONSENSUS I. Eur Heart J 1999;20:136 9.
[5] Stewart JM, Ferreira SH, Greene LJ. Bradykinin potentiating peptide Pca-Lys-Trp-Ala-Pro. Biochem Pharmacol
1971;20:1557 67.
[6] Igic R, Erdos EG, Yeh HSJ, Sorrells K, Nakajima T. Angiotensin I converting enzyme of the lung. Circ Res 1972;31:
S51 61.
[7] Kastis JB. Angiotensin converting enzyme inhibitors. Am Heart J 1998;16:1580 605.
[8] Black HR, Ming S, Poll DS, Wen YF, Zhou HY, Zhang ZQ, Chung YK, Wu YS. A comparison of the treatment of
hypertension with Chinese herbal and western medication. J Clin Hyperten 1996;24:371 8.
[9] Mao HY, Tu YS, Nei FD, Liang GF, Feng YB. Rapid antihypertensive effect of rhomotoxin in 105 hypertension cases.
Chinese Med J 1981;94:733 6.
[10] Rojas A, Bah M, Rojas JI, Gutierrez DM. Smooth muscle relaxing activity of Gentiopicroside isolated from Gentiana
spaathacea. Planta Med 2000;66:765 7.
[11] Cushman DW, Cheung HS. Spectrophotometric assay and properties of the angiotensin converting enzyme of rabbit lung.
Biochem Pharmacol 1970;20:1637 48.
[12] Shuichi S, Yutaka H, Uichi Y. Spectrophotometric determination of glycine with 2,4,6-Trichloro-s-Triazine. Anal Chem
1970;42:101 3.
[13] Makoto H, Yishikazu K, Hiroshi I. A rapid and simple spectrophotometric assay of angiotensin converting enzyme. Anal
Biochem 1978;84:361 9.
[14] Fontes R, Ribeiro M, Sillero A. Inhibition and activation of enzymesThe effect of a modifier on the research rate and on
kinetic parameters. Acta Biochimica Polonica 2000;47:233 57.
[15] Verge S, Richard T, Moreau S, Nurich A, Merillon JM, Vercauteren J, Monti JP. First observation of solution structures of
bradykinin-penta-O-galloyl-D-glucopyranose complexes as determined by NMR and simulated annealing. Biochimica et
Biophysica Acta 2002;1571:89 101.
[16] Inokuchi JI, Okabe H, Yamauchi T, Nagamatsu A, Nonaka GI, Nishioka I. Antihypertensive substance in seeds of Area
catechu L. Life Sci 1986;38:1375 82.
[17] Somanadhan B, Varughese G, Palpu P, Sreedharan R, Gudiksen L, Smitt UW, Nyman U. An ethnopharmacological survey
for potential angiotensin converting enzyme inhibitors from Indian medicinal plants. J Ethnopharmacol 1999;65:103 12.
[18] Duncan AC, Jager AK, van Staden J. Screening of Zulu medicinal plants for angiotensin converting enzyme (ACE)
inhibitors. J Ethnopharmacol 1999;68:63 70.
[19] Nyman U, Joshi P, Marsden LB, Pederson TB, Pinstrup M, Rajasekhararan S, George V, Pushpangdan P. Ethnomedical
information and in vitro screening for angiotensin-converting enzyme inhibition of plants utilized as traditional medicines
in Gujarat, Rajastan and Kerala (India). J Ethnopharmacol 1998;60:257 63.
[20] Hansen K, Nyman U, Smith UW, Adsersen A, Gudiksen L, Rajasekhararan S, Pushpangda P. In vitro screening of
traditional medicines for anti-hypertensive effect based on inhibition of the angiotensin-converting enzyme (ACE).
J Ethnopharmacol 1995;48:43 51.