Search for potential Angiotensin Converting Enzyme

(ACE)-inhibitors from plants

M.A. Lacaille-Dubois
1
, U. Franck
2
and H. Wagner
2
1
Laboratoire de Pharmacognosie, Unit de Molcules dIntrt Biologique, Facult de Pharmacie, Universit de Bourgogne,
Dijon, France
2
Centre of Pharma Research, Pharmaceutical Biology, University of Munich, Munich, Germany
Summary
MeOH extracts, fractions and pure substances from Musanga cecropioides, Cecropia species and
Crataegus oxyacantha /C. monogyna were screened by using an in vitro bio-assay based on the inhi-
bition of Angiotensin Converting Enzyme (ACE), as measured from the enzymatic cleavage of the
chromophore-fluorophore-labelled substrate dansyltriglycine into dansylglycine and diglycine.
Phenolic acids showed no significant ACE-inhibition whereas flavonoids and proanthocyanidins
demonstrated inhibitory activity at 0.33 mg/ml using this test system.
Key words: Musanga cecropioides, Cecropia species and Crataegus oxyacantha/ C. monogyna, angio-
tensin converting enzyme, ACE, hypotensive agents, flavonoids, procyanidins
0944-7113/01/08/01-047 $ 15.00/0
Introduction
Inhibition of Angiotensin I-Converting Enzyme
(ACE) is currently considered to be a useful therapeu-
tic approach in the treatment of high blood pressure.
Within the enzyme cascade of the renin-angiotensin
system, ACE removes histidyl-leucine from an-
giotensin I to form the physiologically active octapep-
tide angiotensin II, one of the most potent vasocon-
strictors known. Angiotensin II also stimulates the
synthesis and release of aldosterone from the adrenal
cortex, which increases blood pressure by promoting
sodium retention (and thereby water retention) in the
distal tubules. Inhibition of ACE will be an effective
screening method in the search for new antihyperten-
sive agents (Wagner, 1993, Hansen et al., 1996a,
1996b, Somanadhan et al., 1999). By using an in vitro
ACE-inhibition assay (Elbl and Wagner, 1991; Wagner
and Elbl, 1992) for a bioguided fractionation of drugs
with known antihypertensive potential, we isolated
some procyanidins and flavonoids from Lespedezae
capitata, Cistus clusii, and Amelanchier ovalis, with
ACE-inhibiting activities. Test compond concentra-
tions from 0.14 to 0.33 mg/ml showed between 37 to
66% inhibition (Wagner et al., 1991).
In continuation of our systematic search for antihy-
pertensive principles in higher plants, we have
screened various plant extracts and isolated com-
pounds for ACE-inhibitory activity, using a purified
enzyme from rabbit lung and dansyltriglycine as sub-
strate (Elbl and Wagner, 1991; Hansen et al., 1995).
Plant species used as diuretic, cardiotonic and antihy-
pertensive remedies in the folk medicine in Central
and South America (Cecropia species, Cecropi-
aceae/Moraceae) and Africa (Musanga cecropioides,
Moraceae) were selected (Franck, 1998). A Crataegus
extract was also chosen in this screening since it was
shown to be active in cardiac insufficiency patients in
comparison with captopril (as a reference ACE-in-
hibitor) (Tauchert et al., 1994). Furthermore ACE-in-
hibitory fractions were found in Crataegus fruits
(Inokuchi et al., 1984).
Phytomedicine, Vol. 8(1), pp. 4752
Urban & Fischer Verlag 2001
http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Material and Methods
Plant Material
Leaves of Musanga cecropioides R. Brown were col-
lected in the North West and Central Province of
Cameroon. They were provided by Dr. Kamanyi (Uni-
versity of Yaounde, Cameroon) and identified by Dr.
G. Achundong of the National Herbarium at Obili
Yaounde. Voucher specimens of the material are de-
posited in the National Herbarium of Yaounde. Leaves
of Cecropia hololeuca Miq., C. pachystachya Trc., C.
glaziovii Sneth. were collected close to Belo Horizonte
(Brazil) and were provided by Pr. A. Bragha de
Oliveira and Dr. C.L. Zani, Laboratory of Chemistry
of Natural Products, Centre Ren Rachou Fiocruz,
Belo Horizonte (Brazil). Voucher specimens were de-
posited in the Herbarium of this Institute. The MeOH
extract was prepared from the flowers and leaves of
Crataegus oxyacantha/ monogyna and provided by the
company Lichtwer (Berlin) under the batch number
461457.
Reference Compounds
Isovitexin, orientin (Extrasynthese, Genay, France),
(+)catechin, chlorogenic acid, ()-epicatechin (Roth,
Karlsruhe), Isoorientin, protocatechic acid, isoquer-
citrin (Institute of Pharmaceutical Biology, Munich).
Instruments and General Methods
TLC was performed on silica gel 60 F 254 (Merck).
1
H- and
13
C NMR-spectra were recorded on an AM
360 and a DRX 500 (Bruker) instrument at 24 C and
on a GSX 400 Jeol instrument. Mass spectra were ob-
tained using a MS 80 RFA (Kratos) and a HSQ 30,
Finnigan MAT instruments.
HPLC analytical scale: HP 1090 Aliquid chromato-
graph with photodiode array detection (Hewlett
Packard, Walbronn). Column: Hibar column, 125 4
mm ID, LiChrospher 100 RP-18 (5 m) with RP-18
precolumn (Merck). Eluent for the separation of the
MeOH extracts of M. cecropioides, C. pachystachya
and Crataegus-extract: Linear gradient from 10 to
30% MeCN-H
2
O with 1% 0.1 N H
3
PO
4
in 030 min,
flow rate: 1 ml/ min; detection wavelength 210 nm.
Eluent for the separation of the MeOH extracts of C.
hololeuca and C. glaziovii: 5 to 20% MeCN-H
2
O with
1% 0.1 N H
3
PO
4
in 020 min, 20 to 40% MeCN-H
2
O
with 1% 0.1 N H
3
PO
4
in 2035 min, 40 to 95% MeCN-
H
2
O with 1% 0.1 N H
3
PO
4
in 3537 min, 95% MeCN-
H
2
O with 1% 0.1 N H
3
PO
4
isocratic in 3540 min,
flow rate: 1 ml/ min; detection wavelength: 210 nm.
HPLC semi preparative scale : L-6200 AIntelligent
Pump Merck-Hitachi (Merck) with a Schoeffel SF 770
Spectrophotometer (Schoeffel) and a Perkin Elmer
561 recorder (Perkin Elmer). Hibar RT column,
250 4 mm ID, LiChrosorb RP-18 with a RP-18 pre-
column (Merck) and Hibar LiChrosorb RP-18 (7 m),
250 10 mm ID, with a RP-18 precolumn (Merck).
Eluent for the isolation of the proanthocyanidin C
1
:
linear gradient from 10 to 50% MeCN-H
2
O in
020 min, 50% MeCN isocratic in 2027 min; detec-
tion wavelength: 200 nm.
Extraction and Fractionation
Musanga cecropioides R. Brown: Powdered leaves
(215 g) were defatted by n-hexane (2l) in a soxhlet and
then extracted with MeOH (2l). After elimination of
the solvent under reduced pressure (T < 40 C), the
residue was suspended in water and heated at 60 C for
60 min. The chlorophyll was removed by filtration and
extraction with n-hexane. The water phase was ex-
tracted with EtOAc (6 200 ml). After removing
the solvent in vacuo (T < 40 C), the EtOAc fraction
(3.5 g) was fractionated over Sephadex LH 20 using
EtOAc/CH
3
CH
2
OH/H
2
O (2:1:2) as an eluent. Seven-
teen fractions were obtained and analysed by HPLC/
UV. By comparison with reference compounds in
terms of retention time and UV spectra (recorded on
line by the photodiode array detection), the presence of
protocatechic acid, chlorogenic acid, catechin, epicate-
chin, isovitexin, orientin and isoorientin was indicated.
Procyanidin B
2
(31 mg) and procyanidin C
1
(30 mg)
were isolated as pure compounds by successive chro-
matography on Sep-Pak and preparative HPLC (elu-
ent: gradient of MeCN-H
2
O) and were identified
by extensive NMR spectral data as (-)-epicatechin-
(48)-()-epicatechin and ()-epicatechin-(48)-
()-epicatechin (48)-()-epicatechin (Franck,
1998, Frank et al., 1999).
Cecropia hololeuca Miq: 6.3 g residue obtained from
concentrating an ethanolic extract were suspended in
water and heated at 60 C for 60 min. The chlorophyll
was eliminated by filtration and further by liquid-liq-
uid extraction with hexane. Liquid-liquid extraction
with CHCl
3
-MeCN-hexane-water (1:3.4:2:1) and fur-
ther partitions with EtOAc (5 100 ml) yielded the
EtOAc fraction. After evaporation of the solvent under
reduced pressure, the residue (139.5 mg) was fraction-
ated over Sephadex LH 20 using EtOAc/CH
3
CH
2
OH/
H
2
O (2:1:2) as an eluent. Five fractions which were
obtained and analysed by HPLC/UV. The constituents
were identified by comparison with authentic samples
in term of retention times and UVspectra as protocate-
chic acid and chlorogenic acid in Fr. 1, epicatechin in
Fr. 2, orientin and isoorientin in Fr. 3. Procyanidin B
2
in Fr. 4 and procyanidin C
1
in Fr. 5 were characterized
by co-TLC with authentic samples.
Cecropia pachystachya Trc: 250 g of the powdered
leaves were extracted and fractionated according to the
procedure described for Musanga cecropioides yield-
48 M. A. Lacaille-Dubois, U. Franck and H. Wagner
(CH
3
)
2
CO/H
2
O (70:30) afforded a mixture of oligo-
meric procyanidins (62 mg). The polymeric com-
pounds were finally eluted by DMF. The high poly-
mers were retained on the column.
ACE-Inhibition Test
The in vitro ACE-inhibitory activity was measured ac-
cording to the method described by Elbl and Wagner
(1991), which was later modified by Hansen et al.
(1995). The chromophore- and fluorophore-labelled
tripeptide dansyltriglycine is used as substrate, which
is cleaved by the enzyme into dansylglycine and the
peptide fragment diglycine. In presence of a specific
ing 6.0 g of an EtOAc fraction. This extract, after pre-
cipitation and recristallisation of isoorientin (3.6 mg)
was fractionated over Sephadex LH 20 using EtOAc-
EtOH-H
2
O (2:1:2) as an eluent. Eight fractions were
obtained and analysed by HPLC/UV. The constituents
were identified by comparison with authentic samples
in term of retention times and UVspectra as protocate-
chic acid, chlorogenic acid in Fr. 2, catechin, epicate-
chin, isovitexin and isoquercitrin in Fr. 3. Furthermore,
isoorientin was characterized by FABMS and
1
H NMR
spectroscopy.
Cecropia glaziovii Sneth: 14.7 g powdered stipules
were extracted in a soxhlet by MeOH (100 ml). After
removing the solvent under
reduced pressure, the MeOH
extract (1.1 g) was fractionat-
ed over Sephadex LH 20
using EtOAc/CH
3
CH
2
OH/
H
2
O (2:1:2) as an eluent. Six
fractions were obtained and
analysed by HPLC/UV. In
the Fr. 2 catechin, epicate-
chin, and isoquercitrin were
characterized by comparison
with authentic samples in
term of retention times and
UV spectra. The presence of
isoorientin, epicatechin and
the procyanidin B
2
was indi-
cated in Fr.3. Procyanidin C
1
was the main constituent of
the Fr. 4.
A methanolic extract was
obtained from the leaves
(12.7 g) and analysed by co-
TLC with authentic samples
and by HPLC/UV. The pres-
ence of chlorogenic acid, cat-
echin, epicatechin, isoorien-
tin, procyanidin B
2
and pro-
cyanidin C
1
was indicated.
Crataegus-extract: An
EtOAc fraction (9.8 g) was
obtained from a MeOH ex-
tract (51.5 g) of Crataegus
oxyacanta/ monogyna from
which a portion (0.85 g)
was fractionated over poly-
amide. Elution with 99%
CH
3
CH
2
OH yielded flavo-
noids. Then, a mixture of
CH
3
CH
2
OH/(CH
3
)
2
CO/H
2
O
(80:14:6) eluted the last
flavonoids and dimers of
procyanidins. A solution of
Search for potential Angiotensin Converting Enzyme-inhibitors 49
inhibitor, the product formation is partially or totally
inhibited.
For these studies, a commercially available angio-
tensin-converting-enzyme preparation from rabbit
lung (EC 3.4.15.1-purchased from Sigma) has been
used. The ACE solution (25 l) was pre-incubated into
microtitre plates with a test or control solution (25 l)
for 5 min at 37 C. The enzyme reaction was started by
adding a combined solution (25l) of the substrate
dansyltriglycin, and the internal standard, dansyl-L-
glutamine. At the end of the incubation time (3035
min at 37 C), the reaction was stopped by addition of
0.1 N Na
2
EDTA(50 l).
The product (dansylglycine) and unreacted substrate
are separated and quantified by reversed phase HPLC
with UV detection at 250 nm. Instrumentation: Merck
Hitachi L 6200 Intelligent Pump; Merck Hitachi AS-
2000 Autosampler; Lambdamax 481 LC spectrometer
(Waters-Millipore); Merck Hitachi D-2500 A integra-
tor; solvent system: Column 120 5.8 mm ID packed
with Spherisorb S5 ODS; mobile phase: 10 mM
Na
2
PO
4
buffer (pH 7)-acetonitrile (88:12), isocratic;
flow rate: 2 ml/min; detection wavelength: 250 nm.
Using this isocratic elution system, the total separation
is achieved within 7 min. The decreased concentration
of dansylglycine in the test reaction compared with the
control reaction was expressed as percentage inhibi-
tion and calculated from the equation:
{dansylglycine} T
Inhibition (%) = 100 100
{dansylglycine} C
where T = test reaction and C = control reaction.
Determination of the Proanthocyanidin Content
According to the procedure of Hiermann et al. (1986),
the powdered drug (b = 1g) was stirred at room tem-
perature with acetone-H
2
O (7:3) for 15 min (4 15
ml). After filtration the mixture was diluted to 100 ml
with 70% acetone. A portion of this solution (10 ml)
was evaporated to dryness at room temperature and n-
BuOH-HCl 37% (95:5) was added. The mixture was
heated under reflux for 2 hr. After cooling, the mixture
was diluted to 100 ml and the extinction E was spec-
trophotometrically measured at 540 nm. The content
of total procyanidins (TP%) was estimated as (TP%) =
E x 4.12 / b.
Results and Discussion
The screening results of several plant methanolic ex-
tracts can be summarized as follows (see Fig. 1): A
complete inhibition of the enzyme activity (100%) was
obtained with the methanolic extract of Musanga ce-
cropioides leaves and a very good ACE-inhibition of
91 9% (s.d.) was obtained with the methanolic ex-
tract of Cecropia glaziovii stipules at a final concentra-
tion of 0.33 mg/ml. The other extracts of Crataegus
aerial parts, Cecropia hololeuca leaves, C. hololeuca
bark, C. pachystachyae leaves showed a moderate
activity between 33 3% (C. hololeuca bark) and
42 2% (C. pachystachyae leaves) at a concentration
of 0.33 mg/ml. All these four extracts showed a con-
tent in procyanidins of about 11%, whereas a higher
content of 13% was determined in Musanga cecropi-
oides leaves. Since this extract was the most active, it
seems as if the content in procyanidins could be partly
responsible for the ACE-inhibitory activity.
The ethanolic extract of C. hololeuca leaves showed
an ACE-inhibition of 40 4% at a concentration of
0.33 mg/ml. Through a bioguided fractionation it was
possible to isolate and identify the flavone-C-glyco-
sides orientin and isoorientin, (+)-catechin, ()-epicat-
echin and two (-)-epicatechin derived oligomeric pro-
cyanidins, chlorogenic acid and other phenolic acids.
Of the substances tested, isoorientin and orientin
showed an inhibition of 48 1% and 20 2% at a con-
centration of 0.33 mg/ml, respectively. The most effec-
tive fraction contained mostly procyanidins with an in-
hibition of 94 4% at a concentration of 0.33 mg/ml.
The similar composition of Cecropia hololeuca and
Crataegus monogyna/ C. oxyacantha suggested a sim-
ilar ACE-inhibitory activity of both extracts. The
Crataegus extract inhibited the enzyme by 33 2% at
a concentration of 0.33 mg/ml and was a little less ac-
tive than the Cecropia hololeuca leaves extract (40%).
The partition of the MeOH extract yielded an EtOAc
extract which was separated by CC on polyamide into
3 fractions of increasing ACE-inhibitory activity: Phe-
nolic acids and flavonoids (327% inhibition, flavane-
3-ols (1530% inhibition), oligomeric procyanidins
with low polymerisation degree (92% inhibition).
The methanolic extract of the leaves of Musanga ce-
cropioides showed an ACE-inhibition of 100% at
0.33 mg/ ml. A bioguided fractionation led to 15 frac-
tions which showed an ACE-inhibitory activity of 22
to 57%. The most effective fraction giving an inhibi-
tion of 57 1% contained chlorogenic acid, flavane-3-
ols (+)-catechin and ()-epicatechin, the flavone isovi-
texin and the flavonol isoquercitrin. The second most
effective fraction giving an inhibition of 53 5% con-
tained the procyanidins B
2
and C
1
and other oligomeric
procyanidins with low polymerisation degree. The re-
sults of the bio-assay on the isolated compounds such
as chlorogenic acid showed a low activity, whereas
flavonoids and procyanidins showed inhibitory activi-
ties dependent on the type of structure (Table 1).
Among the flavonoids, the C-glycosides were more
active than the O-glycosides. Among the C-glyco-
50 M. A. Lacaille-Dubois, U. Franck and H. Wagner
sides, isovitexin and isoorientin showed about the
same activity (46 1% and 48 1%, respectively) and
were more active than their 8C-analogs, vitexin and
orientin (21 1% and 20 2%, respectively).
According to the results of Elbl and Wagner, 1991,
the in vitro activity of flavonoids and procyanidins is
due to the generation of chelate complexes with the
zinc atom within the active centre of the angiotensin
I-converting enzyme. This chelate could be done be-
tween an heterocyclic oxygen and a phenolic hydroxyl
group in its vicinity. The above results showed that the
8-C glycosylation is not favourable for the formation
of a complex with the Zinc atom of the active centre of
the enzyme.
Among the flavan-3-ols, ()-epicatechin was with
an inhibition of 34 1% twice more active than its iso-
mer (+)-catechin (16 3%). Among the proantho-
cyanidins, the inhibition mechanism could also be due
to the complexation of the zinc atom in ACE. This was
demonstrated by Wagner et al., 1991 with an Alchemy
Molecular Modelling programm, which was used to
calculate three-dimensional models of the energy-min-
imised structures of two dimeric C4/C6 and C4/C8
linked procyanidins. The results showed that the two
compounds contain a putative zinc-chelating hetero-
cyclic oxygen atom in the upper part of the
molecule, which is positioned in a way favouring in-
teraction with a phenolic hydroxyl group in the
lower part of the molecule. From literature, it was
assumed that the activity of the oligomeric compounds
increased with the polymerisation degree. Our results
were in good agreement with this conclusion. The
trimeric procyanidin C
1
was more active than the
Search for potential Angiotensin Converting Enzyme-inhibitors 51
Fig. 1. ACE-Inhibition of tested methanolic extracts.
Table 1. ACE inhibitory activity of isolated compounds
(tested concentration: 0.33 mg/ml)
Compound Inhibition,% s.d.
Chlorogenic acid 4 1
Isoquercitrin 32 2
Vitexin 21 1
Isovitexin 46 1
Orientin 20 2
Isoorientin 48 1
(+)-Catechin 16 3
()-Epicatechin 34 1
Procyanidin B
2
25 5
Procyanidin C
1
45 2
The values represents means of two different experiments
under standard assay conditions described in the text.
dimeric procyanidin B
2
(45 2% and 25 5% inhibi-
tion, respectively).
A synergistic effect of flavones and procyanidins in
the Musanga extract such as in Cecropiae species and
Crataegus could be suggested, because none of the
isolated compounds of the effective fractions reached
the activity of the total fractions.
ACE-inhibiting activity was previously reported for
isolated compounds such as vitexin, isovitexin and
(+)-catechin (Arisawa et al., 1989), isoquercitrin
(Kameda et al., 1987) and procanidin B2 (Wagner et
al., 1991). Orientin, isoorientin, (-)- epicatechin, and
procyanidin C1 which are reported here for the first
time with an ACE-inhibitory activity, need further in-
vestigations in order to establish dose-response rela-
tionship.
Since a tetrameric proanthocyanidin glycoside (rats,
3 mg/kg, i.v.) (Terencio et al., 1991) showed in vivo the
same ACE-inhibitory activity as captopril (0.02 mg/
kg, i.v.), further in vivo tests will have to be carried out
in order to demonstrate possible therapeutic relevance
of drugs containing this class of compounds.
Acknowledgments
The authors are most grateful to Dr. Kamanyi and Dr. G.
Achundong, (University of Yaounde, Cameroon) for supply-
ing and identifying Musanga cecropioides from Africa. We
are indebted to Pr. A. Bragha de Oliveira and Dr. C.L. Zani,
University of Belo Horizonte, for supplying Cecropia
species and extracts from Brazil. We also thank the company
Lichtwer (Berlin) for supplying the Crataegus extract.
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Address
M.A. Lacaille-Dubois, Laboratoire de Pharmacog-
nosie, Unit de Molcules dIntrt Biologique, Fac-
ult de Pharmacie, Universit de Bourgogne, BP
87900, 21079 Dijon Cedex, France
Tel.: 0033-3-80 39 32 29; Fax: 0033-3-80 39 33 00;
E-mail: malacd@u-bourgogne.fr
52 M. A. Lacaille-Dubois, U. Franck and H. Wagner