Beruflich Dokumente
Kultur Dokumente
Phytother. Res
.
21
, 3236 (2007)
DOI: 10.1002/ptr
32
M. R. LOIZZO
ET AL.
PHYTOTHERAPY RESEARCH
Phytother. Res.
21
, 3236 (2007)
Published online 27 October 2006 in Wiley InterScience
(www.interscience.wiley.com)
DOI
: 10.1002/ptr.2008
excelsa
(Roxb) (Simaroubaceae)
Monica Rosa Loizzo
1
*, Ataa Said
2
, Rosa Tundis
1
, Khaled Rashed
2
, Giancarlo Antonio
Statti
1
, Antje Hufner
3
Institute for Pharmaceutical Chemistry and Pharmaceutical Technology, University of Graz, Schubertstr.1, A8010
Graz, Austria
arabinopyranoside, kaempferol-3O
-galactopyranoside, quercetin-3O
-glucopyranoside. The
in vitro
hypotensive activities of the MeOH extract and the isolated compounds
were elucidated. All the flavonoids tested exhibited ACE inhibitory activity, in particular the
most active
compound was kaempferol-3O
-galactopyranoside with an IC
50
value of 260
Keywords:
Ailanthus excelsa
(Roxb); flavonoids; angiotensin converting enzyme (ACE); hypertension.
Received 29 May 2006
Revised 3 July 2006
Accepted 13 August 2006
* Correspondence to: Dr M. R. Loizzo, Department of Pharmaceutical
Sciences, University of Calabria, 87036 Rende (CS), Italy.
E-mail: mr.loizzo@unical.it
function (Miller
et al.
, 1994). However, the side effects
such as cough and angioneurotic edema associated
with clinically used ACE inhibitors have been reported
(Israili and Hall, 1992). Thus, the screening and development of new ACE inhibitors would be beneficial
in the treatment of cardiovascular disease without side
effects.
, 2005),
congestive heart failure (Carson, 2000) and renal dysINHIBITION OF ACE BY
AILANTHUS EXCELSA
33
H- and
13
6 and
J
values are given in Hz.
Chemicals.
Chemicals and reagents, used for the study
of antihypertensive activity, were purchased from SigmaAldrich Co. (Milan, Italy) while other chemicals,
solvents and reagents used in chromatography were
purchased from Merck, Egypt.
Plant material.
A. excelsa
(Roxb) leaves were collected
from the Zoo Garden, Giza, Egypt. The plant material
was identified by Dr Kamal El-Batanony, Professor of
Taxonomy and Botany, Faculty of Science, Cairo University. A voucher specimen was deposited in the NRC
herbarium.
Extraction and isolation.
Powdered, air dried leaves of
A. excelsa
(1 kg) were extracted with methanol (70%)
in a Soxhlet apparatus and evaporated to dryness to
give 106.5 g. The residue was dissolved in 1 L of distilled water and then extracted with petroleum ether,
chloroform and ethyl acetate, respectively. Each fraction was dried over anhydrous sodium sulphate and
concentrated under reduced pressure to give 29 g of
petroleum ether, 14 g of chloroform and 10.5 g of ethyl
acetate fractions, respectively. The bioactive ethyl acetate fraction was subjected to column chromatography using silica gel (E. Merck, type 60230 mesh, 300 g)
as adsorbent and elution was carried out with chloroform followed by gradually increasing polarity with
methanol. Elution with CHCl
3
1
,
15 mg), luteolin (
2
, 25 mg),
kaempferol-3O
-
-arabinopyranoside (
3
, 85 mg) and
kaempferol-3O
-
-galactopyranoside (
4
, 35 mg). Pure
compounds quercetin-3O
-
-arabinopyranoside (
5
,
85 mg) and luteolin-7O
-
-glucopyranoside (
6
, 85 mg)
were obtained by column chromatography fractionation
of E and F, respectively, on microcrystalline cellulose
(with water as eluent with gradually increasing amounts
of methanol) and Sephadex LH-20 using water for
elution.
Apigenin (1).
1
12.93
(1H, s, 5-OH), 7.90 (2H, d,
J
=
9.0 Hz, H-2
and H-6
),
6.92 (2H, d,
J
=
9.0 Hz, H-3
and H-5
180.7 (C-4),
164.2 (C-7), 163.2 (C-2), 162.0 (C-4
and C-6
), 120.9 (C-1
), 116.0 (C-3
and C-5
), 7.39 (1H, d,
J
=
2.2 Hz, H-2
), 6.88 (1H, d,
J
=
7.6 Hz, H-5
C
NMR (DMSO-d6, 100 MHz): 181.7 (C-4), 164.2 (C-7),
164.0 (C-2), 161.6 (C-5), 157.4 (C-9), 149.8 (C-4
), 145.9
(C-3
), 121.6 (C-1
), 119.1 (C-6
), 116.2 (C-5
), 113.5
(C-2
-arabinopyranoside (3).
1
H-NMR
(400 MHz, DMSO-d6)
and H-6
), 6.88 (2H, d,
J
=
8.4 Hz, H3
and H-5
), 6.42 (d,
J
=
2.0 Hz, H-8), 6.19 (d,
J
=
2.0 Hz, H-6), 5.33 (d,
J
=
5.2 Hz, H1
), 3.65 (1H, m, H4
=
2.0, 11.6 Hz, H-5
).
13
C NMR
(DMSO-d6, 100 MHz)
,
C6
), 120.8 (C1
), 115.3 (C3
, C5
), 71.7 (C3
), 70.9
(C2
), 66.1 (C4
).
Kaempferol-3O
-
-galactopyranoside (4).
1
H-NMR
(400 MHz, DMSO-d6)
and H-6
), 6.88 (2H, d,
J
=
8.9 Hz, H3
and H-5
), 6.44 (d,
J
=
2.1 Hz, H-8), 6.21 (d,
J
=
2.1 Hz, H-6), 5.34 (d,
J
=
7.8 Hz, H1
), 3.65 (1H, s, H4
), 3.54 (1H, t,
J
=
8.9 Hz, H-2
), 3.33
(1H, m, H-5
), 3.29 (1H, m, H-6
).
13
, C6
), 121.1
(C1
), 115.2 (C3
, C5
), 99.3
(C6), 93.8 (C8), 75.6 (C5
), 73.7 (C3
), 71.2 (C2
), 67.8
(C4
), 60.5 (C6
).
Quercetin-3O
-
-arabinopyranoside (5).
1
H NMR
(400 MHz, DMSO-d6)
), 7.50 (1H, d,
J
=
2.3 Hz, H2
),
6.83 (1H, d,
J
=
8.6 Hz, H5
), 6.38 (1H, d,
J
=
2.3 Hz,
H-8), 6.18 (1H, d,
J
=
2.3 Hz, H-6), 5.26 (1H, d,
J
=
5.3 Hz, H-1
), 3.63
(1H, m, H-4
), 3.60 (1H, d,
J
=
5.9, 11.3 Hz, H-5
), 3.51
(1H, dd,
J
=
2.8, 6.8 Hz, H-3
), 3.21 (2H, d,
J
=
11.3 Hz,
H-5
).
13
177.65 (C4),
164.54 (C7), 161.37 (C5), 156.3 (C2), 156.39 (C9), 148.79
(C4
), 145.16 (C3
), 121.04
(C1
), 115.93 (C2
), 115.54 (C5
), 70.88 (C2
),
66.20 (C4
), 64.39 (C5
).
Luteolin-7O
-
-glucopyranoside (6).
1
H NMR
(400 MHz, DMSO-d6):
34
M. R. LOIZZO
ET AL.
dd,
J
=
2.2, 8.0 Hz, H-6
), 7.41 (1H, d,
J
=
7.8 Hz, H-2
),
6.88 (1H, d,
J
=
7.6 Hz, H-5
), 3.7 (1H, d,
J
=
11.7, H-6
), 3.47 (1H,
m, H-6
), 3.25
(1H, m, H-2
), 3.17 (1H, t,
J
=
7.3, H-4
).
13
C NMR
(DMSO-d6, 100 MHz): 181.9 (C-4), 163.8 (C-7), 164.7
(C-2), 161.3 (C-5), 157.1 (C-9), 150.6 (C-4
), 146.1 (C3
), 121.1 (C-1
), 119.3 (C-6
), 116.2 (C-5
), 113.6 (C-2
),
105.5 (C-10), 103.1 (C-3), 99.6 (C-6), 94.8 (C-8), 100.1
(C-1
), 77.3 (C-5
), 76.56 (C-3
), 73.28 (C-2
), 69.73 (C4
), 60.79 (C-6
).
Bioassay procedures for ACE inhibition.
The
in vitro
ACE inhibitory activity was measured using the
method described by Elbl and Wagner (1991), which
was later modified by Hansen
et al.
(1995). Briefly, the
chromophore-fluorophore labelled substrate dansyltriglycine was cleaved by an angiotensin I-converting
enzyme preparation from rabbit lung (EC 3.4.15.1)
into dansylglycine, which is quantitatively measured by
HPLC. 1 mg of MeOH extract and isolated compounds
was dissolved in 1 mL HEPES assay buffer, to obtain a
final concentration of 330
g/mL.
The ACE solution
(25
L) of
the substrate dansyltriglycine (7.86 m
M
-glutamine (0.353 m
M
) for a time
of incubation chosen by plotting a calibration curve.
The reaction was stopped by adding a solution of 0.1
N
Na
2
EDTA (50
L loop; detector:
Perkin Elmer UV/VIS LC290 spectophotometric;
solvent system: Altech SN 1250-99, Part. N 288215 BIN
II 43; chromatographic column type: Hypersil ODS 5 u
Lot N 5002.150 mm
4.6 m
M
isocratic system 10 m
M
NaH
2
PO
4
SD.
Differences were evaluated by one-way analysis of
variance (ANOVA) test completed by a multicomparison Dunnetts test. Differences were considered
significant at
p
<
0.01.
The inhibitory concentration 50%
(IC
50
) was calculated from a dose-response curve obtained by plotting the percentage inhibition versus the
concentrations with the use of GraphPad Prism 4.0
Software.
RESULTS AND DISCUSSION
As a part of our search for a therapeutic approach
for the treatment of high blood pressure, a MeOH
extract of
A. excelsa
leaves was screened for its inhibitory effect on ACE with 53.78% inhibition at the
screened concentration of 330
g/mL.
The bioactive ethyl acetate fraction of
A. excelsa
was
chromatographed on silica gel followed by successive
separation on Sephadex LH-20 and cellulose affording
six pure known flavonoids identified as apigenin, luteolin,
kaempferol-3O
-
-arabinopyranoside, kaempferol-3O
-
-galactopyranoside, quercetin-3O
-
-arabinopyranoside
and luteolin-7O
-
HNMR,
13
C-NMR and 2D COSY, HSQC, HMBC), identical with those previously described (Nakasugi and
Komai, 1998; Sanbongi
et al.
, 1998; Yun-Lian
et al.
, 2000;
Foo
et al.
, 2000; Flamini
et al.
, 2001). Several flavonoids
g/mL (Lacaille-Dubois
et al
., 2001; Kameda
et al.
, 1987; Wagner, 1993).
Isolated compounds inhibited the ACE activity in a
dose-dependent manner (Fig. 1), and the IC
50
values
are reported in Table 1. The most active compound
was kaempferol-3O
-
-galactopyranoside with an IC
50
of 260
while kaempferol-3O
-
-arabinopyranoside
showed an inhibition of 320
.
The arabinosyl moiety seemed to have a negative
influence on the ACE inhibition, in fact quercetin-3O
-
310
).
Luteolin exhibited an IC
50
of 290
, the introduction
of glucopyranoside moiety in position 7 weakly reduced
the flavonoid activity (IC
50
280
) while apigenin
showed an IC
50
value of 280
on ACE activity.
Our data are in accordance with previous work of Oh
et al.
(2004) that reported the ACE inhibitory activity
of five flavonoids with IC
50
.
Polyphenolic substances such as flavonoids exert a
great variety of physiological actions and are held partly
responsible for the favourable health properties of
some food, including a reduced risk of cardiovascular
diseases. An increasing number of epidemiological
and experimental studies have established a positive
correlation between the consumption of foods and
supplements rich in flavonoids or phytomedicine preparation, and protection against atherosclerosis and
cardiovascular diseases. In addition, flavonoids inhibit
Table 1. Angiotensin converting enzyme inhibitory activity of
flavonoids from
A. excelsa
(Roxb) leaves
Compound IC
50
)
Apigenin 280
3.2
a
Luteolin 290
2.9
a
Kaempferol-3O
-arabinopyranoside 320
4.1
Kaempferol-3O
-galactopyranoside 260
3.0
a
Quercetin-3O
-arabinopyranoside 310
2.2
a
Luteolin-7O
-glucopyranoside 280
3.4
a
Captopril 0.02
0.0004
IC
50
SEM (
n
=
3);
a
p
<
0.01 vs control.
INHIBITION OF ACE BY
AILANTHUS EXCELSA
35
Figure 1.
Dose-dependent inhibition of ACE by isolated flavonoids from
A. excelsa
.
(
1)
apigenin, (
2
) luteolin, (
3
) kaempferol-3O
arabinopyranoside, (
4
) kaempferol-3O
-galactopyranoside, (
5
) quercetin-3O
-arabinopyranoside, (
6
) luteolin-7O
-glucopyranoside.
Each data point represents the mean
SD (
n
=
3).
6
contain aromatic hydroxyl
groups, these free hydroxyl groups may due to the generation of chelate complexes with zinc ions within the
active centre of the enzyme.
Our data demonstrated that flavonoids isolated from
A. excelsa
leaves have similar levels of inhibitory activity toward ACE compared with previously reported
ACE inhibitory flavonoids.
Further work is needed to clarify the mechanism of
ACE inhibition and the relevance of these flavonoids
to the purported antihypertensive activity of the
A.
excelsa.
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