Beneficial effects of different flavonoids, on functional recovery after ischemia and reperfusion in isolated rat heart

Article abstract of DOI:10.1016/S0960-894X(00)00589-8

Three newly synthesised lipid peroxidation inhibitors (7, 11, 14) were evaluated for their effects on myocardial functional recovery during reperfusion after 30 min global ischemia in isolated rat hearts. The flavonoid compounds (7, 11, 14, rutin) reduce ischemia/reperfusion-induced cardiac dysfunction. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00589-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 23±27
Bene®cial E€ects of Di€erent Flavonoids, on Functional Recovery
after Ischemia and Reperfusion in Isolated Rat Heart
Jonathan Lebeau,a Remi Neviereb and Nicole Cotellea,*
a
Â
Equipe Polyphenols, Laboratoire de chimie organique et macromoleculaire, UPRESA 8009, USTL,
Â
59655 Villeneuve d'Ascq, France
Â
b
 Â
Laboratoire: Departement de Physiologie, Faculte de Medecine de Lille, Place de Verdun, 59000 Lille, France
Received 31 May 2000; revised 20 September 2000; accepted 9 October 2000
AbstractÐThree newly synthesised lipid peroxidation inhibitors (7, 11, 14) were evaluated for their e€ects on myocardial functional
recovery during reperfusion after 30 min global ischemia in isolated rat hearts. The ¯avonoid compounds (7, 11, 14, rutin) reduce
ischemia/reperfusion-induced cardiac dysfunction. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
inhibitor (allopurinol),12 a ¯avonol known for its anti-
oxidant properties (rutin: rhamnoside quercetin),13 and
a food additive antioxidant (BHT14) in protecting the
myocardium from the damaging e€ects of ischemia/
reperfusion injury using the isolated perfuse working rat
heart to generate potent cardioprotective therapy
agents.
Flavonoids are polyphenolic compounds which con-
stitute one of the most numerous and ubiquitous groups
of plant metabolites and are an integral part of both
human and animal diets.1 Recent interest has increased
greatly owing to their antioxidant capacity with free
radical scavenging properties attributed to the catechol
or pyrogallol group. The redox properties of poly-
phenols allow them to act as enzyme inhibitors,2,3
reducing agents, hydrogen donating antioxidants and in
some cases they also chelate transition metal ions.4,5
These properties play an important part in reducing the
risk of free radical-related oxidative damage associated
with degenerative disease such as in the treatment and
prevention of cancer4,6,7 and cardiovascular disease.8
However, the compounds with catechol moiety present
Chemistry
The ¯avones were prepared according to the Baker±
Vankataraman procedure15,16 (Scheme 2). 2-Hydroxy-4-
methoxyacetophenone 1 was condensed with 3,5-di-tert-
butyl-4-hydroxybenzoyl chloride in the presence of
DMAP in dry pyridine at 60 ꢀC during 2 h to give the
diester 2 in 57% yield. This result was not surprising
since 3,5-di-tert-butyl-4-hydroxybenzoyl chloride reac-
ted in dry pyridine at 60 ꢀC to give 4-(3,5-di-tert-butyl-4-
hydroxybenzoyloxy)-3,5-di-tert-butylbenzoic acid in
60% yield. Nevertheless, the diester 2 was treated with
sodium hydroxide in dry DMSO to give the diarylpro-
pane-1,3-dione 3 in 48% yield. The cyclisation of the
diarylpropane-1,3-dione 3 was carried out in re¯uxing
acetic acid in the presence of sulfuric acid during 1 h
leading to the ¯avone 4 in 76% yield, which was depro-
tected using boron tribromide in re¯uxing dichloro-
methane to give 5 in 77% yield. The ¯avone 5 was
saponi®ed with sodium hydroxide in dry DMSO for 4 h
at 120 ꢀC to give the target ¯avone 7 in 45% yield.
Alternatively 4 was saponi®ed to give the ¯avone 6 in
54% yield, which was deprotected using boron tri-
bromide to give 718 in 84% yield. Finally, the target
a
potent prooxidant activity in particular con-
ditions.9 11 Therefore, our research interest in this ®eld
has also been focused in the synthesis of modi®ed ¯a-
vonoids. In this report, we reveal the chemical synthesis
of three new ¯avonoids [30,50-di-tert-butyl-7,40-dihy-
droxy¯avone (¯avone 7), 30,50-di-tert-butyl-2,4,40-trihy-
droxychalcone (chalcone 11), 3-(400-hydroxy-300,500-di-
tert-butylbenzylidene)-7,40-dihydroxy-30,50-di-tert-butyl-
¯avanone (arylidene, 14)] (Scheme 1) where the 3,5-di-
tert-butyl-4-hydroxyphenyl moiety was associated to the
¯avonoid backbone and evaluate the possible role of
these ¯avonoids compared with a xanthine oxidase
*Corresponding author. Tel.: 333-20-33-61-35; fax: 333-20-33-61-36;
e-mail: nicole.cotelle@univ-lille1.fr
0960-894X/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00589-8

24
J. Lebeau et al. / Bioorg. Med. Chem. Lett. 11 (2001) 23±27
¯avone 7 was obtained in 6.22% overall yield (calcu-
lated from 2,4-dihydroxyacetophenone) in a six-step
procedure (versus 4.75% yield when the deprotection
was realised before saponi®cation procedure).
The chalcones 9±10 and arylidenes 12±13 were obtained
from the reaction of 1 or 8 with 3,5-di-tert-butyl-4-
hydroxybenzaldehyde (Scheme 3). Best results in chal-
cones were obtained when the acetophenone was treated
with one equivalent of the benzaldehyde and the aryli-
dene with two equivalents of the benzaldehyde. Never-
theless, in all the cases, chalcone, arylidene and starting
material coexist in the ®nal mixture. The reaction of 1
and 3,5-di-tert-butyl-4-hydroxybenzaldehyde led to the
chalcone 9 and arylidene 12, which were isolated and
treated with boron tribromide. Unfortunately, at
78 ꢀC the demethylation did not occur, whereas at
room temperature the target chalcone and arylidene
were totally destroyed. These deceptive results led us to
prepare the 4-benzyloxy-2-hydroxyacetophenone 8 and
reacted it with 3,5-di-tert-butyl-4-hydroxybenzaldehyde.
The resulting chalcone 10 and arylidene 13 were sepa-
rated and readily deprotected using boron tribromide at
78 ꢀC. The target chalcone 1119 and arylidene 1420
were obtained in 8 and 15% overall yield (calculated
from 1 and 8), respectively.
Scheme 1. Chemical structures of BHT, rutin, 7, 11, 14.
Scheme 2. Reagents and conditions: (a) pyridine, DMAP, 2 h, 60 ꢀC; (b) DMSO, NaOH, 2 h, 60 ꢀC; (c) AcOH, H2SO4. (d) BBr3, CH2Cl2, 24 h,
re¯ux; (e) DMSO, NaOH, 4 h, 120 ꢀC.

J. Lebeau et al. / Bioorg. Med. Chem. Lett. 11 (2001) 23±27
25
Scheme 3. Synthesis of chalcones and arylidenes.
Biological Activities
homogenate and enzymatic antioxidant systems22 (GSH
peroxidase, superoxide dismutase and catalase) were
determined in the centrifuged supernatant. In the
experimental protocol studied in this work, there was no
signi®cant increase in the activity of enzymatic anti-
oxidant systems (Table 1) or in the level of peroxidation
compared to control hearts.
Male Sprague±Dawley (300±350 g) were studied
according to the Langendor€ method.17 Brie¯y, the
heart was rapidly excised, placed in a cold (4 ꢀC) perfu-
sion bu€er then immediately cannulated at the aorta
root and perfused with a modi®ed Krebs±Henseleit
bicarbonate bu€er (37 ꢀC) gassed with 95% oxygen and
5% carbon dioxide (10 mL/min). DMSO solutions of
compound were prepared at 10 2 M and diluted in
Krebs to give ®nal concentration of 5±10 mM in a total
DMSO concentration of 0.01% (v/v). The same
amounts of pure DMSO were added to the control. A
compliant water-®lled latex balloon was inserted into
the left ventricle through the mitral valve and connected
to a pressure transducer. The ®lling pressure was then
adjusted to 5 mmHg. Left ventricular developed pres-
sure (LVDP, mmHg) was recorded continuously.
The process of lipid peroxidation, assessed by assaying
thiobarbituric acid reactants, is not a predominant phe-
nomenon of reperfusion-induced injury, at least in the
experimental model used here. However, enzymatic
antioxidant systems investigated in this study do not
seem modi®ed. This could mean that the small quantity
of oxygen free radicals produced does not overwhelm
the enzymatic antioxidant systems of myocardium that
is in agreement with lipid peroxidation results.
Table 1. Preparation and measurement of antioxidant enzymes in the
heart were performed according to the method described by Brown et al.22 a
Five di€erent drug regimes were studied according to
the following protocol. After a 10 min initial period,
isolated hearts were perfused with either Krebs alone,
Krebs+BHT (10 mM), Krebs+rutin (10 mM), Krebs
+allopurinol (10 mM), ¯avone 7 (5 mM), chalcone 11
(5 mM), arylidene 14 (5 mM) in a recirculating manner
for 15 min. Hearts were then subjected to a 30 min
ischemic period followed by 30±40 min reperfusion using
Krebs and the assigned drug regime at the same dose.
SOD
(U/mg proteins) (U/mg proteins) (U/mg proteins)
GPX
CAT
Controls
BHT
Allopurinol
Rutin
7
11
8035Æ767
9636Æ327
9800Æ450
9077Æ667
8433Æ1061
8740Æ1056
8935Æ989
1834Æ229
1803Æ112
1912Æ234
1829Æ233
1982Æ525
1879Æ678
1906Æ634
20Æ5
20Æ3
21Æ1
21Æ6
21Æ3
20Æ7
21Æ6
14
Biochemical analysis
aInhibition of the rate of reduction of cytochrome C by superoxide
radicals was used as an indirect measurement of SOD activity (1
U=SOD activity required to inhibit cytochrome C reduction by
50%). Disappearance of hydrogen peroxide was monitored at 240 nm
as a measure of catalase activity (1 U=1 mmol of H2O2 degraded/min
per mg of protein). Glutathione peroxidase activity was determined by
the disappearance of NADPH is monitored at 340 nm (1 U=1 mmol
of NADPH converted to NADP/min per mg of protein). All bio-
chemical analyses were standardised per gram of protein content, as
determined by Bio-Rad protein assay (Richmond, CA, USA). Values
are mean+S.D. not signi®cantly di€erent from control; *p>0.05
The levels of lipid peroxides and enzymatic antioxidant
systems in isolated rat heart muscle subjected to
global ischemia followed by 30 min of reperfusion were
measured.
After each experimental ischemia/reperfusion sequence,
the homogenate heart was frozen in liquid nitrogen.
Lipid peroxides (TBARS) were assayed in the cardiac

26
J. Lebeau et al. / Bioorg. Med. Chem. Lett. 11 (2001) 23±27
Functional recovery
Conclusion
The results demonstrated that ¯avonoids at the con-
centration of 5 mM and more especially chalcone 11
exhibited signi®cant myocardial protection. This was
evidenced by improved recovery of post-ischemic left
ventricular function (left ventricular developed pressure;
LVDP: Fig. 1) and diastolic function (left ventricular
end-diastolic pressure, LVEDP: Fig. 2) as compared to
the control and BHT groups. Values for developed
pressure in the ¯avonoid-treated groups were sig-
ni®cantly higher than those in the control and BHT
group throughout the reperfusion period (rutin:
81.3Æ3.8 mmHg, 7: 87.12Æ9.21, 11: 102.25Æ1.17, 14:
87.12Æ9.2 versus control: 38Æ6, BHT: 29.79Æ4.25). In
contrast to the control group and the BHT treated
group, the ¯avonoid groups and allopurinol group dis-
played signi®cant reduction in diastolic developed pres-
sure (allopurinol: 5Æ2, rutin: 9.33Æ3.78, 7: 10.5Æ1.9,
11: 9.5Æ1.73, 14: 14.5Æ2 versus control: 30Æ5, BHT:
21.5Æ0.5).
In this paper, we provide evidence that ¯avone 7, chal-
cone 11, arylidene 14, where the BHT moiety and the
¯avonoid backbone were associated, possess potent
cardioprotective properties more important than rutin
and allopurinol. Flavonoid-treated hearts and more
especially chalcone-treated hearts were resistant to
ischemia reperfusion injury, as shown by the improve-
ment of the post-ischemic ventricular function and
myocardial stunning. Previous in vitro study21 has
revealed that new ¯avonoid compounds (7, 11, 14) are
powerful lipid peroxidation inhibitors and poor xan-
thine oxidase inhibitors. These results suggest that the
same ¯avonoids possess cardioprotective e€ects that
may be attributed to their peroxyl radical scavenging
activities although no di€erence has been observed in
homogenate heart TBARS measurements. This may
provide a potential therapeutic approach for the
prevention of post-ischemic myocardial dysfunction.
References and Notes
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D. W.; Buhler, D. R. Food Chem. Toxicol. 1999, 37, 271.
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9. Rhaman, A.; Hadi, S. M.; Parish, J. H.; Ainley, K. Carci-
nogenesis 1989, 10, 1833.
Figure 1. E€ects of ¯avonoids on post-ischemic recovery of left ven-
tricular developed pressure (LVDP) in the isolated heart. Post-
ischemic LV recovery was expressed as the ratio 100Âpost-ischemic
LVDP/pre-ischemic LVDP. Results are expressed as meanÆSEM of
six rats per group. *p< 0.01 compared with controls.
10. Cao, G.; So®c, E.; Prior, R. L. Free Radical Biol. Med.
1997, 22, 749.
11. Sugihara, N.; Arakawa, T.; Ohnishi, M.; Furuno, K. Free
Radical Biol. Med. 1999, 27, 1313.
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13. Manach, C.; Morand, C.; Texier, O.; Favier, M.-L.;
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14. Babich, H. Environ. Res. 1982, 29, 1.
15. Baker, W. J. Chem. Soc. 1933, 1381.
16. Mahal, H. S.; Venkataraman, K. J. Chem. Soc. 1934,
1767.
17. Neviere, R.; Cepinskas, G.; Madorin, W. S.; Hoque, H.;
Karmazyn, M.; Sibbald, W. J.; Kvietys, P. R. Am. J. Physiol.
1999, 277, H885.
1
18. 7, H NMR (DMSO-d6, 200 MHz, d in ppm): 1.41 (18H,
s, di-tert-butyl), 6.77 (1H, s, H3), 6.92 (1H, dd, J=8.60 Hz,
3
Figure 2. E€ects of ¯avonoids on left ventricular post-ischemic LV
end-diastolic developed pressure (LVEDP, mmHg) in the isolated
heart. Results are expressed as meanÆSEM of six rats per group.
*p<0.001 compared with controls.
4J=2.20 Hz, H6), 6.99 (1H, d, J=2.20 Hz, H8), 7.70 (1H, s,
4
3
OH), 7.76 (2H, s, H cycle B), 7.88 (1H, d, J=8.60 Hz, H5),
10.72 (1H, s, OH). 13C NMR (CDCl3, 50 MHz, d in ppm):

J. Lebeau et al. / Bioorg. Med. Chem. Lett. 11 (2001) 23±27
27
1
30.05 (C±CH3), 34.67 (C±CH3), 102.50 (C8), 105.04 (C3),
0
20. 14, H NMR (CDCl3, 200 MHz, d in ppm): 1.32 (18H, s,
di-tert-butyl), 1.40 (18H, s, di-tert-butyl), 5.24 (1H, s, OH),
5.50 (1H, s, OH), 6.38 (1H, d, H8), 6.56 (2H, m, H6 and H2),
0
114.76 (C6), 116.11 (C4a), 122.26 (C1 ), 122.95 (C2 ), 126.47
0
0
(C5), 139.21 (C3 ), 157.33 (C4 ), 157.40 (C8a), 162.52 (C7),
163.32 (C2), 176.26 (C4).
6.53 (1H, s, H2), 7.11 (2H, s, H2 ), 7.30 (2H, s, H200), 8.09 (1H,
s, Hb), 10.80 (1H, s, OH).
0
1
19. 11, H NMR (CDCl3, 300 MHz, d in ppm): 1.50 (18H, s,
di-tert-butyl), 5.64 (1H, s, OH in ortho position of di-tert-
butyl), 7.00 (2H, m, H5), 7.45 (1H, d, H in a of CO,
3J=15.3 Hz), 7.55 (2H, s, H aromatic of B ring), 7.85 (1H,
21. Lebeau, J.; Furman, C.; Duriez, P.; Bernier, J. L., Teissier,
E.; Cotelle, N. Free Radical Biol. Med. 2000, 29, in press.
22. Brown, J. M.; Grosso, M. A.; Terada, L. S.; Whitman, G.;
Banerjee, A.; White, C. W.; Harken, A. H.; Repine, J. E. Endo-
toxin pretreatment increases endogenous myocardial catalase
activity and decreases ischemia-reperfusion injury of isolated
rat hearts. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2516.
3
d, H3, J=9.4 Hz), 7.90 (1H, s, OH in ortho position of CO).
13C NMR (CDCl3, 50 MHz, d in ppm): 30.18 (C-(CH3)), 34.39
(C±(CH3)), 116.78 (C6), 120.24 (C4), 126.11 (CHˆ), 156.95
(C1), 193.75 (CˆO).