Diet enriched with procyanidins enhances antioxidant activity and reduces myocardial post-ischaemic...

ELSEVIER PII s0024-3205(98)oo6o5-5

Life Sciences, Vol. 64, No. 8, 627442, 1999 pp. Copyright 0 1999 EIscvicr Science Inc.

Printed in the USA. All rights resewed 0024-3205/99 S19.00 + .OO

DIET ENRICHED WITH PROCYANIDINS ENHANCES ANTIOXIDANT ACTIVITY AND REDUCES MYOCARDIAL POST-ISCHAEMIC DAMAGE IN RATS

R. Maffei Facino*, M. Carini*, G. Aldini*, F. Berti **, G. Rossoni**, E. Bombardelli” and

P. Morazzoni’

* Istituto Chimico Farmaceutico Tossicologico, University of Milan, Viale Abnuzi 42 - 2013 1 Milan, Italy

* * Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, University of Milan, Via Vanvitelli 32,20129 Milan, Italy

’ Indena S.p.A. - Viale Ortles 12 - 20139 Milan, Italy

(Received in final form November 10, 1998)

Summary

Aim of this work was to study the efficacy of procyanidins from Vitis vinifera seeds, a standardized mixture of polyphenol antioxidants, on cardiac mechanics following ischemia/reperfusion stunning in the rat, after 3 weeks supplementation. Young and aged male rats were fed a diet enriched with procyanidins complexed (1:3 w/w) with soybean lecithin (2.4%); control animals (CTR-young and CTR-aged) received an equal amount of lecithin and 2 additional groups of animals the standard diet. At the end of the treatment, the total plasma antioxidant defense (TRAP), vitamin E, ascorbic acid and uric acid were determined in plasma and the hearts from all groups of animals subjected to moderate ischemia (flow reduction to 1 ml/min for 20 min) and reperfusion (15 ml/min for 30 min). In both young and aged rats supplemented with procyanidins the recovery of left ventricular developed pressure (LVDP) at the end of reperfusion was 93% (p < 0.01) and 74% (p < 0.01) of the preischemic values and the values of coronary perfusion pressure (CPP) were maintained close to those of the preischemic period. Also creatine kinase (CK) outflow was restrained to baseline levels, while a 2-fold increase in prostacyclin (6-keto- PGF,J in the perfusate from hearts of young and aged rats was elicited during both ischemia and reperfusion. In parallel, procyanidins significantly increased the total antioxidant plasma capacity (by 40% in young and by 30% in aged rats) and the plasma levels of ascorbic acid, while tend to reduce vitamin E levels; no significant differences were observed in uric acid levels. The results of this study demonstrate that procyanidins supplementation in the rat (young and aged) makes the heart less susceptible to ischemia/reperfusion damage and that this is positively associated to an increase in plasma antioxidant activity.

Key Words: viris viniferu, procyanidin, supplementation, young rats, aged rats, ischemia/reperfusion SI cardioprotection, plasma antioxidant defense

Correspondence to: Roberto Maffei Facino, Istituto Chimico Farmaceutico Tossicologico, V.le Abruzzi 42, 20131 Milan, Italy. Tel. +39-02-29510823; Fax +39-02-29514197; Email: [email protected]

628 Procyanids as Antioxidants in Heart Vol. 64, No. 8, 1999

Ischemia-reperfusion damage of the heart is a deleterious situation encountered when an abrupt readmission of oxygenated blood floow is reinstituted to the ischemic tissue. This takes place in a variety of clinical situations ranging from thrombolysis, coronary angioplasty, coronary by- pass surgery to organ transplantation (1). A sudden burst of free radicals (basically oxygen- centered species) arising from the oxygen readmission to the coronary vasculature and to the activation of a) endothelial xanthine dehydrogenase/xanthine oxidase system, b) arachidonate pathway, c) “leaky” mitochondria, is the agent incriminated in the onset of arrhythmias and of ventricular dysfunction which precipitate the pathophysiological situation of the heart, far exceeding the damage caused by ischemia alone (2). Antioxidants can ameliorate myocardial stunning/reperfusion dysfunction through several mechanisms (3): free radical scavenging, xanthine oxidase and lipooxygenase inhibition, saving of endogenous antioxidants and antioxidant enzymes, iron and copper chelating properties (transition metals are potent catalists of free radical reactions and are mobilized during ischemia/reperfusion) and for this reason the search is now focusing on new, even more potent and pluricentric natural or synthetic antilipoperoxidant agents. Procyanidins from Vitis viniferu, a highly standardized mixture of oligomeric catechins, strong antioxidants (4) widely employed in the pharmacological treatment of free radical-mediated microvascular pathologies, have been recently shown, when added to the reperfusion medium, to protect rabbit hearts from ischemia/reperfusion injury in isolated Langerdorffs preparations (5), attenuating myocardial dysfunction and preventing ventricular arrhythmias occurring during early reperhtsion when an increased generation of toxic free radicals, mainly HO”, takes place (6). At the light of these findings, the aim of the present study was to evaluate the cardioprotective effect of this polyphenol fraction on ex vivo hearts from both young and aged rats (the senescent heart is very sensitive to ischemia/reperfusion damage) supplemented for 3 weeks with a diet enriched with procyanidins. In parallel we evaluated in both the groups of animals the plasma antioxidant capacity, determining the Total Radical-trapping Antioxidant Parameter (TRAP), and the levels of the main hydrophilic (ascorbic acid and uric acid) and lipophilic (vitamin E) antioxidants.

Materials and Methods

Chemicals The organic solvents used were of analytical grade (Carlo Erba, Milan, Italy). Ascorbic acid, 6- hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox), vitamin E, Folin-Ciocalteu reagent, xilenol orange, .5,5-dithiobis(2-nitrobenzoic acid), diethylenetriaminepentaacetic acid (DETAPAC) and 2-thiobarbituric acid were purchased from Fluka Chimica (Milan, Italy); R- phycoerythrin (R-PE) and 2,2’-azobis(2-amidinopropane) dihydrochloride (ABAP) from Molecular Probes and Wako respectively (Sociem Italiana Chimici, Rome, Italy); soybean lecithin (Lipoid S30, Lipoid GmbH, Ludwigshafen) from Prodotti Giamri (Milan, Italy) and GSH from Boehringer Mannheim Italia (Milan, Italy). Angiotensin II was purchased from Sigma Chemical. Co. (Milan, Italy); the kits for 6-keto- PGF,, and creatine kinase (CK) determinations from Cayman, Chemical Company, (Arm Arbor, MI) and Boehringer-Marmheim Italia (Milan, Italy) respectively. The highly standardized mixture of procyanidins (purity > 96%; CAS 85594-37-2; Indena S.p.A., Milan, Italy) was obtained from Vifis vinifera seeds by a patented process (7) and contains gallic acid, (+)-catechin and (-)-epicatechin (15%), (-)-epicatechin 3-O-gallate, dimers, trimers, tetramers and their esters with gallic acid (80%), pentamers, hexamers, heptamers and their esters with gallic acid (5%) (mean molecular weight, determined by GPC analysis, 1100 f 80 daltons) (8). The complex procyanidins/soybean lecithin (1:3 w/w) was prepared by Indena S.p.A. (Milan, Italy) according to a patented process (9).

Vol. 64, No. 8, 1999 Procyanids as Antioxidants in Heart 629

Apparatus Spectrophotometric and fluorimetric studies were performed using a computer aided Perkin Elmer Lambda 16 spectrophotometer and a computer aided Perlcm Elmer LSSOB luminescence spectrometer (Perkin Elmer, Monza, Italy). High-performance liquid chromatography (HPLC) analyses were carried out with a Hewelett Packard series 1050 modular system equipped with an ultraviolet diode array detector (UV-DAD, HP series 1050) and with a Gilson instrument (pump series 306, Milan, Italy) interfaced with a fluorescence detector (FL3000, Therm0 Separation Products, Milan, Italy). Electron Spin Resonance (ESR) analyses were carried out on a Varian E-line Century series instrument (X-band, 100 kHz field modulation) in the following conditions: microwave power 15 mW; modulation 1 mT; time constant 0.25 s; scan time 1 min.

Animals and Diet The procyanidins/lecithin complex was mixed with food (standard diet, Altromin-R) at 2.4% w/w (Rieper S.p.A., Vandolese, Bolzano), splitted (1 Kg aliquots) in undervacuum aluminum bags and stored in a cold chamber (procyanidins diet). During the experimental period, procyanidms stability was daily checked by measuring the total polyphenol content by the Folin- Ciocalteu method (10). In the control diet, soybean lecithin was mixed with food at 1.6% (w/w) and stored as above described. To check the stability of lecithin and lecithin/procyanidins complex, analyses for thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (orange xilenol method) (11) were daily performed on the food pellets after extraction of the lipids with CHCl, : CH,OH (2: 1) (12). Young (1 months old, 100 f 10 g initial body weight) and aged (20 months, 600 * 30 g) male Wistar rats (Charles River Italia, Calco, Italy) were maintained in compliance with the policy on animal care expressed in the National Research Council guidelines (NRC 1985). The animals were housed individually in stainless steel cages under standard conditions (temperature 22 i 2 ‘C; 65% relative humidity; artificial light from 06.00 to 20.00). Standard food and drinking water were available ad libifum in the first pre-study week; after this time young and aged animals were randomly divided into three groups on the basis of body weight. Young rats: the first group (n = 16, PROC-young) was fed with the procyanidins diet for 3 weeks: at the end of the treatment, 8 animals were sacrified and the remaining 8 animals were maintained on a standard diet for an additional week before sacrifice (washout group). To the second group of animals (controls, CTR-young; n = 8) an equal amount of lecithin was administered through the control diet for the whole experimental period. Additional 8 animals (third group; ST-young) were fed the standard diet without lecithin and procyanidins. Aged rats (n = 24) were fed as young rats: procyanidins diet (PROC-aged; n = 8); control diet (CTR-aged; n = 8) and standard diet (ST-aged; n = 8). Body weight and food consumption were recorded for each animal of the 6 experimental groups at the same time intervals for the 3 weeks of the experiment. At the end, the animals were sacrified after light anesthesia with diethylether: blood was collected into heparinized/EDTA.tubes and the heart excised for perfusion experiments. Plasma was prepared by centrifugation at 2000 x g for 10 min at 4°C and used immediately for determination of all the below reported parameters.

Serum parameters Total cholesterol (TCH), triglycerides (TG) and glucose were enzymatically determined using the commercial kits (TCH n. 1442341, CHOD-PAP method; TG n. 701882, GPO-PAP method; glucose n. 676543; GOD-PAP method) from Boehringer Mannhcim Italia (Milan Italy).

TRAP assay The total plasma antioxidant capacity was determined according to the method of Ghiselli et al. (13) with minor modifications: 8 ~1 of plasma were diluted with 75 mM phosphate buffer pH


7.0, containing 2 pM R-PE (final volume 2.5 ml). The reaction mixture was preincubated at 37OC for 5 min in 10 mm quartz fluorimeter cells before the addition of ABAP (3 mM final concentration) as radical generator. The R-PE fluorescence decay was monitored in continua for 90 min against a blank sample containing all the ingredients except plasma. A triplicate analysis was performed by using a computer-controlled thermostated 4 positions cell-holder. The TRAP values (pM) were determined according to the original method using Trolox at a final concentration of 2.28 nM.

Determination of plasma antioxidants Ascorbic acid (AA) and uric acid were determined in fresh plasma by reverse-phase HPLC analysis. Immediately after preparation, a 400 pl aliquot plasma was deproteinized with 3 M perchloric acid (100 pl) and diluted with the HPLC mobile phase to a final volume of 1.5 ml. The sample was ultracentrifuged at 50,000 ‘pm for 16 minutes at 4°C and the supematant filtered through a 0.2 pm HPLC filter (Supelcosil). A 50 pl volume was injected into the HPLC system using a Supelcosil ABZ column (25 cm x 4.6 mm i.d; 5 pm particle size) equipped with a Supelcosil ABZ column guard (2 cm). The mobile phase was 10 mM phosphoric acid at a flow rate of 1 ml/min. Ascorbic acid and uric acid were detected (UV-DAD) at 260 nm. For determination of vitamin E, 500 pl of plasma were vortexed with 500 pl of ethanol and extracted with n-hexane as described by Lang et al. (14). The extract, concentrated to dryness under nitrogen, was taken up in 250 ul of mobile phase and 20 pl aliquots (filtered on 0.2 pm nylon membrane Supelcosil filters) submitted to HPLC analysis in the following experimental conditions: Supelcosil LC-I 8 (15 cm x 4.6 mm id.; 3 pm particle size) equipped with a guard column (Supelguard LC-18, 2 cm); mobile phase (isocratic elution): 30% isopropanol and 70% acetonitrile/methanol (6:l) at a flow rate of 1 ml/min; fluorimetric detector (h,,,= 290 mu, h,,= 332 run).

Stabilization of ascorbic acid by procyanidins 0.1 ml of a 0.25 M methanol solution of AA were diluted to a final concentration of 1 mM with phosphate buffer, pH 7.4, containing different concentrations of procyanidins, GSH or DETAPAC (0.1-100 pM). The solution was vortexed and 50 pl aliquots immediately transferred into a quartz tube for the ESR analysis. The formation of the ascorbyl radical (AA”) was monitored 100 set after the insertion of the sample into the ESR cavity and the quenching of the ascorbyl radical by the compounds determined as % inhibition of AA” formation, measuring the height of the first line of the doublet (means f SD of 5 determinations). Stabilization of AA (100 yM in 50 mM phosphate buffer, pH 7.4) by equimolar concentrations (20 pM) of procyanidins, GSH and DETAPAC was also evaluated by monitoring the rate of loss of ascorbate by UV spectroscopy (hmax = 265 nm).

Perfused rat heart preparations Hearts isolated from rats of each group were perfused retrogradely at 37°C through the aorta as previously described (16) with a Krebs Henseleit solution @H 7.4) containing (mM): NaCl 118, KCI. 2.8, RI-&PO, 1.2, CaCI, 2.5, MgSO, 1.2, NaHC03 25 and glucose 5.5. The perfusion was maintained at 15 ml/min for 20 min with a roller pump (Minipuls 3, Gilson Villiers, le Bel, France). Left ventricular pressure (LVP) and coronary perfusion pressure (CPP) were recorded using a HP-1280C pressure transducer (Hewlett-Packard, Waltham, MA, USA). LVP was obtained by inserting a small latex balloon filled with saline through the left atrium. Left ventricular end diastolic pressure (LVEDP) was stabilized to 4-5 mm Hg, whereas CPP was maintained at 65-70 mm Hg. All these variables were displayed on a Hewlett-Packard dynograph (HP-7754A). The hearts were electrically paced at the frequency of 300 beats/min with rectangular impulses (1 msec duration, voltage 10% above threshold) by a Grass stimulator


(S-88 model, Grass Instruments, Quincy, Mass, USA). A moderate ischemia (stunning) was induced by reduction of the perfusion flow to 1 ml/min for a period of 20 min. A normal flow rate (15 ml/min) was then restored and reperfusion was continued for 30 min. Left ventricular developed pressure (LVDP = peak left ventricular systolic pressure minus LVEDP) was evaluated during reperfusion. At the beginning of each experiment the activity of angiotensin-II (1 ug as a bolus in the perfusion system) on coronary vasculature was recorded.

6-keto-PGF,, in heartperfusates Prostacyclin (PGI,) generation by the cardiac tissues was measured in heart perfusates as 6-keto- PGF,, according to the enzyme immunoassay method (detection limit 0.05 @ml) of Pradelles et al. (17). The concentration of this stable metabolite was determined collecting the perfusates for 5 min immediately before flow reduction and during the first 10 min of reperfttsion. The rate of formation of 6-keto-PGF,, was expressed in ng/min.

Creatine kinase in heart perfusates The perfusate was collected every 150 set in ice-cooled tubes before flow reduction and during reperfusion, and the activity of creatine kinase (CK) spectrophotometrically evaluated according to the method of Bergmeyer et al. (18). The enzyme activity was expressed as mU/min/g wet tissue.

Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA), followed by the Tukey’s test for statistical significance among groups and by Student’s I-test (two tailed) for unpaired samples. Differences were considered significant when p < 0.05. Statistical analyses were performed using the Prism software package (GraphPad Software, Inc). The area under the curve (AUC) was assessed by the trapezoid method using a computerized programme Microcal Origin. All data are expressed as mean f SEM.

Results

Animals and diet The daily analysis of rat feed shows no alteration in the polyphenol content throughout the experiment. No oxidation products were found in lecithin and in the procyanidins-lecithin complex (peroxides value was below 10 meq/Kg for the overall experimental period; data not shown). The young and aged rats of the four experimental groups receiving lecithin alone or in combination with procyanidins were all in good health conditions after three weeks of treatment. As shown in Tab. 1, no differences were observed between groups of young animals in food intake, body weight gain and growth performance. Animals of the PROC-young group received 0.763 f 0.024 g/Kg/day of procyanidins and 1.43 f 0.048 g/Kg/day of lecithin, and animals of the CTR-young group 1.442 f 0.05 1 g/Kg/day lecithin. Feed conversion efficiency (body weight gained/food intake; g/g) did not differ between groups, ranging from 0.290 to 0.297. The food intake in aged rats was not significantly different in the 3 groups of animals (Table I); although the higher intake of food in aged vs young rats, the PROC-aged and CTR-aged groups received an amount of procyanidins and lecithin/Kg body weight comparable to that of the corresponding young animals (PROC-aged: 0.711 f 0.034 g/Kg/day procyanidins and 1.423 f 0.088 g/Kg/day lecithin; CTR-aged: 1.412 f 0.090 g/Kg/day lecithin). As shown in Table II, no significant differences between groups of both young and aged rats were observed at the end of the treatment in the plasma levels of total cholesterol, triglycerides and glucose.

632 Procyanids as Antioxidants in Heart Vol. 64, No. 8,1999

TABLE I

Body Weight Gain, Food Intake and Feed Efficiency in Young and Aged Rats Fed Procyanidins

Initial body Final body Body weight Food Intake Feed

weight (g) weight (g) gain (g/21 days) (g/21 days) Efficiency”

Young rats

Standard 108.0 f 4.3 249.2 f 5.8 141.2 f 5.6 478.0 f 26.5 0.295 f 0.008

Control 106.5 f 3.4 247.8 f 6.4 141.3 f 6.2 475.4 f 27.5 0.297 f 0.011

Procyanidins 107.7 f 2.9 248.8 f 6.2 141.2 f 6.4 486.9 f 27.5 0.290 f 0.006

Aged rats

Standard 608 f 40 620 f 38 12*41 n.s 1111.9*36 --

Control 585 f 22 601 f 26 16 f 27 n.s 1126.4 f 21 --

Procyanidins 618*25 600*21 18 f 32 n.s. 1120.3 f 45 --

Values are the mean f SEM for 8 animals; n.s. not significant; “Body weight gain(g)/Food intake(g)

TABLE II

Plasma Lipids and Glucose in Young and Aged Rats Fed Procyanidins

Total Cholesterol (mmol/L)

Triglycerides (mmol/L)

Glucose (mmol/L)

Young rats

Standard

Control

Procyanidins

1.588 f 0.062 0.824 f 0.157 9.445 f 0.325

1.742 f 0.016b 0.888 f 0.1 68b 8.348 f 0.344b

1.638 f 0.050 0.870 f 0.116” 9.940 f 0.211”

Aged rats

Standard 1.915 f 0.063 1.272 f 0.155 8.446 f 0.303

Control 2.265 f 0.222b 1.205 f 0.172b 10.35 f 0.404b

Procyanidins 2.040 f 0.209” 1.146 i 0.092 8.985 f 0.426”

Values are the mean f SEM for 8 animals; ‘not significant vs CTR groups; b not significant vs ST groups.

Ischemia and reperfusion in isolated rat heart The reduction of flow from 15 ml/min to 1 ml/min for 20 min in isovohunic left heart preparations from both CTR-aged and CTR-young rats induced a clear-cut decrease of left ventricular function at reperfusion, with a remarkable increase in coronary resistance.


After 30 min of reperfusion, a recovery in LVDP values was observed which, as reasonably expected, was greater in hearts from CTR-young rats than in hearts from CTR-aged rats (5 1% vs 39 %) (Fig. 1). A similar trend was for CPP at the end of reperfusion, where the values of coronary resistance were still increased over the basal values, by 34 % in CTR-young (89 f 8.5 vs66.5+ 5.2 mm Hg; p=zO.Ol)and by 62% inCTR-aged (111 f 5.2vs68.4* 1.7 mmHg; p<

0.01) heart preparations (Fig. 1). When hearts from PROC-young and PROC-aged rats were subjected to ischemia-repetision, the recovery of LVDP at the end of reperfusion was 93 % (p < 0.01) and 74 % (p < 0.01) of the preischemic values (Fig. 1). Anyway, taking into account that the recovery of LVDP at the end of reperfusion in hearts from CTR-aged rats is lower (39%), the extent of protection by procyanidins in hearts from young and aged rats is of the same order of magnitude (Fig. 1). Furthermore, the protective effects of procyanidins on heart contractility during reperfusion in both young and aged rats was strictly associated to a proportional decrease in coronary resistance. In fact, at the end of reperfusion the CPP values in hearts from PROC-young and PROC-aged rats were increased over the corresponding preischemic values only by 5 % (67 & 5.8vs64i3.9mmHg;p>O.O5)andby 17%(77i 1.2vs66i2.7mmHg;p<O.O5)(Fig. 1). The time-course of LVDP and CPP values in hearts from both young and aged rats fed the standard diet for 3 weeks (ST-young and ST-aged) did not differ significantly from those of the CTR groups, to indicate that lecithin by itself does not induce alteration in the cardiac function during ischemia-reperfusion (data not shown). At the end of reperfusion the values for LVDP and CPP were 51.5 f 3.9 mm Hg and 94 f 5.3 mm Hg in ST-young rats and 32.7 f 2.1 mm Hg (LVDP) and 108 + 5.3 mm Hg (CPP) in ST-aged rats.

Creatine kinase (CK) in heartperfusates With a close parallelism with the LVDP and CPP profiles, the kinetics of CK outflow during repetision in hearts from both CTR-young and CTR-aged rats confirm that a significant ischemia was occurring (Fig. 2). In fact at the peak of the concentration, CK was 5.2 fold (p < 0.001) and 5.9 fold @ < 0.001) increased in pefisates of hearts from CTR-young and CTR-aged rats respectively, with a gradual return to baseline levels at the end of repetision (Fig. 2). The kinetics of CK release did not differ significantly in animals receiving the standard diet (ST- young: 238 f 23 mU/min/g tissue at 30 min; ST-aged: 272 f 25 mU/min/g tissue; data not shown). In line with the recovery of LVDP values, the treatment of the animals with procyanidins caused a marked reduction of CK release. In fact, peak concentrations of CK were only slightly increased over baseline levels, by 1.7 fold (p < 0.01) and 2.3 fold (p < 0.01) in heart perfusates from PROC-young and PROC-aged rats respectively (Fig. 2).

6-keto-PGF,, and angiotensin II activity in perfmed rat hearts During preischemia and repefision the rate of release of 6-keto-PGF,, in heart peAsates from both CTR-young and CTR-aged rats was not statistically different. As expected (19). in these two control groups during reperfusion, the rate of formation of the eicosanoid increased about 4- fold, from 2.15 f 0.25 to 8.02 f 0.78 ng/min in CTR-young hearts and from 1.78 f 0.12 to 7.45 f 0.56 ng/min in CTR-aged hearts (Fig. 3). The same increase was observed in ST-young (from 2.23 f 0.32 to 7.95 f 0.64 ng/min) and in ST-aged hearts (from 1.85 f 0.23 to 7.68 f 0.63 ng/min). The release of 6-keto-PGF,, in perfusates of hearts from PROC-young and PROC-aged rats increased about 2 times during both preischemia and reperfusion periods (p < 0.001 vs control preparations) (Fig. 3). These results strongly suggest that procyanidins enhance the capacity of the coronary endothelium to generate prostacyclin both in normal and in response to ischemic conditions.


25

0

125

_ 100

x”

fl 75

8 50

25

0

t , I , I , , , , , , . , -mO 20 25 30 35 40 45 50

-m- cm-Yomg -o- PROC-Young + CTR-Aged + PROC-Aged

x x T; P L t , I , I , I , , , . , , (

-?-&??O 20 25 30 35 40 45 50

Time(d)

Fig. 1 Left ventricular developed pressure (LVDP) and coronary perfusion pressure (CPP) in isovolumic left heart preparations submitted to low flow &hernia and reperkion

from young and aged rats. Values are the mean f SEM for 8 animals. The areas

under the curves (AUCs) for LVDP were: 1103 f 92 (a); 7 13 f 64 (Cl); 1947 f 122

(0); 1479 f 105 (0). The AUCs for CPP (mm Hg increase over the preischemic

values) were: 959 f 85 (W); 1438 f 122 (Cl); 186 f 16 (0); 564 f 45 (0). Statistical differences for both LVDP and CPP: p < 0.05 CTR-young vs CTR-aged and PROC- young vs PROGaged; p < 0.01 PROC-young vs CTR-young and PROC-aged vs CTR-aged.

Vol. 64, No. 8, 1999 Procyanids as Antioxidants in Heart 63.5

3 350-

.$ ‘j 300-

3 en 8 250-

3 200-

g a, 150- L?a

3 loo-

t, I, - ,a I - I’ I’,

-j’o”s”;lO 20 25 30 35 40 45 50

Time(min)

-m- Cl&Young -o- PROC-Young --(I CTR-Aged -O- PROC-Aged

Fig. 2

Creatine kinase (CK) release during reperfusion in young and aged rat hearts. Values are the mean f SEM for 8 animals. The AUCs for CK release during reperfusion

were: 3883 f 275 (N); 5155 f 324 (Cl); 488 f 52 (0); 1264 i 89 (0). Statistical differences: p < 0.05 CTR-young vs CTR-aged and PROC-young vs PROCaged; p < 0.001 PROC-young vs CTR-young and PROC-aged vs CTR-aged.

25_ h d Young 1 Aged *** ._( -! 20- 0 Standard diet ***

0 Control diet

m Procyanidins diet

I

Preischemia Reperfusion Preischemia Reperfusion

Fig. 3

Prostacyclin (6-keto-PGF,,) release in perfusates of isovolumic left heart preparations from young and aged rats. Values are the mean f SEM for 8 hearts. Per&sates were collected during preischemia (5 min) and reperfusion (first 10 min). **p<O.Ol and*** p < 0.001 vs the corresponding ST/CTR groups. All reperfusion values were statistically different from those of preischemia (p < 0.001).


The response to the vasopressor activity of angiotensin II in this set of experiments was inversely related to the generation of prostacyclin by the endothelium of cardiac tissues. When angiotensin II (1 pg as a bolus) was injected through the heart during preischemia, a significant increase in CPP was measured in both ST and CTR animals (Fig.4); the vasopressor activity of the peptide in hearts from ST-aged and CTR-aged rats (25.7 f 1.4 and 25.9 f 2.2 mm Hg) was significantly higher (= 40%; p < 0.05) than that observed in hearts from ST- and CTR-young rats (18.5 f 1.1 and 19.1 f 1.3 mm Hg) (Fig. 4). In perfused hearts from procyanidins-treated young and aged rats (Fig. 4) CPP was reduced approximately by 30% (p < 0.05 vs the corresponding preparations from controls): 13.1 f 0.8 mm Hg (PROC-young) and 18.5 f 1.1 mm Hg (PROC-aged).

30

h

ii? 20

?I

R 0 10

0

Standard diet

Control diet

Procyanidins diet

T

*

I 1

L

l- -L

-

*

I Young

Fig. 4

Aged

Vasopressor activity of angiotensin II (1 pg/bolus) injected in isovolumic left heart preparations from young and aged rats during the preischemic period. Values are the mean i SEM for 8 hearts. * p < 0.05 vs the corresponding ST/CTR groups.

Total plasma antioxidant activity As shown in Fig. 5, the TRAP values determined in ST-young (1136 f 3 8 pM) and CTR-young (1142 f 35 @I) groups did not differ significantly, while in procyanidins-supplemented animals a marked increase, by 40% (1608 f 44 pM) was observed (p < O.OOl), to indicate a strong enhancement of the total antioxidant plasma capacity. Due to the complexity of the measure, to avoid artifactual results, the TRAP value was determined on an additional group of rats that fed the standard diet for a 7 days period after the 3 weeks supplementation with procyanidins. After the washout, the TRAP levels were strictly in the range of the controls (1156 f 34 PM). Similar results were found in the aged animals: no differences between ST-aged and CTR-aged groups. Anyway, TRAP values were significantly lower, by = 35% in respect to the corresponding young groups (724 f 40 pM, p < 0.0001; 766 i 35 PM, p < O.OOOl), indicating a reduced antioxidant plasma capacity associated with aging. Procyanidins supplementation significantly increased the total antioxidant plasma capacity, approximately by 30% (987 f 15 pM; p < 0.01 vs CTR-aged), although to a lesser extent in respect to young animals.


15 1 Ascorbic acid *** 1 TRAP ***

:: 1400 z c=

-

2 1000

2

! 600 n

I **

;I fvi I Young Aged yow3 Aged

m Standard diet 0 Control diet m Procyanidins diet

1 Vitamin E

l * II

n.s. 4.5-

_I -1 - Y 2 E 3.0 1.5

0.0

Uric acid

n.s.

I I o[a Young Aged Young Aged

Fig. 5 Effect of procyanidins supplementation on plasma antioxidant status in young and

aged rats. Values are the mean f SEM for 8 animals. ** p < 0.01 and *** p < 0.001 vs the corresponding STKTR groups.

Plasma levels of vitamin E, mcorbic acid and uric acid No significant differences in the levels of ascorbic acid were found between ST-young (8.74 f 0.30 pg/ml) atld CTR-young (9.88 f 0.47 pg/ml) groups (Fig.S), while in procyanidins-treated animals a significant increase (approximately 20%) was obtained (11.84 f 0.45 &ml; p < 0.01 vs CTR-young). Also ascorbate levels after the washout period returned to the range of the controls (8.25 & 0.33 &ml) (dara not shown). With a good correlation with the TRAP values, in aged rats a marked reduction in ascorbate levels, by 22% in ST-aged (6.33 f 1.15 pg/ml; p < 0.05) and by 27% in CTR-aged (6.65 f 0.63 pg/ml; p < 0.005) groups was observed in respect to the corresponding young groups. After supplementation with procyanidins, we found a 70% increase (12.45 f 0.42 pg/ml; p < 0.001 vs CTR-group) and ascorbate levels closely attained to those of procyanidins-treated young rats. Conversely in young rats procyanidins significantly decreased vitamin E levels, by 20% (5.98 f 0.29 pg/ml; p < 0.01) in respect to both ST (7.50 f: 0.22 pg/ml) and CTR animals (7.52 f 0.30 @ml). In ST- and CTR-aged rats vitamin E was significantly higher than in young rats (11.56 f 0.70 pg/ml and 10.99 f 0.46 @ml), and this in accord with the well documented age-related increase in plasma vitamin E (20, 21); after procyanidins supplementation, vitamin E was slightly but not significantly decreased (9.19 f 0.74 &ml).


No significant differences were observed in the plasma levels of uric acid after procyanidins supplementation both in young (ST-young 3.22 f 0.39 &ml; CTR-young 3.12 f 0.26 @ml; PROC-young 2.98 f 0.28) and aged rats (ST-aged 2.30 f 0.15 ug/mI; CTR-aged 2.26 f 0.20 pg/ml; PROC-aged 2.05 f 0.16 pg/ml), the values in all the aged-groups being slightly, but not significantly lower than those of the corresponding young groups (data not shown).

Stabilization of ascorbic acid by procyanidins When AA was dissolved in phosphate buffer, the characteristic ESR ascorbyl radical signal (a” = 1.76 G) was obtained within 2 min; after this time the signal progressively decreased, due to the spontaneous decay of the radical, mainly due to AA’ self-disproportionation (data not shown) (22). In the presence of procyanidins, a remarkable dose-dependent inhibitory effect was observed (Fig. 6a): the formation of the ascorbyl radical was inhibited by 12.4 f 0.3 % at 1 uM, by 3 1.5 f 1.6 % at 10 pM and by 65.7 f 2.8 % at the highest concentration (100 PM). GSH was slightly less potent than procyanidins, while DETAPAC far more active, since 10 uM totally prevented the radical generation.

0 0.1 1.0 10.0 100.0 @I 0 60 120 180 240 300 mill

Fig. 6 Stabilization of ascorbic acid by procyanidins. (a) Dose-dependent inhibition of ascorbyl radical formation (ESR spectroscopy). (b) Kinetics of ascorbate oxidation (100 uM; UV 265 nm) in the absence (0) or in presence of 20 pM DETAPAC (Cl), 20 pM GSH (0) or 20 pM Procyanidins (W), Values are the mean f SD of 5 determinations.

These results, which indicate that procyanidins are able to stabilize ascorbic acid, through an antioxygen effect and/or an iron/copper sequestering effect (adventitious catalytic metals, iron - 1 PM, copper < 0.1 pM are usually present in buffer and salts) were confirmed by UV monitoring at 37°C the rate of ascorbate disappearance (100 uM in phosphate buffer) under aerobic conditions (Fig. 6b). While in the controls, after 150 min incubation, ascorbate was totally consumed, in the samples protected with 20 pM procyanidins, ascorbate levels decreased only by 37.6% and after 5 h a residual 25% was still observed. The stabilizing effect induced by equimolar concentrations of GSH was slightly less pronounced, since after 150 and 300 min ascorbate levels were decreased by 53% and 85%; by contrast 20 pM DETAPAC totally prevented ascorbate oxidation in the same time interval.


Discussion

The results of this study demonstrate that a 3 weeks supplementation of young rats with antioxidant procyanidins from Vitis vinifera seeds increases the total plasma antioxidant capacity, by approximately 40%, spares/stabilizes the terminal antioxidant ascorbate by 20% and conversely reduces the mobilization of vitamin E from the liver, the preferential site of

delivery to plasma (23). The increase in the TRAP value is due to the burden of reducing species from procyanidins intake (0.7 g/Kg/day) and/or metabolization circulating in plasma and by the rise in ascorbic acid levels. As demonstrated by in vitro evidence (ESR and UV studies), procyanidins exert a direct regenerating effect on ascorbate, which can result in vivo in an increase of the antioxidant. Ascorbic acid in turn may modulate the release/transport of vitamin E from the liver (the synthesis of vitamin E transfer protein in the hepatocyte is a redox-sensitive process) (23). In

our conditions we are just dealing with the opposite situation which takes place in plasma following an oxidative stress, where it is documented that to a fall in ascorbate corresponds a rise in vitamin E, challenged from its binding sites by the change in the oxidative conditions of plasma (24,25). In the aged rats, which at baseline levels show vs young a decreased plasma antioxidant potency (by 30%), procyanidins supplementation is still able to promote a remarkable increase in the TRAP values (by 30%), near to those of unsupplemented young animals. The rise in ascorbate levels is much more pronounced than in the young, while vitamin E shows only a trend for re- equilibration (this very likely due to the slower response mechanisms of the liver of the senescent animal).

Anyway, the finding of major relevance of this study is that procyanidins supplementation induces a greater resistance (better recovery) of the hearts to a typical sturming/reperfusion procedure. In fact in all of the procyanidins fed rats (young and aged) left ventricular function at the end of reperfusion was significantly restored, and CPP values, markedly rised in the controls during reperfusion due to ventricular contracture, were reduced close to those of the preischemic period. The same is for another biochemical marker, CK, whose outflow was drastically restrained in both the groups of animals receiving procyanidins. Recovery from ischemia/reperfusion damage is of much more relevance in the aged rats, at the light of the fact that senescent hearts are very sensitive to ischemia-reperfitsion insults as a consequence of the age-associated physiological and biochemical changes including abnormalities in force production, excitation-contraction coupling, coronary endothelial function, reduction of substrate utilisation and antioxidant capacity (26-29). As shown in Fig. 7, highly significant positive correlations were observed between TRAP and LVDP values relative to the 4 groups of animals (r = 0.7899; p < 0.0001) or between ascorbic acid and LVDP (r = 0.8169; p < O.OOOl), and similar correlations were found between the antioxidant plasma markers and the other cardiac parameters: TRAP vs CPP (r = - 0.7542; p < 0.0001); TRAP vs CK (r = - 0.7124; p < 0.0001); ascorbic acid vs CPP (r = - 0.7932; p <

0.0001); ascorbic acid vs CK (r = - 0.8266; p < 0.0001). Although we have not measured the antioxidant status of the pre-ischemic and post-ischemic hearts from control and supplemented rats, these findings reinforce the view that the improvement in the myocardial function is linked to a preservation/amelioration of the antioxidant defenses of the heart, markedly impaired during the ischemia/reoxygenation stress (myocardial levels of ascorbate during global ischemia in isolated rat heart preparations decrease

by 30% and by 50% during reperfusion) (30). Conceivably, the increase in the plasma antioxidant potential, may result in the heart in the maintenance of adequate levels of reducing equivalents able to prevent, through a direct

640 Procyanids as Antioxidants in Heart Vol. 64, No. 8, 19%

scavenging of free radicals, the inactivation of glutathione peroxidase (one of the major myocardial defence against oxy radicals-driven membrane injury) and the oxidation of critical thiols of the Ca”-ATPase, the enzyme involved in the regulation of cytoplasmic Ca”, and located in the sarcoplasmic reticulum (the oxidation-induced inhibition of the Ca2’ transport system is irreversible and in cardiac muscle has been implicated as the mechanism responsible for ischemia/reperfusion injury) (3 1).

3000- r = 0.7899

2.500- p < 0.0001

9 2000-

S & 1500-

% cl looo-

500-

OJ . 0

, , I 250 500 750 1000 1250 1500 1750

I

2000

3000 1

TRAP (NW

2500-

2000..

1500-

1000-

500-

r = 0.8169 a

p < 0.0001 l : a

0 0.0 2.5 5.0

I 7.5 10.0

0 12.5 15.0

Ascorbic acid (@ml)

Fig. 7 Correlation between plasma antioxidant markers and LVDP in 4 experimental groups (n = 8): CTR-Young (m); PROC-Young (0); CTR-Aged (0); PROC-Aged

(0).

Alternatively the cardioprotective effect of procyanidins in young and aged rats may be due to an intervention on the endothelial cells lining the coronary vasculature, resulting in:

a) preservation of the coronary endothelium-dependent relaxant function (vasodilation) against vasopressor stimuli. This is demonstrated by the significant increase in prostacyclin outflow and the reduction in the coronary perfusion pressure induced by angiotensin II in


procyanidins-supplemented animals (Fig.4). Increased PGI, release, namely of 6-keto-PGF,, was demonstrated in human endothelial cells by several natural polyphenols (32) and more recently (33) in bovine aortic endothelial cells by red wine in toto, in which procyanidins are contained in large amounts (1 g/l). In addition procyanidins may maintain optimal levels of nitric oxide in the coronary vasculature, by preventing its decomposition by superoxide anion or by stimulating the endothelial NO/guanosine-3’,5’-cyclic monophosphate (cGMP) pathway. Grape skin extracts have been shown (34) to induce endothelium-dependent relaxation of phenylephrine-precontracted intact rat aortic rings and a concomitant increase in the vascular tissue levels of cGMP (both the effects were reversed by the NO-synthase inhibitors) and similar effects of endothelium-dependent vasorelaxation have been preliminarily observed by us for procyanidins from T/iris vinifera at 10 PM levels working with the human internal mammary artery.

b) Inhibitory action on endothelial xanthine oxidase. As previously demonstrated in vitro (4), procyanidins non-competitively inhibit xanthine oxidase, with a potency fairly comparable to that of the well know-n competitive inhibitor allopurinol, which is currently prescribed as “rescue” for patients undergoing by-pass surgery (35). This action can take place also in vivo, since procyanidins and the xanthine dehydrogenaselxanthine oxidase assembly bind to glycosaminoglycans (GAGS) of the luminal face of the blood vessels (36). Hence, when located at the endothelium surface procyanidins, since their high reducing properties, can prevent

oxidation of the thiol group of xanthine dehydrogenase, limiting its conversion to xanthine oxidase (37).

In summary, this study is the first to our knowledge which demonstrates that procyanidins from Vitis vinzfkra seeds (in this case suitably embedded within a lipophilic carrier) exert a strong antioxidant and cardioprotective activity. Presently we do not know which constituent/s is responsible for the observed effects (i.e. is preferentially absorbed and bioavailable), but from preliminary HPLC evidences we have indication that in plasma from young rats at the end of the treatment with procyanidins, at least six phenolic compounds (among which catechin and dimeric catechin) are detectable. Finally the results of the present study, coupled with other recent data in vitro which demonstrate that procyanidins at pM levels restrain the oxidative burst of activated human neutrophils and the release of tissue damaging proteases (38) and furthermore bind strongly to iron and copper (5), make this class of polyphenols, present in red wine and in many other vegetables and fruits, of oustanding interest in the therapeutic strategy for cardioprotection.

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