Fruit juice-induced endothelium-dependent relaxations in isolated porcine coronary arteries: evaluation of different fruit juices and purees and optimization of a red fruit juice blend

Abstract

Numerous studies have indicated that several polyphenol-rich sources such as red wine and green tea are potent inducers of endothelium-dependent relaxations in isolated arteries. As various fruits and berries are known to contain high levels of polyphenols, the aim of the present study was to assess the ability of selected pure fruit juices and purees as well as blends to cause endothelium-dependent relaxations in isolated arteries. Vascular reactivity was assessed using porcine coronary artery rings, and fruit juices, purees and blends were characterized for their content in vitamin C, total phenolic, sugar and antioxidant activity. Fruit juices and purees caused variable concentration-dependent relaxations, with blackcurrant, aronia, cranberry, blueberry, lingonberry, and grape being the most effective fruits. Several blends of red fruits caused endothelium-dependent relaxations. Relaxations to blend D involved both a NO- and an EDHF-mediated components. The present findings indicate that some berries and blends of red fruit juices are potent inducers of endothelium-dependent relaxations in the porcine coronary artery. This effect involves both endothelium-derived NO and EDHF, and appears to be dependent on their polyphenolic composition rather than on the polyphenolic content.

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Introduction

Several epidemiological studies have suggested an association between certain diets and a lower risk of cardiovascular diseases. Indeed, a recent meta-analysis has indicated that a greater adherence to a Mediterranean diet is associated with a significant reduction by about 9% in mortality from cardiovascular diseases.¹ Moreover, the Mediterranean diet has also been associated with a beneficial effect on endothelium-dependent vasodilatation, insulin resistance, and myocardial and cardiovascular mortality.^2,3 The Mediterranean diet is characterized by the intake of high amounts of fruits, vegetables, cereals, and olive oil as well as a moderate consumption of red wine, which provide high levels of polyphenols. Polyphenols are thought to mediate, at least in part, the health benefit. Consistent with such a hypothesis, a reduced risk of cardiovascular diseases has been observed with the consumption of numerous polyphenol-rich sources such as fruits and vegetables,^4–6 red wine,^7,8 cocoa and chocolate,^9,10 and green tea.^11,12

Endothelial cells lining the luminal surface of all blood vessels have a key role in the control of vascular tone in part via the release of potent vasodilators including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). Several studies have indicated that polyphenol-rich sources such as red wine and tea catechins are able to induce potent endothelium-dependent relaxations by increasing the NO component and also, in some blood vessels, the EDHF component of the relaxation. For a review, see ref. 13. Moreover, red wine polyphenols caused the PI3-kinase/Akt-dependent activation of endothelial NO synthase by stimulating the phosphorylation of Ser1177 (an activator site) and the dephosphorylation of Thr495 (an inhibitor site).¹⁴ Thus, the ability of polyphenols to stimulate the endothelial formation of major vasoprotective factors NO and EDHF, is likely to contribute to their beneficial effect on the cardiovascular system. Besides red wine and tea catechins, fruits and in particular berries are important sources of polyphenols. Therefore, the aim of the present study was to evaluate the ability of several fruits including different berry juices and purees as well as different blends of red fruits, to induce endothelium-dependent relaxations in porcine coronary arteries. In contrast to a single fruit juice, the development of blends of fruit juices is an interesting approach to optimize the tasting and the biological activity of the beverage.

Materials and methods

Material

Fruit juices and purees, as well as blends, were provided by Eckes-Granini Group GmbH (Nieder-Olm, Germany). Reagents were obtained from Sigma-Aldrich (St. Quentin Fallavier, France) except 9,11-dideoxy-11α,9α-epoxymethano-prostaglandin F_2α (U46619) from Cayman Chemical (Ann Arbor, MI, USA).

Vascular reactivity

Vascular reactivity studies were done in isolated porcine coronary arteries as described previously.¹⁵ Pig hearts were collected from the local slaughterhouse. Left circumflex coronary arteries were excised, carefully cleaned of loose connective tissue and cut into rings (3–4 mm length). Rings were suspended in organ baths containing oxygenated (95% O₂ and 5% CO₂) Krebs bicarbonate solution (mM: NaCl 119, KCl 4.7, KH₂PO₄ 1.18, MgSO₄ 1.18, CaCl₂ 1.25, NaHCO₃ 25, and D-glucose 11, pH 7.4, 37 °C) under a resting tension of 5 g for the determination of changes in isometric tension. All experiments were performed in the presence of indomethacin (10 μM) to rule the formation of vasoactive prostanoids. In some rings, the endothelium was removed mechanically by gently rubbing the lumen of the ring with forceps. Coronary artery rings were contracted with U46619 before a concentration-relaxation curve to a fruit juice was constructed. The NO-mediated component of the relaxation was determined in the presence of charybdotoxin (100 nM) plus apamin (100 nM) to rule out EDHF-mediated responses. The EDHF-mediated component of the relaxation was determined in the presence of N^ω-nitro-L-arginine (L-NA, 100 μM) to rule out the formation of NO.

Determination of fruit juice contents

The total phenolic content of fruit juices was determined in triplicate and expressed as mg of gallic acid equivalents (GAE) using the Folin–Ciocalteu method.¹⁶

The determination of the fruit juice content in ascorbic acid was done by potentiometric titration with dichlorophenolindophenol solution according to the International Federation of Fruit Juice Producers (IFU) methods No. 17,¹⁷ that in sugars by HPLC according to the International Federation of Fruit Juice Producers (IFU) methods No. 67,¹⁷ and antioxidant capacity by TEAC using the method described by Miller et al.¹⁸

Statistical analysis

All values were expressed as means ± a SEM of five different experiments. Statistical analysis was performed with Student's t-test for paired data or ANOVA followed by Fischer's protected least-significant difference test where appropriate. P < 0.05 was considered to be statistically significant.

Results

Composition of fruit juices and purees and red fruit juice blends

Analysis of the individual fruit juices has indicated variations in their total polyphenol concentration as expressed in gallic acid equivalents. The values ranged from 1.70 g l⁻¹ for apple puree to 7.15 g l⁻¹ for aronia juice (Table 1). Most of the fruit juices had only low concentrations of vitamin C, which ranged from 0.019 g l⁻¹ for blackberry puree to 0.430 g l⁻¹ for apple puree. In contrast and as expected, acerola puree contained a very high concentration of vitamin C (13.3 g l⁻¹, Table 1). The antioxidant capacity of the fruit juices determined as TEAC indicated great variations. Indeed, values ranged from 6.8 for apple puree to 92.3 for acerola puree (Table 1). No correlation was observed between the concentration of vitamin C and the antioxidant capacity of the fruit juices. In contrast, a positive correlation was observed between the polyphenol content and the antioxidant capacity of the fruit juices when acerola was not included (R² = 0.8667 without acerola).

Table 1 Antioxidant capacity, vitamin C content and polyphenol content of the individual fruit and berry juices and purees

Fruits and berries	Antioxidant capacity (mmol Trolox/l)	Vitamin C (g l^−1)	Polyphenols (g l^−1 GAE)
Acerola puree	92.29	13.30	3.12
Apple puree	6.80	0.430	1.70
Aronia juice	84.00	0.055	7.15
Blackberry puree	50.84	0.019	4.37
Blackcurrant puree	78.77	0.344	6.87
Blueberry puree	63.47	0.023	5.94
Boysenberry puree	62.00	0.028	3.82
Cranberry puree	23.96	0.037	2.26
Elderberry puree	64.47	0.065	3.86
Grape juice	23.00	0.035	1.99
Lingonberry juice	39.95	0.037	3.64
Raspberry puree	21.90	0.224	2.69
Strawberry puree	17.00	0.110	2.03

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Since individual fruit juices often have an astringent taste mostly due to their high polyphenol content, five different blends of red fruit juices were prepared and evaluated. The shared fruits present in all blends included grape juice (59 to 71%), acerola puree (4%), lingonberry juice (5%), and apple puree (10%). The additional fruits (juices or purees) were mostly from berries as indicated in Table 2. Analysis of the blends (Table 3) indicated that they contained similar concentrations of vitamin C (0.38 to 0.43 g l⁻¹), total polyphenol concentration (2.84 to 3.26 g l⁻¹ GAE) and antioxidant capacity (32.4 to 37.1). All blends were submitted to a panel of 80 consumers to establish a ranking of acceptability in terms of flavor, with blend D ranking first (Table 3).

Table 2 Composition of the fruit and berry blends

Mixture	A	B	C	D	E
Components	Acerola (4%)	Acerola (4%)	Acerola (4%)	Acerola (4%)	Acerola (4%)
	Apple (10%)	Apple (10%)	Apple (10%)	Apple (10%)	Apple (10%)
	Grape (59%)	Grape (71%)	Grape (66%)	Grape (63%)	Grape (62%)
	Lingonberry (5%)	Lingonberry (5%)	Lingonberry (5%)	Lingonberry (5%)	Lingonberry (5%)
	Boysenberry (3%)	Blueberry (6%)	Aronia (4%)	Aronia (4%)	Aronia (4%)
	Cranberry (6%)	Elderberry (3%)	Blackcurrant (6%)	Blueberry (10%)	Blackberry (5%)
	Elderberry (3%)	Raspberry (8%)	Raspberry (10%)	Strawberry (10%)	Blackcurrant (6%)
	Raspberry (15%)

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Table 3 Antioxidant capacity, vitamin C, polyphenol and sugar contents of the different fruit and berry blends, and their evaluation by consumers

Mixture	A	B	C	D	E
Antioxidant capacity (mmol Trolox/l)	32.44	34.97	36.58	36.74	37.10
Vitamin C (g l^−1)	0.38	0.35	0.42	0.40	0.43
Polyphenols (g l^−1)	2.84	3.10	3.15	3.19	3.26
Sugar (g l^−1)	111.2	129.2	123.2	122.4	115.9
Consumer test result (rank)	4	2	3	1	5

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Individual fruit juices and purees cause endothelium-dependent relaxations

The ability of 13 individual different fruit juices or purees to induce concentration-dependent relaxations was assessed in porcine coronary artery rings with endothelium, which were pre-contracted with the thromboxane mimetic U46619 (Fig. 1). The group of fruits including blackcurrant puree, aronia juice, cranberry puree, blueberry puree, lingonberry puree and grape juice caused concentration-dependent relaxations starting at a volume greater than 0.01% and reaching maximal relaxation at 0.5%. The group including apple puree, raspberry puree, and strawberry puree induced concentration-dependent relaxations starting at a volume greater than 0.1% (Fig. 1B). The group including boysenberry puree, blackberry puree, acerola puree and elderberry juice caused only minor relaxations at volumes up to 2% (Fig. 1C). No correlation was found between the vasorelaxant property and the content of vitamin C, the total polyphenol content or the antioxidant capacity.

Fig. 1 Selected fruit and berry juices and purees cause endothelium-dependent relaxations in the porcine coronary artery. Arterial rings with endothelium were contracted with U46619 before the addition of increasing concentrations of either (A) grape juice, cranberry puree, lingonberry juice, aronia juice, blackcurrant puree or blueberry puree, (B) grape juice, apple puree, raspberry puree, blackberry puree or strawberry puree, and (C) grape juice, boysenberry puree, acerola puree or elderberry juice. Results are expressed as means ±SEM of 5 different experiments.

Red fruit juice blends cause endothelium-dependent relaxations

Five blends (A to E) of red fruit juices were evaluated for their potency to induce concentration-dependent relaxations of pre-contracted porcine coronary artery rings with endothelium (Fig. 2). All five blends caused concentration-dependent relaxations with blend D and E being the most active ones. Although blend D and E caused full relaxations in rings with endothelium, no such effect was observed in those without endothelium indicating that they induce endothelium-dependent relaxations (Fig. 2 and data not shown).

Fig. 2 Blends of red fruit juices (A to E) induce endothelium-dependent relaxations in the porcine coronary artery. Arterial rings with or without endothelium were contracted with U46619 before the addition of increasing concentrations of a blend of red fruit juices. Results are expressed as means ±SEM of 5 different experiments.

Further characterization of the endothelium-dependent relaxation was done with blend D (Fig. 3). Relaxations to blend D were not affected by apamin plus charybdotoxin (inhibitors of EDHF-mediated relaxation), slightly but significantly reduced by L-NA (a competitive inhibitor of NO synthase), whereas the combination of L-NA, charybdotoxin plus apamin reduced the maximal relaxation to about 80% (Fig. 3). These findings indicate that blend D induced endothelium-dependent relaxations of coronary arteries, which include a NO-mediated component and also, to some extent, an EDHF-mediated component.

Fig. 3 Characterization of the endothelium-dependent relaxations induced by blend D in coronary artery rings. Coronary artery rings with endothelium were contracted with U46619 before the addition of increasing concentrations of blend D. Some rings with endothelium were incubated with either N^ω-nitro-L-arginine (L-NA, 100 μM, an inhibitor of endothelial NO synthase), charybdotoxin (100 nM) plus apamin (100 nM; two inhibitors of EDHF-mediated relaxations) or the combination of L-NA, charybdotoxin and apamin for 30 min before addition of U46619. Results are expressed as means ±SEM of 5 different experiments. p < 0.05 vs. respective control.

Blends A to E contained similar amounts of grape juice (59–71%), apple puree (10%), lingonberry juice (5%) and acerola puree (4%). Therefore, the biological activity of this mixture of 4 juices (blend 1), which represents between 78 to 82% of blend A–D, was determined. Blend 1 induced a concentration-dependent relaxation curve, which was slightly but significantly shifted to the right compared to blend D, and was similar to that induced by its main constituent grape juice (Fig. 4A). Altogether, these findings suggest that the biological activity of blend D is mostly dependent on its major constituent grape juice and that it is further increased by the addition of small volumes of blueberry puree (10%) and aronia juice (4%), two highly active products, but not by the addition of a small volume of strawberry puree (10%), a less active product (Fig. 4B).

Fig. 4 Comparison of blend D and its fruit products on the induction of endothelium-dependent relaxations in the coronary artery. Blend 1 contains grape juice, apple puree, lingonberry juice, and acerola puree as in blend D. Coronary artery rings with endothelium were contracted with U46619 before the addition of increasing concentrations of a fruit juice. Results are expressed as means ±SEM of 5 different experiments.

Discussion

The evaluation of 13 different fruits juices or purees has revealed that several of them are potent inducers of endothelium-dependent relaxations in porcine coronary arteries. These active products include cranberry, lingonberry, aronia, blackcurrant, blueberry, and grape. In contrast, strawberry, apple and raspberry showed only some activity at high concentrations whereas blackberry, boysenberry, elderberry and acerola were mostly inactive. The determination of the total phenolic content of the different products has indicated that there is no correlation between the total phenol content as assessed by the Folin–Ciocalteau method and the vasorelaxant activity. However, the total phenol content correlated with the antioxidant capacity of the different products as assessed by TEAC when acerola, a rich source of vitamin C, was excluded. Altogether, these findings indicate that the vasorelaxant activity of the fruit products is mostly dependent on their qualitative polyphenolic composition rather than on their quantitative polyphenolic content.

Amongst the various fruits investigated, the most active ones were predominantly berries including cranberry, lingonberry, aronia, blackcurrant, and blueberry. Berries are characterized by the presence of high levels of anthocyanins, which are pigments responsible for the blue-red colors of fruits, and procyanidins.¹⁹Anthocyanins are a group of phenolic compounds which share the same basic flavonoid skeleton but differ in the number and position of hydroxyls on the B ring, and also in the nature of the sugar moiety attached in position C3. Anthocyanins are likely to contribute to the endothelium-dependent relaxations induced by the active berries and this effect may depend on the hydroxylation of the B ring and the nature of the sugar moiety attached in position C3. Indeed, delphinidin, but not malvidin and cyanidin, elicited endothelium-dependent relaxations of rat aortic rings.^20,21 In addition, cyanidin-3-glucoside has been shown to stimulate endothelial NO synthase activity by increasing the phosphorylation of Ser1179 and the dephosphorylation of Ser116.²² Although petunidin and petunidin-3-glucoside were inactive, petunidin-O-coumaroyl-glucoside caused the phosphorylation of eNOS at Ser1177 and increased the formation of NO.²³ Moreover, the stimulatory effect of petunidin-O-coumaroyl-glucoside was not shared by malvidin-coumaroyl-glucoside, which differs from the former by the presence of a methoxy group instead of a hydroxyl group on the B ring.²³ The possibility that hydroxyl groups have an important role in the polyphenol-induced activation of endothelial NO synthase is further supported by the fact that methylation of all hydroxyl groups of (−)-epigallocatechin-3-O-gallate and of a grape seed extract resulted in the loss of their vasorelaxant activity.^24,25 Besides anthocyanins, procyanidins are also likely to contribute to the endothelium-dependent vasorelaxation induced by the active berries. Indeed, procyanidins from grape-derived products have been shown to stimulate the endothelial formation of NO.^24,26–29 In addition, the fact that several berries including raspberry, blackberry, boysenberry and elderberry had little biological activity despite having a high level of polyphenols indicates that the qualitative composition of the polyphenolic content is important for the biological activity.

The present study has also evaluated the vasorelaxant activity of blends of various fruits and berries. These blends were developed in order to obtain mixed fruit juices with a high level of biological activity as assessed by endothelium-dependent relaxations associated to a pleasant taste. All five blends elicited potent endothelium-dependent relaxations with blend D and E being the most actives ones. These blends contained all a high proportion of grape (59 to 71%), smaller proportions of apple (10%), lingonberry (5%) and acerola (4%), and small proportions of different kinds of berries. The combination of grape, apple, lingonberry, and acerola elicited also endothelium-dependent relaxations of coronary arteries but to a lesser extent than blend D and E. Therefore, the addition of small amounts of blueberry and aronia in the case of blend D, and cranberry in the case of blend E most likely contributed to increase the vasorelaxant activity of the blends since these berries elicited potent endothelium-dependent relaxations. Since the taste of blend D was well appreciated by 80 potential consumers (Table 3), this blend was selected for further evaluation.

Relaxations to blend D were observed in rings with endothelium but not in those without endothelium. Moreover, these relaxations were significantly reduced by N^ω-nitro-L-arginine (a competitive inhibitor of NO synthase), not affected by charybdotoxin plus apamin (inhibitors of EDHF-mediated responses), and markedly reduced by L-NA, plus the combination charybdotoxin plus apamin. Altogether, these findings indicate that blend D-induced vasorelaxation is strictly dependent on the endothelium and involves an increased endothelial formation of NO and also, to some extent, EDHF.

Although anthocyanin- and procyanidin-rich products such as red wine, grape juice, and berries can elicit potent endothelium-dependent relaxations of isolated blood vessels, the bioavailability of these polyphenols still remains unclear. Indeed, previous studies have indicated that anthocyanins have a low absorption and bioavailability, and that the structure of the anthocyanin aglycon and the attached glucoside moiety seem to have a strong effect on their absorption, metabolism and excretion.³⁰ Similarly, procyanidins have been shown to be absorbed to some extent since low levels have been detected in blood.^31,32 Despite their relatively poor apparent bioavailabiltity, numerous studies have reported that intake of polyphenol-rich products has a beneficial effect on the endothelial function in experimental animals and humans. For a review, seeref. 13. Indeed, addition of freeze-dried blueberries in the diet of spontaneously hypertensive rats reduced significantly their systolic blood pressure.³³ Moreover, several clinical studies have indicated that oral consumption of anthocyanin- and procyanidin-rich products such as red wine,^34–36 grape juice^37–39 and cocoa^40–44 improved the endothelial function in healthy subjects and in patients with cardiovascular diseases.

Recent studies have indicated that unabsorbed procyanidins and anthocyanins reach the large intestine where they can be catabolised by the colic flora into small ring fission compounds such as hydroxycarboxylic acids, hydroxyphenylacetic acids or phenylalkyl acids.^45–49 The possibility that these small phenolic compounds might, in turn, be absorbed in the large intestine and reach the bloodstream to affect the endothelial function still remains to be determined.

Conclusions

In conclusion, the present study indicates that various fruit and berry products have different abilities to elicit relaxation of porcine coronary artery rings. The most active products include blackcurrant, blueberry, aronia, cranberry, lingonberry and grape. In addition, the vasorelaxant activity seems to depend mostly on the qualitative composition of the phenolic compounds rather than on the total phenolic content. The present data indicate also that blends of fruit products can be developed to combine a high level of vasorelaxant activity and an enjoyable taste. The possibility that the regular intake of such active fruit juices may improve the endothelial function and, hence, vascular health still remains to be determined.

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