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Endothelium-Dependent Vascular Effects of Pycnogenol

Fitzpatrick, David F.; Bing, Bettye; Rohdewald, Peter

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Journal of Cardiovascular Pharmacology: October 1998 - Volume 32 - Issue 4 - p 509-515
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The nutritional supplement Pycnogenol (P) contains compounds capable of producing a variety of potentially protective effects against chronic age-related diseases such as atherosclerosis and the cardiovascular consequences of this disease. These compounds consist of catechin, taxifolin, procyanidins of various chain lengths formed by catechin and epicatechin units, and phenolic acids and their glucose esters or glucosides, extracted from the bark of the French maritime pine (1).

Many of the beneficial effects of P appear to be related to the antioxidant properties of its components. These properties include inactivation of the superoxide radical (2-5) and the hydroxyl radical (2,5,6) and inhibition of singlet oxygen formation (6). With respect to the vascular system, prevention of lipid peroxidation, especially of low-density lipoprotein (LDL), would lead to a reduced risk of atherosclerosis. In vitro, P inhibits peroxidation of LDL (7), lipid peroxidation in phospholipid liposomes (2), lipid peroxidation caused by t-butylhydroperoxide (8), and UV-B-induced lipid peroxidation in cells (4). An intact capillary system is one of the prerequisites for optimal functioning of the microcirculation. Components of P protect collagen (9) and elastin (10) against enzymatic attack. Treatment of animals with pathologically low capillary resistance (e.g., spontaneously hypertensive rats) with P enhanced capillary resistance significantly (11). In humans, edema formation in the lower legs resulting from venous insufficiency could be reduced significantly by oral intake of this supplement (12).

Pycnogenol also is reported to exert a weak inhibitory effect on the angiotensin-converting enzyme (ACE; 13). The mild hypotensive effect observed in Sprague-Dawley rats after i.v. administration of the supplement may be related to this effect. Taking all of these beneficial effects together, P may prevent damage to the vascular system.

Another property of plant extracts in general is endothelium-dependent relaxing (EDR) activity. As was demonstrated earlier (14), red wines, grape juice, and grape-skin extracts cause EDR in isolated rat aortic rings (and in canine coronary artery rings; unpublished results). Subsequently, extracts of other commonly consumed vegetables, fruits, spices, and several types of tea were shown to exhibit EDR in varying degrees (15). In the EDR response, the constitutive endothelial nitric oxide synthase (eNOS) enzyme is stimulated to produce NO plus citrulline from the amino acid L-arginine. The NO then diffuses abluminally to the smooth-muscle layer of the vessel to cause vasorelaxation (dilation), and also luminally, where it protects the vessel wall by inhibiting platelet aggregation (16) and neutrophil adhesion to endothelium (17) and by inhibiting oxidation of LDL (18). This latter effect is thought to be important in prevention of plaque formation during development of atherosclerosis. Conceivably, therefore, several mechanisms could work in concert to maintain open arteries, to inhibit thrombus formation, and to prevent plaque formation during atherogenesis. In addition, NO was recently shown to inhibit smooth-muscle cell proliferation, another characteristic of atherosclerosis (19). These effects on NO enhancement would be in addition to any antioxidant benefits of the compounds. The aims of our study were to determine whether P has EDR activity, to characterize any such activity, especially the ability to counteract vasoconstrictions induced by sympathomimetic agents, and to obtain information regarding the identity of the compounds responsible for the EDR effect.


Aortic ring preparation and testing for EDR

The procedure previously described (15) was used. In brief, male Sprague-Dawley rats, 200-250 g, were killed with an overdose of sodium pentobarbital (100 mg/kg, i.p.), bled, and the thoracic aorta excised, cleaned, and rings (3-4 mm in length) were cut, taking care not to disturb the endothelium. In some instances, the endothelium was deliberately removed by gently rubbing the lumen with a curved forceps. The rings were then suspended in tissue baths containing a physiologic salt solution (PSS) with the following composition (in mM): 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 MgSO4, 1.2 NaH2PO4, 0.026 EDTA, 1.5 CaCl2, and 11 glucose. The buffer was bubbled continuously with O2/CO2 (95%:5%), and maintained at 37°C. Mechanical activity was recorded on a Grass polygraph. After equilibration for ≥1 h under 1.5 g of tension, tissues were contracted submaximally with epinephrine (E), norepinephrine (NE), or phenylephrine (PE). (Our early experiments used PE. We then learned that Pycnogenol blocks E-induced platelet aggregation and became interested to see whether we could observe the relaxing effects of P by using all these sympathomimetics as contractile agents.) Then acetylcholine (ACh, 3 μM), a known EDR-active compound, was added to the bath to test for intactness of the endothelium. Rings were washed with PSS 3 times over the next 1 h before the next sequence. To test for EDR activity by P, rings were contracted with E, NE, or PE, and then the supplement was added in increasing concentrations. In some experiments, the effects of N-methyl-L-arginine (NMA) and superoxide dismutase (SOD) were tested both by pretreating rings with these agents and by addition of each during the response sequence, as described in the figure legends.

Inhibition of E- and NE-induced contractions was accomplished by preincubating rings with a particular concentration of P (1, 3, or 10 μg/ml) for 10 min, followed by performance of concentration-effect curves for the catecholamines. In separate experiments, the three Sephadex LH-20 (Sigma Chemical Co., St. Louis, MO, U.S.A.) fractions of P were tested for EDR activity by using PE-contracted rings.

Fractionation of Pycnogenol

P (350 mg) dissolved in 5 ml ethanol was placed on top of a column of 26 g Sephadex LH-20 in ethanol in a glass column, inner diameter 20 mm, with porous glass filter and stopcock. The first fraction, containing phenolcarbonic acids in their glucose derivatives, vanillin, catechin, and taxifolin, was eluted with 240 ml ethanol. Fractions were monitored by thin-layer chromatography (TLC). The second fraction, containing dimers, trimers, and tetramers consisting of catechin and epicatechin units in different proportions, was eluted with 1,000 ml ethanol. Oligomeric procyanidins from tetramers up to heptamers were eluted with 250 ml of a mixture of ethanol/acetone.


Pycnogenol was a gift from MW International Inc. (Hillside, NJ, U.S.A.). N-methyl-L-arginine, epinephrine, norepinephrine, phenylephrine, acetylcholine, and Sephadex LH-20 were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Other chemicals were obtained from Fisher Scientific, Pittsburgh, PA, U.S.A.


Data are presented either as representative experiments (which were repeated at least 2 other times), or as mean ± SEM for n (number of rats). Comparisons between concentration-effect curves were made by using a one-way analysis of variance followed by Dunnett's multiple comparison test. Maximum responses were compared by using a two-tailed unpaired Student's t test. A value of p < 0.05 was considered statistically significant.


Addition of Pycnogenol to precontracted intact rat aortic rings in tissue baths resulted in relaxations that were enhanced with increasing concentration of the supplement (Fig. 1). Rings in which the endothelium had previously been rubbed off did not relax. The NOS inhibitor NMA reversed the relaxation to a contraction equal to, or greater than, the original epinephrine-induced tone. The normal NOS substrate, L-arginine, by competing with NMA, rerelaxed the ring, demonstrating that the original P-induced relaxation was, indeed, caused by enhanced levels of NO in the tissue. Pretreatment of rings with the NOS inhibitor was also shown to prevent the endothelium-dependent P-induced and ACh-induced relaxations, as shown in Fig. 2, but the inhibitor did not affect relaxation caused by nitroprusside (NP), an endothelium-independent vasodilator.

FIG. 1:
Effect of cumulative increases of Pycnogenol (Pyc) on epinephrine (E)-contracted endothelium-intact (+ENDO) and endothelium-denuded (No ENDO) rat aortic rings. Relaxation of intact ring is reversed by N-methyl-L-arginine (NMA), which, in turn, is reversed by L-arginine (L-Arg). In this particular ring, basal (or E-stimulated) increase in nitric oxide (NO) apparently blunted the initial E-induced contraction, because the contraction after NMA was greater than the original contraction. Finally, the endothelium-independent relaxing agent nitroprusside (NP) is shown completely to relax both intact and denuded rings.
FIG. 2:
Effect of nitric oxide synthase (NOS) inhibitor pretreatment on P-induced relaxations in endothelium-intact rat aortic rings. Preexposure of aortic ring to N-methyl-L-arginine (NMA, bottom tracing) blocked relaxations induced by P and by the known endothelium-dependent relaxing agent, acetylcholine (ACh). Nitroprusside (NP)-induced relaxations (endothelium-independent) were not inhibited by the NOS inhibitor.

To determine whether the known effect of P as a superoxide scavenger is involved in the mechanism of EDR, SOD (10 and 100 U/ml) was first added to intact, precontracted rings (Fig. 3A). The 10 U/ml concentration caused a slight relaxation, which was not increased substantially by increasing the SOD concentration to 100 U/ml. The presence of SOD did not alter the subsequent relaxation response to P. Pretreatment of rings with SOD before contraction likewise did not significantly alter P-induced vasorelaxations (Fig. 3B). These results indicate that scavenging of superoxide anions does not play a major role in P-induced EDR.

FIG. 3:
Effect of superoxide dismutase (SOD) on Pycnogenol (P)-induced relaxation. A: Superoxide dismutase (SOD; 10 U/ml, bottom tracing) produced some relaxation, but further increasing the concentration to 100 U/ml produced little further relaxation. P caused the ring to relax to near maximum, similar to the control relaxation (top tracing). Similar results were obtained in two other experiments. B: Pretreatment of rat aortic rings with SOD (50 U/ml) did not significantly alter the concentration-effect curve to P. This figure represents means ± SEM (n = four rats).

Figure 4 shows concentration-dependent relaxations induced by the supplement in rat aortic rings contracted with E (Fig. 4A) and NE (Fig. 4B). The median effective concentrations (EC50s) of Pycnogenol for relaxation of E- and NE-induced tone were similar (2.73 ± 1.16 μg/ml and 3.54 μg/ml, respectively), and maximal relaxations produced by the supplement were greater against NE (−93.0 ± 2.5%) than against E contractions (−78.3 ± 3.0%).

FIG. 4:
Relaxation of epinephrine-(E) (A) and norepinephrine (NE) (B) -induced contractions of intact (+Endo) and deendothelialized (De-endo) rat aortic rings by cumulative increases in Pycnogenol (P) concentration. E and NE concentrations were 0.3 μM. Relaxation data are expressed as percentage of maximal relaxation to 3 μM acetylcholine (ACh), added at the end of the experiment. Values are expressed as mean ± SEM (n = four rats).

The effect of preincubating aortic rings in the presence of P on subsequent contractions brought about by the catecholamines is illustrated in Fig. 5A and B and Table 1. Pycnogenol produced concentration-dependent inhibitions of both E and NE cumulative contractile responses. EC50s and maximal responses to the catecholamines were shifted to the right and downward, respectively, suggesting a physiological antagonism. The 3- and 10-μg/ml concentrations of P significantly shifted the curves in both E and NE experiments. With regard to maximal contractile responses, the 3- and 10-μg/ml concentrations of the P significantly inhibited E-induced contractions, whereas all concentrations of the supplement significantly decreased maximal contractions induced by NE (Table 1).

FIG. 5:
Inhibition of epinephrine (A) and norepinephrine (B) concentration-contractile effect curves by various concentrations of Pycnogenol (P). Each aortic ring was exposed to a different concentration of the supplement for 10 min before concentration-effect sequences to the catecholamines. Contractile data are expressed as percentage of a maximal contraction of each ring induced by 1 μM catecholamine, obtained 30 min before P addition (not shown). Values are expressed as mean ± SEM (n = six rats).
Inhibition of epinephrine and norepinephrine concentration-effect curves by different concentrations of Pycnogenol

The three fractions obtained by Sephadex LH-20 chromatography of P were tested for EDR activity by using PE-contracted aortic rings. Figure 6 shows that fraction 1 exhibited no relaxing activity in concentrations ≤150 μg/ml, and that fraction 2 relaxed the vessel ring, but only at concentrations of ≥30 μg/ml. On the other hand, fraction 3 potently and almost maximally relaxed (91.5 ± 3.1%) endothelium-intact aortic rings, with an EC50 of 2.18 ± 0.49 μg/ml in four experiments. Again, no relaxation was seen in deendothelialized rings.

FIG. 6:
Effect of cumulative increases of three Sephadex LH-20 fractions of Pycnogenol on phenylephrine (PE)-induced contraction in endothelium-intact (top three tracings) and deendothelialized rat aortic rings (bottom). Acetylcholine (ACh) was added at the end to determine extent of endothelium present in each ring.


Results of this study clearly demonstrate that Pycnogenol can induce endothelium-dependent vasorelaxation in vitro by a mechanism involving increased levels of NO: (a) The relaxations are not seen in deendothelialized vascular rings; (b) The relaxations are converted back to the contracted state (or prevented) by inhibition of NO synthase (20); and (c) This inhibitory effect is, in turn, reversed by the normal substrate, L-arginine (Figs. 1 and 2). These results, therefore, confirm that the P-induced relaxation involves increased NO levels produced by endothelial NOS.

A logical question arises: Is the increase in NO level a consequence of increased NO synthesis, or is it a result of protection of already synthesized NO, caused by antioxidant effects of phenolic components of P? The antioxidant effects of P are well known. It has been shown to exert antioxidant activity in a variety of systems. It inactivates the superoxide radical (2-5) and the hydroxyl radical (2,5,6) and inhibits singlet oxygen formation (6). Additionally, P induces intracellular synthesis of antioxidative enzymes such as SOD and catalase and increases the intracellular glutathione content (7). This combined extra- and intracellular antioxidative action of the supplement is complemented by its inhibitory effect on the respiratory burst of macrophages (21), thereby reducing the amount of cytotoxic oxygen radicals. Conceivably, such antioxidant activity could account for increased levels of NO in blood vessel walls, because superoxide can inactivate NO (22). However, SOD did not significantly alter P-induced relaxations (Fig. 3), indicating that the EDR is not a result of superoxide scavenging by P. Furthermore, Andriambeloson et al. (23) recently showed that wine phenolics, many of which are similar to those in P, induce EDR by increasing synthesis of NO. Thus it appears that increased NO synthesis, rather than an antioxidant action, accounts for most of the increase in NO levels produced by the EDR-active compounds in P, suggesting that these two separate mechanisms may contribute to the beneficial effects of this product on the cardiovascular system.

The finding that P can counteract the contractile effects of catecholamines (Fig. 5, Table 1) suggests that this nutritional supplement could be of benefit in checking the unwanted consequences of increased levels of such hormones, which can occur in response to a wide variety of mental and physical forms of stress. The value of this would be to maintain a patent vessel, even in the presence of increased hormone secretion, and in vessels already narrowed by atherosclerosis. Another important function of NO is inhibition of platelet aggregation. Oral intake of P is able to counteract smoking-induced platelet aggregation (1). Because increased platelet reactivity may be attributable to nicotine-enhanced epinephrine output in smokers (24), and because P has been shown to inhibit epinephrine-induced platelet aggregation in vitro (25), it follows that the mechanism of this effect could be by stimulation of NO production by this supplement.

Fractionation of P by Sephadex LH-20 liquid chromatography yielded three fractions: fraction 1 contains phenolic acids, catechin, and taxifolin; fraction 2 contains dimeric, trimeric, and a few tetrameric procyanidins; and fraction 3 contains higher molecular weight procyanidins. The compounds in P responsible for EDR were primarily localized in fraction 3 (Fig. 6). This fraction was shown in an earlier study (13) to inhibit ACE to a greater extent than the other fractions. ACE inhibition does not appear to play a role in P-induced EDR, however, because the ACE inhibitor captopril did not appreciably affect relaxations caused by P (unpublished observation).

The question of bioavailability of these procyanidin oligomers is difficult because of problems related to measuring the compounds in plasma. However, as shown by Inokuchi et al. (26), a high-molecular-weight procyanidin fraction derived from Areca catechu L. seeds and administered orally to spontaneously hypertensive rats, produced an antihypertensive response equal to that of the ACE inhibitor, captopril, at equivalent doses. This indicates that these compounds were absorbed orally, although whether absorption was of the intact molecule or of a metabolite formed in the gastrointestinal tract is not known. Further experiments are needed to verify absorption of EDR-active compounds of P and the NO-stimulating activity of these compounds.

In conclusion, Pycnogenol has been shown to cause endothelium-dependent vasorelaxation and to protect against contractile responses to E and NE in intact isolated rat aortic rings. The compounds responsible for these effects appear to be higher molecular weight procyanidins. These effects are a result of increased production of NO by endothelial calls. Because NO exhibits other vasoprotective effects as well (inhibits platelet aggregation and platelet and leukocyte adhesion, decreases lipid oxidation, and possibly reduces vascular smooth-muscle cell proliferation), it is not unreasonable to suggest that the cardiovascular system could be a target of the beneficial effects of Pycnogenol.

Acknowledgment: We thank Horphag Research Ltd. for financial support of this study.


1. Rohdewald P. Pycnogenol. In: Rice-Evans CA, Packer L, eds. Flavonoids in health and disease. New York: Marcel Dekker, 1997:406-20.
2. Sibbel RP. Untersuchungen zu Radikälfangereigenschaften, Analytik und Herstellungsverfahren eines Trockenextraktes aus der Rinde der Meereskiefer Pinus pinaster Ait. [PhD dissertation]. Münster, Germany: Westfälische Wilhelms-Universität, 1996.
3. Blazso G, Gabor M, Sibbel R, Rohdewald P. Antiinflammatory and superoxide radical scavenging activities of a procyanidins containing extract from the bark of Pinus pinaster Sol. and its fractions. Pharm Pharmacol Lett 1994;3:217-20.
4. Gouchang Z. Ultraviolet radiation-induced oxidative stress in cultured human skin fibroblasts and antioxidant protection. Biol Res Rep Univ Jyväskylä 1993;33:1-86.
5. Noda Y, Anzai K, Mori A, Kohno M, Shinmei M, Packer L. Hydroxyl and superoxide radical scavenging activities of natural source antioxidants using the computerized JES FR30 ESR spectrometer system. Biochem Mol Biol Int 1997;42:35-44.
6. Elstner EF, Kleber E. Radical scavenger properties of leucocanidine. In: Das NP, ed. Flavonoids in biology and medicine III: current issues in flavonoid research. Singapore: National University of Singapore Press, 1990:227-35.
7. Wei Z, Peng Q, Lau BHS. Pycnogenol enhances endothelial cell antioxidant defenses. Redox Rep 1997;3:219-24.
8. Rong Y, Li L, Shah V, Lau BHS. Pycnogenol protects vascular endothelial cells from t-butyl hydroperoxide induced oxidant injury. Biotech Ther 1995;5:117-26.
9. Kuttan R, Donnely PV, Di Ferrante N. Collagen treated with (+)-catechin becomes resistant to the action of mammalian collagenase. Experientia 1981;37:221.
10. Tixier JM, Godeau G, Robert AM, Hornebeck W. Evidence by in vivo and in vitro studies that binding of Pycnogenols to elastin affects its rate of degradation by elastases. Biochem Pharmacol 1984;24:3933-9.
11. Gabor M, Enge E, Sonkodi S. Die Kapillarwandresistenz und ihre Beeinflussung durch wasserlosliche Flavonderivate bei spontan hypertonischen Ratten. Phlebologie 1993;22:67-9.
12. Schmidtke I, Schoop W. Das hydrostatische ödem und seine medikamentöse Beeinflussung. Swiss Med 1984;6:67-9.
13. Blazso G, Gaspar R, Gabor M, Rüve H-J, Rohdewald P. ACE inhibition and hypotensive effect of a procyanidins containing extract from the bark of Pinus pinaster Sol. Pharm Pharmacol Lett 1996;6:8-11.
14. Fitzpatrick DF, Hirschfield SL, Coffey RG. Endothelium-dependent vasorelaxing activity of wine and other grape products. Am J Physiol 1993;265:H774-8.
15. Fitzpatrick DF, Hirschfield SL, Ricci T, Jantzen P, Coffey RG. Endothelium-dependent vasorelaxation caused by various plant extracts. J Cardiovasc Pharmacol 1995;26:90-5.
16. Yao S, Ober JC, Krishnaswami A, et al. Endogenous nitric oxide protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured arteries. Circulation 1992;86:1302-9.
17. Provost P, Lam J, Lacoste L, Merhi Y, Waters D. Endothelium-derived nitric oxide attenuates neutrophil adhesion to endothelium under arterial flow conditions. Arterioscler Thromb 1994;14:331-5.
18. Hogg N, Kalyanaraman B, Joseph J, Stuck A, Parthansarathy S. Inhibition of low-density lipoprotein oxidation by nitric oxide. FEBS Lett 1993;334:170-4.
19. Scott-Burden T, Vanhoutte P. The endothelium as a regulator of vascular smooth muscle proliferation. Circulation 1993;87:V51-5.
20. Radomski MW. Nitric oxide: biological mediator, modulator and effector. Ann Med 1995;27:321-9.
21. Nelson AB, Lau BHS, Nagatoshi I, Rong Y. Pycnogenol inhibits macrophage oxidative burst, lipoprotein oxidation and hydroxyl radical-induced DNA damage. Drug Dev Ind Pharm 1998;24:1-6.
22. Rubanyi G, Ho E, Cantor E, Lumma W, Botelho L. Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes. Biochem Biophys Res Commun 1991;181:1392-7.
23. Andriambeloson E, Kleschyov AL, Muller B, Beretz A, Stoclet JC, Andriantsitohaina R. Nitric oxide production and endothelium-dependent vasorelaxation induced by wine polyphenols in rat aorta. Br J Pharmacol 1997;120:1053-8.
24. Folts JD, Bonebrake FC. The effects of cigarette smoke and nicotine on platelet thrombus formation in stenosed dog coronary arteries: inhibition with phentolamine. Circulation 1982;65:465-70.
25. Rüve H-J. Identifizierung und Quantifizierung phenolischer Inhaltsstoffe sowie pharmakologisch-biochemische Untersuchungen eines Extraktes aus der Rinde der Meereskiefer Pinus pinaster Ait. [PhD dissertation]. Münster, Germany, Westfalische Wilhelms-Universität, 1996.
26. Inokuchi J-I, Okabe H, Yamauchi T, Nagamatsu A, Nonaka G-I, Nishioka I. Antihypertensive substance in seeds of Areca catechu L. Life Sci 1986;38:1375-82.

Blood vessel; Endothelium; Nitric oxide; Nutritional supplement; Procyanidins; Pycnogenol

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