Nutrition Research 21 (2001) 12511260

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A randomized, double-blind, placebo-controlled, prospective, 16 week crossover study to determine the role of Pycnogenol in modifying blood pressure in mildly hypertensive patients
Saeed Hosseinia, Jeongmin Leea, Ramon T. Sepulvedaa, Peter Rohdewaldc, Ronald R. Watsona,b,*
a

College of Public Health, Health Sciences Center, University of Arizona, Tucson, AZ 85724, USA b School of Medicine, Health Sciences Center, University of Arizona, Tucson, AZ 85724, USA c Westfalische Wilhelms-Universitat, Munster, Germany Received 8 February 2001; accepted 26 June 2001

Abstract Background: Pycnogenol is a bark extract from the French maritime pine (Pinus pinaster) consisting of a mixture of bioavonoids. Bioavonoids are also components of a wide variety of edible plants, fruits, and vegetables and act as antioxidants and iron chelators. Pycnogenol is a mixture of watersoluble procyanidins, catechin, taxifolin, and phenolcarbonic acids. It has been used as a dietary supplement for years. Hypertension, or a blood pressure higher than 140/90 mm Hg, is the most common risk factor for cardiovascular and cerebrovascular morbidity and mortality. Purpose: The aim of the study was to test a possible protective effect of oral Pycnogenol, administrated for eight weeks to non-smoking, mildly hypertensive patients. Methods: Pycnogenol, 200 mg/day, or placebo was provided to eleven subjects (seven men and four women) with systolic blood pressure of 140 159 mm Hg in a double blind, randomized, cross-over study and/or diastolic blood pressure of 90 99 mm Hg for eight weeks. The subjects blood pressure was taken during supplementation, and the serum level of thromboxane was measured. Results: A signicant decrease in the systolic blood pressure was observed during Pycnogenol supplementation. However, Pycnogenols lowering of diastolic blood pressure did not reach statistical signicance when compared to placebo. Serum thromboxane concentration was signicantly (p 0.05) decreased during Pycnogenol supplementation.

* Corresponding author. Tel.: 1-520-626-2850; fax: 1-520-626-6093. E-mail address: Rwatson@U.Arizona.edu (R.R. Watson).
0271-5317/01/$ see front matter 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 3 4 2 - 6

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Conclusion: Supplementation of Pycnogenol is effective in decreasing systolic blood pressure in mildly hypertensive patients. 2001 Elsevier Science Inc. All rights reserved.
Keywords: Pine bark; Bioavonoids; Thromboxane; Blood pressure

1. Introduction Extracts from pine tree bark have been used in traditional medicine. Pycnogenol is a proprietary bark extract of the French maritime pine tree (Pinus pinaster). The extract is prepared by a standardized procedure that includes an extraction of fresh pine bark with ethanol and water. The resulting product is a mixture of avonoids as monomers: catechin and taxifolin, and condensed polymers (85%), up to dodeoameric avanols, designated as procyanidins. Additionally, the extract contains phenolic acids as gallic, caffeeic, and ferulic acid as minor constituents. Pycnogenol also contains glycosylation products, i.e., sugar derivatives of phenolcarbonic acids and taxifolin [21]. These molecules have antioxidant properties [21] and may act as modulators of metabolic enzymes and other cellular functions. Pycnogenol has prevented pathologic symptoms such as chronic inammation and increased platelet aggregation, a risk factor for cardiovascular diseases [21]. Pycnogenol prolonged the ascorbate radical lifetime to the greatest extent by regenerating ascorbic acid [18]. In vitro Pycnogenol inhibits LDL peroxidation, lipid peroxidation in phospholipid liposomes [21], and lipid peroxidation caused by t-butylhydroperoxide. Pycnogenol has an efcient antioxidant activity [18]. Inhibition of vasoconstriction caused by adrenalin is produced by stimulation of nitric oxide synthesis under Pycnogenol [6]. Hypertension, or a blood pressure higher than 140/90 mm Hg, is the most common risk factor for cardiovascular and cerebrovascular morbidity and mortality [14]. In the United States, high blood pressure is responsible for 40,000 deaths annually in the U.S., while being the most modiable risk factor for stroke. Hypertension affects about one in four adults, or almost 50 million people, in the United States [2]. The Framingham Study showed that as people aged from thirty to sixty-ve years, their blood pressure increased an average 20 mm Hg systolic and 10 mm Hg in diastolic pressure with systolic blood pressure continuing to rise up to age ninety. While higher blood pressure increases the likelihood of a cardiovascular event, hypertension is not often well controlled, and too few patients are adequately treated [5]. Epidemiologic studies predict that reduction of the systemic blood pressure by the amount usually achieved in major clinical trials could reduce cerebrovascular events by 42% and cardiac events by 24% [8]. Although our understanding of the pathophysiology of elevated arterial pressure has increased, in most cases, the etiology, and thus potentially the means to be employed to prevent or cure it, are still largely unknown. In consequence, hypertension is frequently treated nonspecically, resulting in a large number of minor side effects and a relatively high rate of non or inadequate treatment [9]. In hypertensive patients, the release of vasoconstrictor peroxides derived from the activity of cyclo-oxygenase in the endothelium and in the vascular smooth muscle is important. Excess free radicals released by the dysfunctional endothelium also stimulate the synthesis of contracting agents. The therapeutic benet of some anti-hypertensive drugs, such as

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calcium antagonists and angiotensin-converting enzyme inhibitors, could be in part due to their inhibition of the production of free radicals. Pycnogenol is a strong antioxidant [21], inhibits ACE [4], and produces anti-hypertensive effects in rats [18,21]. The inhibition of thromboxane has been shown previously [3]. Blood Pressure of 140 159/90 99 mm Hg should have blood pressure continued within 2 months, and if no more than 1 other risk factor is present, pharmacological therapy may be delayed for up to 6 months [9]. Therefore Pycnogenols actions on patients with mild hypertension, not routinely treated with standard drugs, were studied.

2. Materials and methods 2.1. Subjects Eleven patients (four women and seven men) with an average age of fty years (mean SD 50.3 9.3 years), systolic blood pressure of 140 159 mm Hg, and/or diastolic blood pressure of 90 99 mm Hg were investigated. The exclusion criteria included those taking antihypertensive medication; taking non-steroidal, anti-inammatory drugs, including aspirin, use of tobacco, or taking any vitamin supplements other than a single, daily multivitamin tablet. These items were assessed using the Arizona Cancer Center Food Frequency Questionnaire. The subjects selected were stage I hypertensive [9]. Most subjects asked for participation were using medication and had more severe hypertension. Thus, forty were screened to select the eleven participants, none of which dropped out during the study. To eliminate the effects of bioavonoids in wine, the eleven were also asked to exclude wine from their diets for the duration of the study. The University of Arizonas Institutional Review Board approved the study, and informed written consent was obtained prior to participation. 2.2. Procedure Study was performed as a randomized, double-blind, placebo controlled cross-over study. At the rst visit, non-smoking, mildly hypertensive patients were recruited and screened for the study. Each patient signed a consent form, had a medical history and physical examination performed including height, weight, blood pressure, ECG and heart rate, had blood collected for laboratory safety and thromboxane assays, and lled out the Arizona Cancer Center Food Frequency Questionnaire. Blood pressure was measured three times in sitting patients after a fteen-minute rest. The second visit was one week after screening. At this time, blood pressure and heart rate were measured again and patients began taking placebo pills for a one-week run-in period. If any of the subjects had not taken all of the placebo pills, they would have been excluded from the study. However, all complied fully during the rst week The third visit occurred one week after the second visit, and again, serum was collected for analysis, blood pressure and heart rate were assessed. The data for week 1 and week 0 were combined as baseline values. Each patient was then randomized using a statistical

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formula prepared by the Biostatistics department to either four pills of Pycnogenol (200 mg) or placebo daily for eight weeks. After seven weeks on double-blind drug or placebo pill consumption, each patient returned to the clinic for another blood pressure and heart rate assessment, and blood collection. Eight weeks after the initiation of the study, each patient again had blood pressure and heart rate measured and recorded and an ECG performed. The values for weeks seven and eight were combined, and the study drug was collected and exchanged. No washout period was used, as we have previously shown that Pycnogenols effects on platelet aggregation in smokers [3] disappeared three days after the cessation of its use. Each patient was questioned about changes in concomitant medications and adverse events. Seven weeks after the second study drug (Pycnogenol or placebo) was provided, each patient returned to the clinic. Blood pressure and heart rate were recorded. In the nal visit at week sixteen, blood pressure and heart rate were measured and ECG performed. The values of weeks fteen and sixteen were combined. Blood was collected again for safety labs, and thromboxane assay at both visits. Each patient was questioned about changes in concomitant medications and adverse events with none reported. The Pycnogenol pills and placebo were obtained from the Cognis Corporation (LaGrange, Illinois). Compliance was initially assessed during the run-in period of one week during which all of the patients took placebo pills. During each eight-week period of treatment, all unused pills were collected. All of the subjects took at least ninety percent of the pills provided in a blinded fashion. A registered nurse took each subjects blood pressure, according to American Heart Association guidelines [19]. Subjects were encouraged to take their pills in the morning. All tests were two to four hours after the last consumption of pills. The composition of the placebo was: dicalcium phosphate, wheat our, stearic acid, croscarmellose sodium, silicon dioxide, magnesium stearate, FDC Yellow #6 Lake, and cellulose coating. 2.3. Determination of serum thromboxane After blood was drawn and centrifuged, the serum was separated. Then 0.2 ml of methanol was added to 1 ml of serum and vortexed. After centrifuging for ten minutes at 1200 rpm, the serum was poured into preconditioned C18 Sep-Pak columns (Waters Corporation) with a ow rate of 1 ml per minute. Columns were washed with 15% methanol in water, petrol ether and nally thromboxane B2 was eluted by methyl formate, evaporated with nitrogen gas. After dissolving the residue in 1 ml diluted extraction buffer, 50 l samples were assayed in triplicate by Neogen Corporation (Lexington, KY) thromboxane B2 ELISA kit. 2.4. Statistics The effects of Pycnogenol treatment on hypertension compared to placebo, and were assessed using the t-Test: Paired Two Sample for means placebo and baseline were not signicantly different. Data are presented, as mean SEM. Test statistics were considered signicant at the p 0.05 level.

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Fig. 1. Effect of Pycnogenol, 200 mg/day or placebo, for 8 weeks on systolic blood pressure in mildly hypertensive patients. Subjects received placebo or Pycnogenol (200 mg)/day for eight weeks in a double-blind fashion. The data are presented as mean SE with p 0.05 compared to placebo was signicant. After eight weeks subjects were assigned to the other arm, placebo or Pycnogenol for eight more weeks. As Pycnogenol was previously shown to loose activity in vivo three days after cessation of treatment, no washout period was employed. In part A of this gure, all eleven subjects data are analyzed. In part B of this gure, those four subjects with the highest blood pressure were separately analyzed.

3. Results Pycnogenol or placebo pills were given in a randomized, double-blind, cross-over fashion for eight weeks to non-smoking, mildly hypertensive patients (7 females and 4 males, all Caucasians). They had an average systolic blood pressure of 140 (mean SEM 139.9 3.3). Pycnogenol treatment decreased systolic blood pressure signicantly (p 0.05) to 133 (mean SEM 132.7 4.18) as compared to placebo supplementation in the same individuals for 8 weeks (Fig. 1A). Our data also demonstrated that Pycnogenol supplementation did decrease diastolic blood pressure in hypertensive patients with an average diastolic blood pressure of 94 (mean SEM 93.8 1.23), however, the difference did not reach statistical signicance to 92 (mean SEM 92.0 1.7) (Fig. 2A). Review of the four subjects whose baseline systolic blood pressure was greater than 140 mm Hg showed that Pycnogenol supplementation was effective at decreasing systolic blood pressure in these subjects with higher pressure, while it was less effective in those who had a systolic blood

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Fig. 2. Effect of Pycnogenol, 200 mg/day or placebo, for 8 weeks on diastolic blood pressure in hypertensive patients. Subjects received placebo or Pycnogenol (200 mg)/day for eight weeks in a double-blind fashion. Data are presented as mean SE with p 0.05 compared to placebo was signicant. After eight weeks subjects were assigned to the other arm, placebo or Pycnogenol for eight more weeks. In part A of this gure, all eleven subjects data are analyzed. In part B of this gure, those four subjects with the highest blood pressure were analyzed.

pressure that was less than 140 mm Hg (Fig. 1B). We did not nd similar effects for those subjects who had higher diastolic blood pressure (Fig. 2B). We measured the serum thromboxane B2 levels prior to and post supplementation. Serum thromboxane B2 levels showed a signicant (p 0.05) decrease after Pycnogenol supplementation, but not after placebo (Fig. 3).

4. Discussion Our data show a signicant decrease in systolic blood pressure and serum thromboxane B2 levels after eight weeks of Pycnogenol supplementation in non-smoking, mildly hypertensive patients who did not require standard pharmacological drug therapy. Pycnogenols antioxidant effects could protect cell membranes against oxidative stress, which could ultimately promote the production and release of arachidonic acid mediators including thromboxane. By protecting cell membranes, Pycnogenol should preserve the

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Fig. 3. Effect of Pycnogenol, 200 mg/day or placebo, for 8 weeks on serum thromboxane level in mildly hypertensive patients. Subjects received four pills of placebo or Pycnogenol (200 mg)/day for eight weeks. Data are presented as mean SE with p 0.05 compared to placebo was signicant. After eight weeks subjects were assigned to the other arm, placebo or Pycnogenol for eight more weeks. In this gure, all eleven subjects data are analyzed.

activities of Na -K ATPase, Na -Ca 2 exchange, and Ca 2 binding, known to contribute to the Ca 2 movements across the sarcolemmal membrane. These enzymes are susceptible to alteration by a Reactive Oxygen Species (ROS) [10 12]. Mitochondria are important cellular sources of oxygen radicals, however at the same time prone to damage. ROS reduce the ability of mitochondria to synthesize ATP [17] as well as utilize substrates. ROS generation also depressed mitochondrial cytochrome oxidase and glucose-6-phosphatase, causing a decrease in ATP [10]. Population studies reveal that hypertensive patients generally have a lower intake of ascorbic acid and possibly other antioxidants. Inadequate antioxidant intake may lead to defective vasodilatation and increased blood pressure due to destruction of endothelium-dependent relaxing factors by free radicals [13]. Pycnogenol stimulates the production of the endothelium-dependent relaxing factor, nitric oxide, thus counteracting the effect of vasoconstriction caused by stress hormones [6]. We have also shown previously that Pycnogenol decreased platelet aggregation [3,20,22] and serum thromboxane B2 levels in smokers after smoking [3]. Here we conrm that observation in non-smokers. We showed that Pycnogenol decreased thromboxane B2 levels in mildly hypertensive patients as the pine bark extract modulated their systolic blood pressure. Thromboxane A2 is a vasoconstrictor that shifts the vascular musculature to vasoconstrictor state, thereby possibly augmenting the effects of other vasoconstrictors. Although the thromboxane A2 synthesis occurs mainly in platelets, thromboxane A2 can also be generated by the cells in the blood vessel wall and by intestinal, pulmonary, and renal tissues [1,15].

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Thromboxane A2 alters GFR by potentiation of the tubuloglomerular feedback mechanism, thereby contributing to salt retention and vasoconstriction [23]. In angiotensin II-salt-induced hypertension, increased thromboxane A2 production from vascular and renal tissues accompanies the elevation of blood pressure [16]. The increased formation of thromboxane A2 would result in renal and extrarenal vasoconstriction, and expansion of the blood volume through a reduction in water and sodium excretion and increased blood pressure. However, the action of thromboxane A2 in hypertension is expressed not only via altered kidney function. Thromboxane A2 could potentiate the growth of vascular smooth muscle cells. Hypertensive patients generally show hyperplasia or hypertrophy in various tissues of the cardiovascular system. Thickening of the vascular wall increases wall stiffness and narrows the vascular lumen, thereby elevating vascular resistance and decreasing compliance, which contribute to elevation and maintenance of blood pressure. The high levels of endogenous thromboxane A2 observed in hypertension could stimulate the rapid growth of vascular smooth muscle cells in vivo and produce hypertrophy in vascular tissues. Thus, enhanced thromboxane A2 generation could participate in the development and maintenance of hypertension through action on the growth of vascular smooth muscle cells as well as through action on the kidney [7]. In humans and animals with normal blood pressure, prostaglandin I2 and thromboxane A2 are involved in the homeostatic regulation of blood pressure via effects on renal hemodynamics and the vascular system. Metabolic abnormalities of prostaglandin I2 and thromboxane A2 exist in hypertensive humans and animals. The role of thromboxane A2 is expressed partly by thromboxane A2s vasoconstricting properties and partly by thromboxane A2s stimulating effect on the rapid growth of vascular smooth muscle cells. Prostacyclin and thromboxane A2, products of separate branches of the arachidonic acid cascade, can have opposing effects on kidney function and on the vascular musculature. Prostacyclin acts as a vasodilator, while thromboxane A2 acts as a vasoconstrictor. The balance between these two compounds appears to contribute to the homeostatic regulation of normal blood pressure. In the hypertensive situation, this balance is disrupted and, at least in animal models, there is excessive production of both [21]. The increase in prostacyclin formation may be a reaction to the elevated blood pressure, possibly due to mechanical stimulation of the vascular smooth muscle cells in the blood vessel wall. However, the thromboxane A2 increase may be more directly involved in the development and maintenance of hypertension. Not only is thromboxane A2 a vasoconstrictor, but it can also stimulate the growth and proliferation of vascular smooth muscle cells. This may partially account for the vascular hypertrophy seen in hypertension. While there is no ideal anti-hypertensive drug, our data suggest a role for Pycnogenol with its high antioxidant activity, ACE inhibitor, increased no production and inhibitor of Thromboxane synthesis. Since hypertension is a relatively symptom-free disease, initially, it is important that any anti-hypertensive agent develops be efcacious and safe, with minimal side effects [18,21]. The subjects in our study, with mild hypertension, must not necessarily be treated but are at risk of progression. Their hypertension beneted from the natural bark extract, Pycnogenol. Further studies with Pycnogenol in patients with higher blood pressures are warranted especially as they are routinely treated currently.

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Acknowledgments We greatly appreciate the assistance of Valerie Butler for study coordination and assessment, and Timothy Fagan, M.D. for clinical evaluation. This research was supported by a grant from Horphag, Inc.

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