Pycnogenol does not impact the antioxidant or vitamin C status of healthy young adults

Methods 

Results 

The characteristics of the subjects are shown in Table 1.There was no change in subjects' body weights from the start of the study to the end of the study, indicating that subjects maintained energy balance throughout the study. Subjects maintained a consistent dietary pattern for both the baseline and PYC phase of the study (Table 2).There was no change in subjects' dietary intakes between the baseline and PYC phases of the study. There was no apparent increase in fasting vitamin C concentration of plasma 2 weeks after supplementing the diet with PYC (Table 3).There was a significant (P<.001) association between dietary intake of vitamin C and the concentration of vitamin C in plasma (r=.60). There was also no apparent increase in the total antioxidant capacity of serum 2 weeks after supplementing the diet with PYC (Table 3). One hour after the ingestion of a load dose of placebo or 200 mg of PYC, the antioxidant capacity of serum increased by an average of 15% to 19%. The total serum antioxidant capacity increased only 3% more than the apparent meal-induced increase in total serum antioxidant capacity 1 hour after consuming 200 mg of PYC. This increase did not reach statistical significance. Results from univariate ANOVA found that gender had no effect on the change in baseline or PYC values for any of the variables listed in Table 2. The total antioxidant capacity of the PYC used in the present study was also measured using the ORAC assay and was found to contain 3003 TE/g, giving a daily ORAC intake of 600 TE.

Table 1. Characteristics of subjects

		Male (n=12)	Female (n=15)
		←mean±SD→
	Age (y)	27±7	24±5
	Height (cm)	178.6±3.6	166.6±6.6
	Initial weight (kg)	80.5±11.4	61.4±10.0
	Final weight (kg)	80.9±11.8	60.9±9.6
	Initial body mass index	25.3±3.5	22.0±3.0
	Final body mass index	25.7±3.6	21.9±2.8

SD=standard deviation.

Table 2. Average daily dietary intake

		Male (n=12)		Female (n=15)
		Baseline	Pycnogenol	Baseline	Pycnogenol
		←mean±SD→		←mean±SD→
	Energy (kcal)	2,748±479*	2,603±529*	1,738±325	1,797±345
	Protein (% kcal)	16.2±3.4	15.6±2.9	15.6±2.7	14.7±2.0
	Carbohydrate (% kcal)	52.2±5.5	54.6±5.4	57.6±8.0	57.9±5.7
	Fat (% kcal)	26.6±2.9	25.4±2.7	26.6±6.1	26.1±5.5
	Alcohol (g)	7.9±5.7	5.1±5.0	4.6±2.6	3.7±2.5
	Vitamin C (mg)	104±57	116±56	123±51	134±49
	Vitamin E (ATE)	9.4±4.7	11.2±6.9	7.5±1.9	8.5±3.5
	Selenium (μg)	145±68	144±99	117±112	83±16

ATE=alpha-tocopherol equivalents=1 mg. SD=standard deviation. *t test, male>female, P<.05.

Table 3. Fasting and 1-hour plasma/serum values

		Male (n=12)		Female (n=15)
		Baseline	Pycnogenol	Baseline	Pycnogenol
		←mean±SD→		←mean±SD→
	Fasting vitamin C (μM)	48±5	50±7	59±3	64±5
	Fasting ORACa	274±58	261±66*	225±52	204±54*
	1-hour ORAC	293±49	282±55	266±46	248±46
	Change in ORACa,b	19.5±38	20.9±19.4	41.2±30	44.2±36.5
	Percent change in ORACc	8.8±14.3	10.0±11.2	20.8±18.5	25.6±24.2

SD=standard deviation. aORAC results expressed as Trolox equivalents (TE=1 μM). bChange=1-hour value−fasting value. cPercent change=[(1-hour value−fasting value)/fasting value]×100. *PYC<baseline, paired t test, P=.005.

PYC did not significantly increase the level of total phenolics excreted in the urine (Table 4).When measuring antioxidant potential of urine, there was no measurable increase as a result of supplementation.

Table 4. Urinary excretion of total phenolics, FRAP, and ORAC

		Male (n=11)		Female (n=14)
		Baseline	Pycnogenol	Baseline	Pycnogenol
		←mean±SD→		←mean±SD→
	Phenolics, mg/mg creatinine	0.55±0.13	0.54±0.17	0.63±0.26	0.73±0.22
	FRAP,a μmol/mg creatinine	0.51±0.13	0.51±0.27	0.67±0.22	0.75±0.26
	ORAC,a μmol/mg creatinine	3.95±1.29	3.08±0.90	4.94±2.02	4.64±1.42

FRAP=ferric-reducing antioxidant potential. ORAC=oxygen radical absorbance capacity. SD=standard deviation. aFRAP and ORAC results expressed as Trolox equivalents (TE=1 μmol).

Male subjects had significantly greater fasting serum antioxidant capacity than female subjects at baseline and after the intervention (Student t test, P<.05). Because of the gender difference in fasting serum ORAC values, associations between dietary factors and fasting ORAC values were analyzed by gender. To assess total nutrient antioxidant intake for each subject, the average vitamin C, vitamin E, and selenium intake for each subject was expressed as the percentage of the dietary reference intake (DRI) for the subject's age and gender ((9)). An average percent of the DRI for all nutrient antioxidants was then determined. In female subjects, there were weak associations between the fasting ORAC concentration and the percent DRI for vitamin C (r=.30, P=.11), percent DRI for selenium (r=.27, P=.15), and average percent DRI for all nutrient antioxidants (r=.35, P=.06), but not for vitamin E (r=−.04, P=.91). Surprisingly, in male subjects, there were weak negative associations between the fasting ORAC values and the percent DRI for vitamin C (r=−.49, P=.16) and the average percent DRI for all nutrient antioxidants (r=−.39, P=.06), and no associations for selenium (r=−.23, P=.28) and vitamin E (r=−.03, P=.87). Interestingly, there was a significant negative association between fasting ORAC values and alcohol among men (r=−.44, P=.03) but not among women (r=.105, P=.58).

Discussion 

PYC is used throughout Europe as a dietary supplement and is readily available to Americans on the internet ((10)). The claims surrounding the beneficial aspects of PYC are numerous. There is a large body of literature on this supplement; however, the majority of studies published to date are in vitro or cell culture studies. These studies suggest that PYC may enhance immune cell function [11], [12], act to control the inflammation response ((13)), and help protect endothelial cells against injury [14], [15], [16], [17], [18]. Among the few human studies, Virgili and colleagues ((19)) investigated the urinary excretion of a minor constituent of PYC, ferulic acid, after giving subjects doses of 100 to 400 mg. They found that subjects excreted significant but highly variable amounts of ferulic acid, providing evidence that at least part of the phenolic compounds in PYC are absorbed and excreted. Petrassi and colleagues ((20)) reported that PYC, at a dose of 300 mg/day for 2 months, significantly reduced ambulatory venous pressure and patients' perception of leg heaviness and subcutaneous edema in patients with chronic venous insufficiency. Two studies [21], [22] reported that PYC reduces platelet aggregation, but not blood pressure, in smokers 2 hours after receiving doses of 100 to 200 mg. Saliou and colleagues ((23)) found that PYC consumed at a dose of 1.10 mg/kg body weight for 4 weeks followed by a dose of 1.66 mg/kg for an additional 4 weeks reduced skin cell damage in a group of fair-skinned subjects. In a small study with 40 subjects, Spadea and Balestrazzi ((24)) reported that PYC, at a dose of 150 mg/day for 2 months, maintained retinal function and improved visual acuity in patients with diabetes atherosclerosis or other vascular diseases.

The present study explored whether PYC interacts with vitamin C to increase its concentration in the body. Previously, Cossins and colleagues ((25)) studied the effects of various flavonoids and PYC on the ascorbate radical lifetime using electron spin resonance spectroscopy. These investigators theorized that flavonoids could recycle vitamin C from dehydroascorbic acid, the fully oxidized form of vitamin C, by donating two hydrogen atoms and reforming ascorbic acid. Their results showed that PYC was the most powerful compound, prolonging the ascorbate radical lifetime by 200%. Although this research showed a potential interaction between PYC and vitamin C in vitro, the present study failed to detect measurable increases in the fasting levels of plasma vitamin C in a group of healthy young men and women consuming 200 mg of PYC daily for 2 weeks along with their usual diet.

The present study also explored whether PYC has antioxidant potential in vivo. Several research groups have reported that PYC shows antioxidant properties in vitro. PYC seems to be effective against reactive oxygen [26], [27] and nitrogen [28], [29] species and reduces lipid peroxidation ((30)) and protein modification by malondialdehyde ((31)). Despite these findings, the present study failed to detect any measurable increases in fasting serum total antioxidant capacity 2 weeks after supplementation or in the antioxidant capacity 1 hour after consuming a daily dose with a shake, beyond the increase observed with the shake alone. This study is the first to obtain a measure of total antioxidant capacity excreted in the urine. None of the three measures used in the present study noted a significant increase in urinary antioxidant capacity after PYC supplementation. This is also the first study to note a possible gender difference in fasting serum total antioxidant capacity. There were weak associations between dietary antioxidants and serum ORAC values in females but not in males. However, there was a significant negative association with alcohol consumption among males, possibly indicating that alcohol negates any positive benefit of nutrient antioxidants on ORAC values ((32)).

Only a few studies have measured the in vivo fasting antioxidant response after the short-term ingestion (5 days to 28 days) of food or beverages known to be rich in phenolic compounds. Significant increases in the fasting antioxidant response are reported for red wine [33], [34], red grape juice ((35)), or controlled diets high in fruits and vegetables ((36)). However, other studies have reported no change in the fasting antioxidant response after consumption of red wine [32], [37], grape seed extract ((38)), black tea ((39)), controlled diets high in fruits and vegetables ((40)), or a spray-dried extract made from fruits and vegetables ((40)). Although the results of studies examining the impact of the short-term ingestion of phenolic compounds on the fasting antioxidant response remain conflicting, several other studies suggest that phenolic compounds increase the antioxidant capacity of serum or plasma after acute ingestion. The total antioxidant capacity of serum increased and peaked within 30 minutes to 2 hours after ingestion of red wine or whiskey [41], [42], [43], [44], grape seed extract ((38)), red grape juice ((35)), green or black tea [44], [45], semisweet chocolate ((46)), and cranberry juice ((47)). In the present study (and unlike those previously conducted), the acute dose of PYC was administered with a beverage to mimic the consumption of the dietary supplement with a meal. The total serum antioxidant capacity increased only 3% more than the apparent meal-induced increase in total antioxidant capacity 1 hour after ingestion. This meal-induced increase in serum antioxidant capacity has been previously reported [48], [49]. It is not known why this phenomenon occurs, but it may be the result of an increased internal release of antioxidants into the blood caused by oxygen consumption and energy expenditure increases after a meal ((49)).

This study had some limitations. First, our subjects were healthy young adults. We cannot rule out the possibility that this supplement may be of benefit to older adults or individuals with altered antioxidant status because of smoking or disease. Second, there is no single measure that provides a complete picture of antioxidant status, although plasma ORAC measures have proven successful in assessing changes in the order of 10% to 15% in two studies [36], [48]. The ORAC assay, as used in this study, measures changes in the aqueous compartments of the cell and thus may not reflect changes in lipophilic antioxidants. To get a complete picture of antioxidant status, a battery of assays is needed. In a recent compilation by Pryor ((50)), many of these assays are discussed. Third, this study collected only one sample of blood after the ingestion of the daily dose. It may be that ORAC values after the load dose of PYC continue to increase beyond 1 hour after ingestion. Fourth, our sample size was small and dietary intake was not under our direct control.

The antioxidant results of this study are not surprising after comparing the antioxidant potential (ORAC assay) of the daily dose of PYC with common servings of fresh fruits and vegetables. The daily dose of PYC provides only 600 ORAC units (1 unit=1 μM TE), whereas 6 prunes provides 2,885, 2/3 cup of blueberries provides 2,400, and cup of spinach provides 1,260 ((51)). The total observed urinary output of approximately 6,000 units per day suggests that normal intake is in that range. Previously, intake from fruits and vegetables (2-3 servings) has been estimated to be 1,640 units ((36)), and by increasing the servings of fruits and vegetables to 10 per day, ORAC intake was approximately 3,700 units per day. Thus the increase of about 600 units from the PYC in this study is a relatively low fraction of the total potential intake. Increases in ORAC intakes of 3,000 units have produced increases in in vivo antioxidant capacity as indicated by ORAC and FRAP measures [36], [48]. In conclusion, PYC, at a dose of 200 mg per day, does not appear to enhance the in vivo concentration of vitamin C or act as a potent antioxidant in healthy young adults.

Applications 

■Most claims made by the distributors of dietary supplements have not been substantiated by well-controlled human research studies. Dietetic professionals can disseminate to clients and consumers accurate and scientifically verifiable information concerning the validity of claims for dietary supplements. Our study found nothing to support distributors' claims. We suggest that consumers eat fruits and vegetables rich in nutrient and nonnutrient antioxidants ((52)) to improve their vitamin C and antioxidant status, rather than a dietary supplement with questionable health claims.