Effects of Blueberry and Cranberry Juice Consumption on the Plasma Antioxidant Capacity of Healthy Female Volunteers

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Effects of blueberry and cranberry juice consumption on the plasma antioxidant capacity of healthy female volunteers

CB Pedersen1, J Kyle2, A McE Jenkinson2, PT Gardner2 DB McPhail2 and GG Duthie2*

1Technical University of Denmark, 2800-Lyngby, Copenhagen, Denmark; and 2Rowett Research Institute, Aberdeen, AB21 9SB, Scotland, UK.

Objective: To assess whether consumption of 500 ml of blueberry juice or cranberry juice by healthy female subjects increased plasma phenolic content and antioxidant capacity. Design: Latin square arrangement to eliminate ordering effects. After an overnight fast, nine volunteers consumed 500 ml of blueberry juice, cranberry juice or a sucrose solution (control); each volunteer participated on three occasions one week apart, consuming one of the beverages each time. Blood samples were obtained by venipuncture at intervals up to four hours after consumption of the juices. Urine samples were also obtained four hours after consuming the juice. Results: Consumption of cranberry juice resulted in a signi®cant increase in the ability of plasma to reduce potassium nitrosodisulphonate and Fe(III)-2,4,6-Tri(2-pyridyl)-s-triazine, these measures of antioxidant capacity attaining a maximum after 60 ± 120 min. This corresponded to a 30% increase in vitamin C and a small but signi®cant increase in total phenols in plasma. Consumption of blueberry juice had no such effects. Conclusion: The increase in plasma antioxidant capacity following consumption of cranberry juice could mainly be accounted for by an increase in vitamin C rather than phenolics. This also accounted for the lack of an effect of the phenolic-rich but vitamin C-low blueberry juice. Sponsorship: Funded by the Scottish Executive Rural Affairs Department and the Danish Government. Descriptors: vitamin C; phenolics; cranberry; blueberry; antioxidant capacity European Journal of Clinical Nutrition (2000) 54, 405±408

Introduction Many berries are rich in both phenolics and vitamin C and are a relatively unexploited source of antioxidants in Diets rich in fruits and vegetables are believed to protect Northern European populations with habitually low anti- against various diseases common in the western world such oxidant intakes and high rates of heart disease (Heinonen as cardiovascular disease and several forms of cancer. This et al, 1998). Consequently, we have assessed whether bene®cial effect has been ascribed, in part, to high levels of consumption of berry juices by human volunteers increases the antioxidants, vitamin C, vitamin E and carotenoids in plasma antioxidant capacity. The aim was to compare the these foods (Williams, 1995). However they often make short-term response, in terms of plasma antioxidant capa- only a minor contribution to the total antioxidant potential city, to ingestion of juices with different relative and of fruits and vegetables (Robards & Antolovich, 1997; absolute amounts of phenolics and vitamin C. Wang et al, 1996), and attention has therefore turned to the role of phenolics as a source of dietary antioxidants. Many of these compounds have potent hydrogen donating abilities in vitro and contribute to the antioxidant capacity Subjects and methods of teas, wines, and a range of fruit and vegetables (Benzie An organic blueberry juice (Beutelsbacher, Germany) and a et al, 1999; Shahidi et al, 1992; Gardner et al, 1997; cranberry juice (Cranberry Classic, Ocean Spray, UK) were Gardner et al, 1999; Hertog et al, 1992). However, their obtained from local retailers. Ascorbic acid, EDTA, acetic nutritional relevance is still unclear as little is known about acid, sodium hydroxide (all Analar grade), metaphosphoric their absorption, metabolism and antioxidant ef®cacy in acid (general purpose reagent grade), acetonitrile (HPLC vivo (Robards & Antolovich, 1997; Peterson & Dwyer, grade) were obtained from Merck (Poole, UK). All other 1998; Duthie, 1999). reagents were obtained from Sigma-Aldrich (Poole, UK). The trial was approved by the joint ethical committee of the Grampian Health Board and the University of Aber- deen. Nine healthy female volunteers (23 ± 41 y) were *Correspondence: Dr GG Duthie, Rowett Research Institute, Greenburn Road, Aberdeen, AB21 9SB, Scotland. recruited to the study. All were normotensive, free from E-mail: [email protected] clinical disorders and had taken no vitamin=mineral sup- Guarantor: Dr GG Duthie. plements for at least three weeks before the start of the Contributors: All authors contributed to the design and expedition of the study. After an overnight fast, the volunteers consumed study and to the writing of the paper. Any mention of brand names does not imply product endorsement. 500 ml of either blueberry juice, cranberry juice or a control Received 29 July 1999; revised 24 November 1999; accepted solution containing a similar sugar content to the fruit 14 December 1999 juices (9% (w=v) sucrose in water) within 10 min. This Blueberry and cranberry juice and plasma antioxidant capacity CB Pedersen et al 406 was done as a Latin square format; on three occasions one Results week apart a different preparation was consumed each Anthropometric characteristics of the volunteers are in time. Blood samples (18 ml) were collected by venipunc- Table 1. The FRAP value of the blueberry juice was ture into evacuated tubes containing EDTA as anticoagu- twice that of the cranberry juice although the quenching lant (Evacuette, Greiner Labortechnik, Austria) 5 min of Fremy's radical by the juices was similar. Moreover, before and 0.5 h, 1 h, 2 h and 4 h after consumption of the total phenol content was three-fold greater in the blueberry beverage. A single urine sample was collected from each compared with the cranberry juice although only the latter volunteer 4 h after consumption of the juice. All samples contained vitamin C (Table 2). were kept on ice and blood samples centrifuged (4C, Plasma volume did not change during the course of the 2400 g, 15 min) within 45 min. The harvested plasma and trial (data not shown). Despite it being rich in phenolics, the urine were aliquoted, snap frozen in liquid nitrogen and consumption of the blueberry juice did not signi®cantly stored at 7 80C. For analysis of vitamin C, an aliquot of increase plasma concentrations of total phenols (Figure 1) plasma (0.6 ml) was stabilised with an equal volume of or vitamin C (Figure 2). However, following consumption 10% (w=v) meta-phosphoric acid (MPA) and snap-frozen of cranberry juice there was a signi®cant increase in plasma as above. concentrations of total phenols after 1 h (P < 0.05) and a Two methods were employed to measure antioxidant marked 30% increase in vitamin C from 0.5 to 4.0 h activity. The ability of the plasma or juice to donate a (P < 0.001) (Figures 1 and 2). Moreover, only consumption hydrogen atom or electron to the synthetic free radical, of the cranberry juice was associated with a signi®cant potassium nitrosodisulphonate (Fremy's salt) was moni- increase (P < 0.001) in antioxidant capacity of plasma as tored by electron spin resonance spectroscopy (ESR) determined by both ESR spectroscopy and change in FRAP (Gardner et al, 1997; Gardner et al, 1999). A 10-fold value (Figure 3). These increases were still apparent 4 h dilution of plasma or juice (3 ml) was mixed with an after consumption of the juice. Total phenol contents in equal volume of Fremy's salt (50 mM and 1.0 mM solutions urine were similar for all treatments (mean Æ s.e.m.: 40 Æ 6, for plasma and juice, respectively) and kept for 5 min at 34 Æ 3 and 37 Æ 5 GAE=ml subsequent to consumption of room temperature before measurement. The spectrum of blueberry, cranberry and sucrose solution, respectively). the low ®eld resonance of the Fremy's radical was recorded after 5 min by ESR. Signal intensity was obtained by double integration and the concentration calculated by comparison with a control reaction with distilled water Table 1 Anthropometric characteristics of the volunteers instead of fruit juice. Spectra were obtained at 21Cona 2 Bruker ECS 106 spectrometer working at ca 9.5 GHz (X- Age (y) Height (cm) Weight (kg) Body Mass Index (kg=m ) band frequency) and equipped with a cylindrical (TM110 Mean 31 164 64 24 mode) cavity. The microwave power and modulation s.e.m. 2 2 4 4 amplitude were set at 2 mW and 0.01 mT, respectively. Range 23 ± 41 155 ± 169 52 ± 82 21 ± 31 Antioxidant capacity was expressed as the number of Fremy's radicals reduced by the plasma or juices. Antioxidant potential of the plasma and juices was also Table 2 Antioxidant capacities and content of vitamin C and total estimated from their ability to reduce Fe(III)-2,4,6-Tri(2- phenols of the juices pyridyl)-s-triazine (TPTZ) complex to Fe(II)-TPTZ, the Blueberry juice Cranberry juice resulting intense blue colour being linearly related to the amount of reductant (antioxidant) present (Benzie and FRAP value (mMFe2) 21.46103 9.966103 No. of Fremy's radicals reduced=litre 5.1161021 5.2561021 Strain, 1996). The ferric reducing antioxidant potential Vitamin C (mM) 0 1.53 (FRAP value) was measured at 593 nm 7 min after 30 ml Total Phenols (mg GAE=ml) 2589 893 of plasma or a 10-fold dilution of the juices in distilled water was added to 900 ml of Fe(III)-TPTZ by which time FRAP, ferric reducing antioxidant potential; GAE, gallic acid equivalents. the reaction was complete at 37C. Results were interpo- lated from a standard curve prepared from a stock solution (0.278 g FeSO4 7H2O to 1 litre distilled H2O). Total phenol content in the juice samples was determined as gallic acid equivalents (GAE) using Folin-Ciocalteu reagent (Singleton & Rossi, 1965). Total phenols in plasma and urine were also determined with Folin-Ciocalteu reagent, using a modi®cation to remove protein interference in the samples (Sera®ni et al, 1998). Vitamin C concentration in plasma and juice was measured by high pressure liquid chromatography (Ross, 1994). Haematocrit and total haemoglobin were measured by routine procedures to allow correction for any changes in plasma volume (Dill & Costill, 1974) arising from consumption of 500 ml of ¯uid. Results were subjected to repeated measures analysis of variance using antedependence modelling (Kenward, Figure 1 Changes in total phenol levels in plasma after consumption of 1987). Having obtained initial tests of signi®cance from 500 ml of blueberry juice, cranberry juice or 9% (w=v) sucrose in water these analyses, speci®c comparisons were made by (control). Data is presented as mean Æ s.e.m. *P < 0.05. Consumption of the cranberry juice resulted in a weak but signi®cant increase (P < 0.05) in ANOVA of means between treatments and paired t-tests total phenols after 60 min, whereas the change for blueberry juice did not across time using GenStat 3.2 software. achieve statistical signi®cance compared with control.

European Journal of Clinical Nutrition Blueberry and cranberry juice and plasma antioxidant capacity CB Pedersen et al 407 juice (Whitehead et al, 1995; Benzie et al, 1999; Kivits et al, 1997; Sera®ni et al, 1998; Duthie et al, 1998). This effect has been ascribed to the low molecular weight phenolics in many plant-based foods which can act as antioxidants because their extensive conjugated p-electron systems allow ready donation of electrons or hydrogens from the hydroxyl moieties to free radicals (Scott, 1997). In the present study consumption of phenolic-rich blueberry juice failed to increase the total phenol content or antioxidant capacity of plasma. As renal excretion of phenols was not increased following blueberry juice consumption, such compounds may not be readily absorbed from the gut. The small increase in plasma total phenols following consump- Figure 2 Changes in plasma vitamin C concentrations after consumption tion of the cranberry juice may indicate the presence of of 500 ml of blueberry juice, cranberry juice or 9% (w=v) sucrose in water other more bio-available phenolics not present in the blue- (control). Data as mean Æ s.e.m., ***P < 0.001. There is a signi®cant berry juice. However, their absorption would appear to be increase in vitamin C concentration following consumption of cranberry juice but not blueberry juice compared with the control. insuf®cient to result in an increase in total phenols in the urine. Moreover, our in-house experiments suggest that vitamin C may also react to a limited degree in the total phenol assay. This may therefore account for the small increase in total phenolics observed following consumption of the vitamin C-rich juice. The blueberry juice used was an organic product whereas the cranberry juice was a vitamin C-forti®ed and more widely available commercial product. Different methods of juice production may alter phenolic pro®les by, for example, the oxidative degradation of anthocyanins to free benzoic acid (Rommel & Wrolstad, 1993). Such oxidative products may have limited bio-availability and may account in part for the limited uptake of phenolics from the blueberry juice despite its greater total phenolic content. However, as post-ingestion responses re¯ect a combination of the constituents of a food or beverage and different constituents may not necessarily be absorbed at the same rate (Paiva et al), it is also possible that the 4 h duration of the trial may have been insuf®cient to allow signi®cant detection of phenolics in plasma. Increased antioxidant capacity in plasma following consumption of the cranberry juice was associated with the marked rise in vitamin C concentration and is consistent with the ability of ascorbate to reduce the Fe(III) chelate and Fremy's salt in vitro. As one molecule of vitamin C reacts with 2.0 and 2.4 molecules of Fe(III) and Fremy's salt respectively (Benzie & Strain, 1996; Mitchell et al, 1998), this implies that 86 ± 89% of the increased antioxidant capacity 2 h after drinking the cranberry juice was due to vitamin C. However, the response of Fremy's appears to mirror that of vitamin C more closely than that of FRAP. The reason for this is unclear although it is possible that the Figure 3 Changes in antioxidant capacity of plasma measured by (a) change in Fremy's re¯ects the change in ascorbate only Fremy's radical reduction and (b) Fe3 reduction after consumption of whereas the change in FRAP re¯ects an increase in plasma 500 ml blueberry juice, cranberry juice or 9% (w=v) sucrose in water concentrations of both ascorbate and phenolics. Moreover, (control). Data as mean Æ s.e.m., **P < 0.01, ***P < 0.001. Only con- the possibility of artefactual loss of ascorbate in the sumption of the cranberry juice resulted in an increase in plasma unacidi®ed plasma samples, thus decreasing the estimate antioxidant capacity. of antioxidant capacity, cannot be excluded. The measurement of antioxidant potential by ESR spectroscopy has several advantages. Reactions are only likely Discussion to be signi®cant with good-atom donors, a necessary Nutritional guidelines indicate that an increase in the requirement for antioxidant function in biological systems. consumption of foods rich in antioxidant nutrients may In addition, the radicals have well-de®ned spectra which decrease the risk of developing many diseases including will allow them to be clearly resolved from other radical coronary heart disease and certain cancers (The Scottish intermediates which may be formed during oxidation Of®ce, 1993). Several studies have reported increases in processes. In addition, the technique is very sensitive, antioxidant capacity of plasma following consumption of allowing detection at the sub-micromolar level and can antioxidant-rich fruit, vegetables, teas, wines and grape- be used with turbid, solutions. Results are presented in this

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