Exotic fruits like açaí, camu camu, and blackberries rich in natural antioxidants (ascorbic acid, anthocyanins) are marketed as “functional” food supporting a pro-/antioxidant balance.
Confirming data from human studies are lacking. Within a randomized controlled crossover trial, 12 healthy non-smokers ingested 400 mL of a blended juice of these fruits or a sugar solution (control). Blood was drawn before and afterwards to determine antioxidants in plasma, markers of antioxidant capacity [Trolox equivalent antioxidant capacity, Folin-Ciocalteu reducing capacity, total oxidant scavenging capacity (TOSC)] and oxidative stress [isoprostane, DNA strand breaks (SB) in leukocytes in vivo], and their resistance vs. H2O2-induced SB. Compared with sugar beverage, juice consumption increased plasma ascorbic acid and maintained TOSC levels (p < 0.05). SB in vivo increased after ingestion of both beverages (p < 0.001), probably due to postprandial and/or circadian effects. Exotic fruit juices cannot further improve a stable pro-/antioxidant balance in healthy non-smokers.
G.2 Introduction
There is convincing epidemiologic evidence that regular fruit and vegetable consumption contributes to decrease the risk of several chronic diseases like coronary heart disease (Nikolic et al.) and probably certain kinds of cancer (AICR, 2007). It is hypothesized that antioxidative ingredients like polyphenols and water-soluble vitamins are the decisive factors explaining the health promoting properties of fruits and vegetables (Liu, 2003).
This scientific evidence is worldwide translated into public health initiatives like “5 a day” and school fruit programs with the goal to increase daily consumption of fruits and vegetables.
Encouraged by these policy-driven actions, food industry is strongly engaged to launch novel fruit (and vegetable) based products on the market. Blended juices, juice concentrates and smoothies rich in polyphenols and water-soluble vitamins like ascorbic acid are marketed as health supporting food specifically preventing from radical-driven chronic diseases.
In this respect, economic and scientific interests are focused on fruits frequently consumed in South America. Camu camu (Myrciaria dubia) grows in the Amazon region and contains anthocyanins [30 – 50 mg/100 g, with cyanidin 3-O-glucoside as major (89% of total) anthocyanin], and an extraordinary high content of ascorbic acid (up to 3.0 g/100 g pulp) (Rodrigues and Marx). Another popular fruit in Central and South America is açaí (Euterpe oleraceae Mart.). It is also rich in anthocyanins, especially in cyanidin 3-O-glucoside (up to 456 mg/L pulp) and exhibit high antioxidant capacity in vitro (World Cancer Research Fund, 2007;
Lichtenthäler et al., 2005). Andean blackberries similarly present a high antioxidant capacity because of their high content in ellagtannins (Acosta-Montoya et al., 2010).
Controlled clinical trials to evaluate the protective effects of these fruits are scarce. Daily intake of 70 mL camu camu juice for one week reduced urinary 8-hydroxydesoxyguanosine, a biomarker of DNA damage, which did not occur after ingestion of equal amounts of isolated ascorbic acid (1050 mg/d) (Inoue et al., 2008). Intervention studies in healthy non-smokers have shown that bolus consumption of açaí (pulp or juice) (Mertens-Talcott et al., 2008) or a juice blend with açaí as predominant ingredient (Jensen et al., 2008) increased antioxidant capacity in plasma (Mertens-Talcott et al., 2008) and erythrocytes (Jensen et al., 2008), respectively, and decreased lipid peroxidation (Jensen et al., 2008). Even if individual markers on antioxidant capacity and oxidative stress improved in these studies, a comprehensive picture concerning antioxidant defense is lacking.
The aim of our study was, thus, to investigate the effects of a bolus consumption of a blended juice made of açaí, Andean blackberries and camu camu on the concentrations of plasma antioxidants, plasma antioxidant capacity, and markers of oxidative stress in healthy non-smokers. Secondary goal was to detect and characterize metabolites of anthocyanins in plasma using a newly developed HPLC technology.
G.3 Materials and methods 1. Subjects and study design
To reach these study goals, we performed a randomized, controlled trial with crossover design.
The primary endpoint was the assessment of total antioxidative capacity in blood. Sample size calculation was based on data from a preliminary trial with three healthy non-smokers ingesting a bolus of 400 mL of the juice blend under standardized conditions. Trolox equivalent antioxidant capacity (TEAC) increased from 1.25 mmol/l Trolox equivalents (TE; baseline) to 1.36 mmol/l TE (0.5 h; maximal TEAC). Considering a standard deviation of 0.075 mmol/l TE, a difference of 0.11 mmol/l TE could be detected with α = 0.05 and a power of 90% if 11 participants were recruited. To account for dropouts, 12 participants were included in our study.
Participants (18 - 50 years, body-mass-index between 18.5 - 24.9 kg/m2, non-smokers for at least 6 months) were recruited within our staff. Exclusion criteria (questionnaire) were: known hepatic/gastrointestinal disorders, pregnancy or breastfeeding, regular use of vitamin or flavonoid-rich supplements. Participants were asked on their usual intake of fruit and vegetables (portions per day).
The randomization into two equal groups was stratified by sex and was done by lots. Group A first ingested 400 mL of a fruit juice blend after 12 h overnight fast; after a wash-out period of 2 - 3 weeks, they received 400 mL of a solution with equal amounts of monosaccharides (13.0 g glucose, 7.2 g fructose) as control. Group B consumed the test beverages in reversed order.
The fruit juice blend consisted of 44% açaí, 12% camu camu and 44% blackberry juice (Rubens ssp.). The fruit juice was produced according to the technological standards for the production of customary in trade from a commercially available frozen açaí puree (açaí juice pads; Açaí GmbH, Berlin, Germany), camu camu pulp [Brazilian Agricultural Research Corporation (Embrapa)] and Costa Rican blackberry juice [Centro National de Investigacion en Tecnologia de Alimentos (CITA), Costa Rica]. Ingredients of the juice blend are presented in table G.3.1.
Juice Control
Anthocyanins (mg CE) 276 n.d.
Ascorbic acid (mg) 936 n.d.
Total phenolic content (mg GAE) 1612 n.d.
Glucose (g) 13.0 13.0
Fructose (g) 7.2 7.2
Table G.3.1. Ingredients of a single portion (400 mL) of the study drinks. CE: cyanidin 3-O-glucoside equivalents, GAE: gallic acid equivalents, n.d.: not determined.
Fructose is degraded endogenously to uric acid and can, thus, exhibit antioxidative effects (Hallfrisch, 1990, Livesey, 2009). The control beverage, thus, contained amounts of fructose equal to the test juice. To avoid artefacts by other foods, subjects were instructed to abstain from foods rich in polyphenols (hand-out) starting 24 h before the first study day until completion. On the study day, participants received a standardized meal (two bread rolls with butter and cheese) 3 and 5 hours after consumption of the study beverages. Water was allowed to drink ad libitum.
Written informed consent was obtained by all participants. The study was conducted according to the Declaration of Helsinki and approved by the ethical Committee of the University of Bonn (No. 207/08).
2. Blood sampling
Blood samples (7.2 mL each) were collected before and 30, 60, 90, 120, 180 and 360 min after consumption of the study drink using tubes (S-Monovette, Sarstedt, Nümbrecht, Germany) coated with EDTA [analyses of ascorbic acid, fat soluble pro-/vitamins, antioxidant capacity, 8-isoprostaglandine F2α (8-iso PGF2α) and phenolic acids in plasma], heparin (determination of
DNA strand breaks in leukocytes), and tubes without anticoagulant (analysis of cholesterol, triglycerides, and uric acid).
3. Preparation of plasma samples
After blood withdrawal, EDTA tubes were placed on ice immediately. Then, blood was centrifuged at 3,000 × g for 20 min at 4 °C. For the determination of ascorbic acid, 500 µL of a cold 6% perchloric acid/2% metaphosphoric acid solution (v/v) was added to 500 µL fresh EDTA plasma in order to precipitate proteins and to stabilize ascorbic acid. After centrifugation (3,000 × g, 10 min, 4 °C), the supernatant was aliquoted and stored at -80 °C until analysis. To avoid oxidation, 10 µL butylhydroxytoluol (0.5% w/v in ethanol) was added to 1000 µL fresh EDTA plasma for later analysis of 8-iso PGF2α and fat soluble pro-/vitamins. Ten µL of a solution of 0.4 M NaH2PO4 with 20% ascorbic acid and 0.1% EDTA (pH 3.6) were added to 500 µL plasma in which phenolic acids should be detected. Heparinized blood was used immediately for the determination of DNA strand breaks in leukocytes.
4. Dietary intake of energy and nutrients
The intake of energy, macronutrients, dietary fiber and antioxidant pro-/vitamins on the day before the study was calculated using Ebis Pro 4.0 software (University of Hohenheim, Stuttgart, Germany) based on German Nutrient database, version II.3. The flavonoid intake was calculated by using the USDA database (USDA, 2007).
5. Analytical methods
Plasma antioxidant capacity
Since no single assay truly reflects overall antioxidant capacity, multiple assays with different radicals and mechanisms (hydrogen or electron transfer which reflect radical quenching and radical reduction, respectively) should be used (Prior et al., 2005). Thus, TEAC (Miller et al., 1993) (CV 1.2%) was measured and expressed as Trolox equivalents. Furthermore, the Folin-Ciocalteu reducing capacity (FCR) (Prior et al., 2005), was assessed with the modifications of Arendt et al.
(2005) to avoid interferences with plasma proteins (CV 2.0%). The FCR of plasma was expressed as catechin equivalents. Additionally, total oxidant scavenging capacity (TOSC) in plasma (diluted 1:20 (v/v) with aqua dest.) was determined against peroxyl radicals (CV 3.6%) according to Lichtenthäler et al. (2003).
Concentrations of antioxidants in plasma/serum
Ascorbic acid in plasma was measured by HPLC with UV/VIS detection at 243 nm (CV 1.8%) according to Steffan (1999).
α-Tocopherol and β-carotene were also determined by HPLC. The protocol of Erhardt et al.
(1999) was modified by using apocarotenal as internal standard, Nucleosil® 100-5 CN (Macherey-Nagel, Düren, Germany) as column and a solution of 98% hexane and 2% isopropanol as mobile phase. α-Tocopherol was detected at 292 nm (CV 4.1%) and β-carotene at 450 nm (CV 3.5%).
Uric acid in serum was determined photometrically within routine analysis (Urea Flex® reagents cassette, Siemens Healthcare Diagnostics, Eschborn, Germany) (CV 1% according to manufacturer).
Phenolic acids in plasma
A solid phase extraction using Supel® – Select HLB SPE tubes (bed wt., 60 mg, volume 3 mL) (Supelco, Steinheim, Germany) was performed to eliminate plasma proteins. After equilibration with 0.1% formic acid, the cartridge was loaded with plasma (450 µL). After washing with aqua dest., a solution of methanol, acetonitrile and formic acid (50 + 49.9 + 0.1, v/v/v) was used for elution. The eluate was evaporated under nitrogen to dryness and reconstituted with a solution (50 µL) of methanol, water and trifluoroacetic acid (20 + 79.9 + 0.1, v/v/v). Thereafter, single compounds with reducing capacity were determined in the samples by HPLC-CEAD detection at 100, 200, 300 and 400 mV using the conditions (instrument settings, elution) previously described by Ritter et al. (2010). The analytical column was an Aqua 3 µm C18, 150 mm, 4.6 mm i.d. with a guard column (Security Guard, Aqua RP-18, 4 mm, 3 mm i.d.) (both from Phenomenex, Aschaffenburg, Germany). For analysis, 20 µL of the sample (plasma, standard and juice) were injected. Protocatechuic acid (≥ 97%) was obtained from Merck (Darmstadt, Germany), gallic acid (≥ 97.5%), vanillic acid (≥ 97%) and ferulic acid (≥ 99%) from Sigma-Aldrich (Steinheim, Germany) and caffeic acid (purum) from Serva (Heidelberg, Germany).
Lipid peroxidation
Total 8-iso PGF2α concentration was determined as sum of free plus esterified 8-iso-PGF2α in EDTA-plasma by an ELISA kit (Cayman Chemical, Ann Arbor, MI, USA, CV 10%) as described previously (Roggenbuck et al., 2008).
DNA strand breaks
DNA single strand breaks (SB) were measured in leukocytes in vivo and after 20 min incubation at 4 °C with 300 µM H2O2 ex vivo using the single cell gel electrophoresis assay (also called Comet Assay). The protocol of Arendt et al. (2005) was used, however, electrophoresis period was extended to 20 min. Fifty nuclei per slide were evaluated for DNA damage by calculating tail moment with the Comet Assay III software (Perceptive Instruments, Suffolk, UK). The
difference in tail moment between untreated cells (SB in vivo) and cells challenged with H2O2 was calculated to determine the resistance of DNA vs. H2O2 ex vivo (CV 22%).
Triglycerides
Triglycerides in serum were measured within routine procedures by using Flex® reagent cartridges and the Dimension Vista® System (Siemens Healthcare Diagnostics; CV 3% according to manufacturer).
Statistics
Since data were normally distributed according to Kolmogorov-Smirnow-test, parametrical tests were used.
The effects of beverage, time, interactions of beverage and time (beverage × time) and the order of beverage intake on laboratory parameters were investigated with repeated measures ANOVA.
In case of significant effects, a paired or unpaired t-test was performed subsequently.
Area under the curve (AUC) was calculated for all parameters by the trapezoidal rule for non-uniform intervals. AUC obtained after consumption of juice and sugar solution was compared with each other by an unpaired t-test.
Results are shown as mean and standard deviation. Statistical evaluation was performed with PASW Statistics, version 17.0 (SPSS Inc., Chicago, IL, USA).