Effect of juice processing on the cancer chemopreventive effect of cranberry

Food Research International 44 (2011) 902–910

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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Effect of juice processing on the cancer chemopreventive effect of cranberry S. Caillet a, J. Côté a, G. Doyon b, J.-F. Sylvain c, M. Lacroix a,⁎ a b c

INRS-Institut Armand-Frappier, Research Laboratory in Sciences Applied to Food, 531 des Prairies, Laval, Québec, Canada H7V 1B7 Agriculture and Agrifood Canada, 3600 Casavant West, Saint-Hyacinthe, Québec, Canada J2S 8E3 Atoka Cranberries Inc., 3025 route 218, Manseau, Québec, Canada G0X 1V0

a r t i c l e

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Article history: Received 10 November 2010 Accepted 20 January 2011 Keywords: Cranberry products Phenolic extracts Quinone reductase Cancer chemopreventive effect

a b s t r a c t Cancer chemopreventive properties were evaluated in cranberries and cranberry products (mash, depectinized mash, pomace, raw juice, clarified juice and juice concentrate). Three extracts isolated from frozen cranberries and cranberry solids (mash, depectinized mash and pomace) containing anthocyanins, water-soluble and apolar phenolic compounds were tested. Cranberry juices and extracts were screened for their ability to induce the phase II xenobiotic detoxification enzyme quinone reductase (QR). The results showed that there was no cytotoxicity against the cells used in the test. All samples stimulated quinone reductase activity except the highest concentrations of the anthocyanin-rich extract of pomace, which inhibited QR activity. Also, the results showed that the QR induction for all samples varied with concentration and that there was an optimal concentration for which the QR induction was maximal. Although the three cranberry extracts were good QR inducers, our results indicated that the phenols present in aqueous extract showed QR inductions which were more important than those obtained with phenols present in solvent extracts. Also, the ability of phenols to stimulate the QR activity has been reduced continuously and significantly (P ≤ 0.05) during the technological process. Especially, it appears that conditions of the evaporation to obtain a juice concentrate exerted a significant effect (P ≤ 0.05) on inducer potencies of bioactive molecules. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Cranberry fruits are excellent raw materials for juice production, as they contain numerous antioxidants including phenolic compounds, vitamin C, minerals and many others. Compounds present in the fruits of the Vaccinium species are reported to play several roles in human health maintenance (Kahlon & Smith, 2007). Effective inhibition of urinary tract infections was recently attributed to the presence of high molecular weight constituents, present in Vaccinium macrocarpon (cranberry) juices, which act as anti-adhesive agents preventing bacterial colonization (Avorn et al., 1994). Health benefits, including reduced risks of cancer and cardiovascular disease, are believed to be due to the presence of various polyphenolic compounds, including anthocyanins, flavonols, and procyanidins (Chu & Liu, 2005; Seeram, Adams, Hardy, & Heber, 2004). The potent antioxidant properties of Vaccinium fruits have been well documented (Wang, Cao, & Prior, 1997). Biological properties of the fruit extract, rich in anthocyanins, include antioxidant capacity, astringent and antiseptic properties, ability to decrease the permeability and fragility of capillaries, inhibition of platelet aggregation, inhibition of urinary tract infection and ⁎ Corresponding author at: Research Laboratory in Sciences Applied to Food, INRSInstitut Armand-Frappier, Université du Québec, 531 Boul. des Prairies, Laval, Québec, Canada H7V 1B7. Tel.: +1 450 687 5010x4489; fax: +1 450 687 5792. E-mail address: [email protected] (M. Lacroix). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.01.055

strengthening of collagen matrices via cross linkages (Côté, Caillet, Doyon, Sylvain, & Lacroix, 2010; Pappas & Schaich, 2009). A study demonstrated that specific fractions isolated from fruits of Vaccinium myrtillus (bilberry) possess potential anticarcinogenic constituents (Bomser, Madhavi, Singletary, & Smith, 1996). Cranberry and cranberry extracts have been shown to have anticancer properties. Cranberry extracts showed in vitro antitumor activity by inhibiting the proliferation of MCF-7 and MDA-MB-435 breast cancer cells. Cranberry extracts also exhibited a selective tumor cell growth inhibition in prostate, lung, cervical, colon, and leukemia cell lines (Krueger, Porter, Weibe, Cunningham, & Reed, 2000; Seeram et al., 2004). Solid-state bioprocessing of natural products including cranberry pomace has been shown to enhance its functionality. Phenolic extracts from berries of the Vaccinium species were able to modulate the induction and repression of ornithine decarboxylase (ODC) and quinone reductase that critically regulate tumor cell proliferation (Bomser et al., 1996). Fruit extracts were screened for their ability to induce the phase II xenobiotic detoxification enzyme quinone reductase (QR), an enzyme involved in the detoxification of potential carcinogens and xenobiotics (Prochaska, 1994). A specific fraction isolated from the fruits was an active QR inducer, indicative of the potential to inhibit the initiation stage of chemical carcinogenesis (Bomser et al., 1996). It is generally accepted that carcinogenesis is a multistage process that may be caused by carcinogen-induced genetic and epigenetic

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damage in susceptible cells (Sporn, 1996). The first stage of the carcinogenic process, tumor initiation, involves exposure of normal cells to electrophilic carcinogen metabolites or reactive oxygen species (Prochaska & Talalay, 1991). One of the major mechanisms to protect against the toxic electrophilic metabolites of carcinogens and reactive oxygen is the induction of phase II detoxification enzymes such as glutathione S-transferases, UDP-glucuronosyltransferases, and NAD (P)H:quinone reductase (NQO1) (Cantelli-Forti, Hrelia, & Paolini, 1998; Gutierrez, 2000; Kang & Pezzuto, 2004). NAD(P)H:quinone reductase (NQO1), one of the phase II drug-metabolizing enzymes, plays an important role in the mechanism of cancer chemoprevention, presumably at the initiation stage of carcinogenesis. The induction of phase II enzymes can offer protection against toxic and reactive chemical species (Prochaska, Santamaria, & Talalay, 1992; Talalay, 2000). Several studies have shown that elevation of phase II enzymes correlates with protection against chemically-induced carcinogenesis in animal models (Boone, Steele, & Kelloff, 1992; Kang & Pezzuto, 2004). Enzyme inducers are of two types: monofunctional and bifunctional (Prochaska & Talalay, 1988). Bifunctional inducers increase phase II enzymes as well as phase I enzymes such as aryl hydrocarbon hydroxylase, and bind with high affinity to the aryl hydrocarbon receptor (Yang, Smith, & Hong, 1994). Monofunctional inducers induce phase II enzymes selectively and are independent of the aryl hydrocarbon receptor. Since phase I enzymes can activate procarcinogens to their ultimate reactive species, monofunctional agents that induce phase II enzymes selectively would theoretically appear to be more desirable candidates for cancer chemoprotection (Talalay, 2000) In addition, selective phase II enzyme inducers would be anticipated to serve as anticarcinogens early in the process of carcinogenesis. Bearing in mind the importance of phase II enzyme induction in cancer chemoprevention, methods to determine phase II enzyme inducer potencies of pure compounds and extracts of natural products are necessary. Quinone reductase is a flavoprotein that catalyzes the two-electron reduction of electrophilic quinones into stable hydroquinones and reduces oxidative cycling (Prochaska & Talalay, 1991). It is a representative phase II detoxifying enzyme based on a wide distribution in mammalian tissues. The enzyme shows a strong induction response and is easily measured by a coupled tetrazolium dye reduction assay (Talalay, 2000). In addition, with in vitro and in vivo systems, induction has been shown to correlate with the elevation of other protective phase II enzymes (Pezzuto, 1995), and induction provides a reasonable biomarker for the cancer. The murine hepatoma cell line Hepa 1c1c7 contains inducible quinone reductase that is easily measurable and provides a reliable, highthroughput system for the detection of induction (Prochaska et al., 1992). This cell line has been used for the discovery of novel natural product anticarcinogens (Talalay, 2000). The aim of the present study was to evaluate the potential cancer chemopreventive effect in cranberry fruits and cranberry processing products (mash, depectinized mash, pomace, raw juice, clarified juice and juice concentrate), and demonstrate the technological process effect on their ability to induce enzyme quinone reductase. In this work, three extracts isolated from frozen cranberries and cranberry solids (mash, depectinized mash and pomace) respectively containing anthocyanins, water-soluble and apolar phenolic compounds were tested. 2. Materials and methods

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until used. Cranberry samples were collected at each steps of the juice process which is illustrated in Fig. 1. The initial processing steps to make juice involve reducing frozen cranberries to a mash using a fruit mill. Following the milling step, mash heating is required at 55 °C for extracting the valuable color (plasmolysis). The heated mash is then allowed to macerate under minimal agitation. At the same time, enzymes are added to provide acceptable yields and throughputs on the press and improve color extraction. The aim of the mash enzyme treatment is more or less to completely degrade soluble pectin. The raw juice recovery from depectinized mash was done using a fruit press at 1.90 bar. During the juice pressing step, high amounts of press cake were obtained: cranberry pomace is the main byproduct of the cranberry processing industry. It is composed primarily of skin, seeds, and stems left over after pressing the fruit for juice. During the filtering process, a cross-flow membrane filtration was used to remove colloids and generate a clear juice from raw juice. Then the clarified juice was concentrated by evaporation to obtain a juice concentrate at 50° brix. 2.2. Extraction of phenolic compounds and sample preparation The extraction conditions employed were as mild as possible to avoid oxidation, thermal degradation and other chemical and biochemical changes in the sample. Extraction of phenolic compounds from frozen cranberries and cranberry solids (mash, depectinized mash and pomace) was achieved according to three methods using solvents of different graded polarity for the recovery of specific classes of phenolics which have different solubility. The most water soluble phenolic compounds (E1) were extracted with water/methanol (85:15, v/v) (Seeram et al., 2004), the most apolar phenolic compounds (E2) (flavonols, flavan-3-ols and proanthocyanidins) were extracted with acetone/methanol/water (40:40:20, v/v), modified from a method described by Neto et al. (2006), and the anthocyanins (E3) were extracted with methanol/water/acetic acid (85:15:0.5, v/v/v) as described by Wu and Prior (2005). Frozen cranberries and cranberry solids were crushed at 4 °C for 40 s in a Waring commercial blender (Waring Laboratory, Torrington, CT) to obtain a fine powder. Immediately after crushing samples, extractions have been performed at 4 °C under agitation and nitrogen for 40 min by macerating 300 g of the fruit powder with extracting solvents. Three successive extractions in each extracting solvent were performed using the same procedure. The first extraction was done using 700 mL of solvent, but for the two last ones, 500 mL was used instead. The solvent containing the phenolic compounds was recuperated after each extraction and the solvents from the successive extractions were combined, then filtered on Whatman paper no. 4 (Fisher Scientific, Nepean, ON, Canada). The filtrate was concentrated by the evaporation of solvent using the SpeedVac Automatic evaporation system (Savant System, Holbrook, NY), then dry matter was determined by freeze-drying the extracts for 48 h with a Virtis Freeze mobile 12 EL (The Virtis Co., Gardiner, N.Y), and stored at − 80 °C until used. Prior to the experiment, the freeze-dried extracts were weighed and redissolved to a specified volume in 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Oakville, ON, Canada) solution to avoid the cellular cytotoxicity of hepa 1c1c7 cells used for the quinone reductase assay, then the cranberry juices (20 mL) and phenolic extracts were adjusted to pH 2.5 (natural fruit pH) with 1 mol/L HCl.

2.1. Raw material and cranberry processing 2.3. Total phenol concentration Frozen cranberries (V. macrocarpon) and six cranberry processing products (mash, depectinized mash, pomace, raw juice, clarified juice and juice concentrate) were used to determine their cancer chemopreventive effect. These samples were provided by Atoka Cranberries Inc. (Manseau, QC, Canada) and were stored at − 80 °C

Total phenolic compound content in each cranberry extract or juice was determined by spectrophotometry (absorbance at 760 nm) according to the Folin–Ciocalteu procedure (Singleton & Rossi, 1965). The total phenolic compound content of samples was estimated from

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Fig. 1. Flow chart for the juice processing of cranberry (Vaccinium macrocarpon) fruit.

a calibration curve (r2 = 0.9886) by plotting known solutions of gallic acid (10, 20, 40, 60, 80, 100 and 500 μg/mL).

with 10% fetal bovine serum, at 37 °C in a humidified atmosphere at 5% CO2 in air.

2.4. Quinone reductase (QR) assay

2.5.2. Assay procedure Hepa lc1c7 cells were seeded in 96-well plates (Cellbind surface, Corning Life Sciences, Lowell, MA, USA) at a density of 10,000 cells/mL in 200 μL of α-minimal essential medium supplemented with 10% fetal bovine serum. The cells were grown for 24 h in a humidified incubator in 5% CO2 at 37 °C. The medium was decanted after a 24 h incubation, 190 μL fresh medium and 10 μL of 10% DMSO containing test phenolic extracts or cranberry juices were added to each well. Ten final concentrations for the phenolic extracts (0.39, 0.78, 1.56, 3.12, 6.25, 12.50, 25.00, 50.00, 100.00 and 200.00 mg/mL) and six final concentrations for the cranberry juices (1.56, 3.12, 6.25, 25.00, 50.00 and 100% (v/v)) were tested. The cells were incubated for an additional 48 h. After the cells were treated with test samples for 48 h, the medium was decanted and the cells were incubated at 37 °C for 10 min with 50 μL of 0.8% digitonin and 2 mM EDTA solution (pH 7.8). The plates were then agitated on an orbital shaker (100 rpm) for 10 min at room temperature and 200 μL of reaction mixture (the following stock solution (150 mL) was prepared for each set of assays: 7.5 mL of 0.5 M Tris–HCl (pH 7.4), 100 mg of bovine serum albumin, 1 mL of 1.5% Tween 20, 0.l mL of 7.5 mM FAD+, l mL of 150 mM glucose 6-phosphate, 90 μL of 50 mM NADP+, 300 U of yeast glucose 6-phosphate dehydrogenase, 45 mg of MTT, 150 μL of 50 mM menadione and distilled water to volume) were added to each well. Menadione solution (1 μL of 50 mM menadione dissolved in acetonitrile per mL of reaction mixture) was added just before the mixture was dispensed into the microtiter plates. The reaction generated a blue color, and this was arrested after 5 min by the addition of 50 μL of a solution containing 0.3 mM dicoumarol in 0.5% DMSO and 5 mM potassium phosphate, pH 7.4. The plates were then scanned at 595 nm

The assay for detecting the induction of cellular quinone reductase was based on the methods described by Prochaska and Santamaria (1988) and by Kang and Pezzuto (2004) with some modifications. This system uses 96-well plates and provides a highly quantitative and reproducible method for determining inducer potencies of plant extracts. Quinone reductase-specific activity is determined by measuring NADPH-dependent menadiol-mediated reduction of 3(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan. Protein is determined by using the bicinchoninic acid (BCA) protein assay kit with an identical set of test plates. Details of the procedure follow. 2.5. Materials Chemicals (BSA, dicoumarol, digitonin, DMSO, EDTA, FAD+,, fetal bovine serum (FBS), glucose 6-phosphate, glucose 6-phosphate dehydrogenase, menadione, 3-(4,5-dimethylthiazo-2-yl)-2,5diphenyltetrazolium bromide (MTT), NADP+, β-naphthoflavone, SDS, tris–base, Tween 20) were purchased from Sigma-Aldrich (Oakville, ON, Canada), and cell culture media and supplements (HBSS, α-MEM) were obtained from Invitrogen Inc. (Burlington, ON, Canada). 2.5.1. Cell cultures Hepa 1c1c7 murine hepatoma cells (ATCC CRL-2026) were maintained in α-minimal essential medium (MEM) (containing αMEM Glutamax, non-essential amino acids, sodium pyruvate, but without ribonucleosides or deoxyribonueleosides) supplemented

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with a microplate spectrophotometer (ELx800 BioTech Instruments Inc., Vinooski, VT, USA). The first column of wells in each plate normally contained the reaction mixture only and served as the nonenyzmatic blank. The second column of wells was the control cells treated with medium containing 0.5% DMSO. The third column of wells contained the solvent of samples (10% DMSO) to verify the solvent effect on cells. The fourth column of wells contained βnaphthoflavone (bifunctional inducer) that was used as positive control groups. 2.5.3. Protein determination Since some compounds can be tested if inducer activity can depress the rate of cell growth, it is desirable to relate the observed quinone reductase activity to the number of cells or to the amount of protein in each microtiter well. This normalization can be accomplished by protein dosage with a set of microtiter plates treated identically to those used for the MTT assay. The protein quantification was performed using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's specifications. A 20 μL aliquot of lysed cells of each well was transferred to the new plate and 300 μL of bicinchoninic acid (BCA) was added to each well. Plates were then incubated in a humidified incubator in 5% CO2 at 37 °C and after 30 min of incubation, absorbance was read at 562 nm with a microplate spectrophotometer. Standard curve of BSA was used. 2.5.4. Data analysis The specific activity of QR is defined as nmol MTT blue formazan formed per mg protein and per min: Specific activity = ðabsorbance change of MTT at 595 nm = min × 3247 nmol = mgÞ = ðabsorbance of BCA protein assay at 562 nmÞ where the reaction time was 5 min and MMTT = 3247 was the molar mass of MTT. The ratio of quinone reductase specific activities of sample-treated cells to solvent-treated control cells as a function of inducer concentration permits the determination of the Induction Index (or fold of induction): Induction indexðIIÞ = specific activity of sample−treated cells= specific activity of control cells Then, data were reported to the quantity of dry matter of each sample and the quantity of phenolic compounds and results were expressed as II(QR)/mg of dry matter or II(QR)/mg of phenol. When the induction index was greater than 1 this indicates that the compound stimulates the quinone reductase activity whereas a result below 1 indicates that the compound inhibits quinone reductase. Also, the induction of QR activity was expressed as CD value, concentration required to double QR specific activity, and the chemoprevention index (CI) is obtained by dividing IC50 value with CD value. CI is a well known screening tool for finding potential chemopreventive agents. IC50 value is the half maximal inhibitory concentration of cell viability, and the cell cytotoxicity was evaluated by using the Cell Proliferation Reagent WST-1 produced by Roche Diagnostic (Mannheim, Germany) as described by the manufacturer. 2.6. Statistical analysis of data A random block consisting of a 3 × 3 factorial design was used: for each analysis, three separate replicates (i.e. three different batches) were analyzed and three samples were tested in each replicate. Analysis of variance and Duncan's multiple-range was done using Stat-Packets Statistical Analysis software (Walonick Associates Inc.,

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MN, USA) for the quinone reductase assay. Differences between means were considered significant when P ≤ 0.05. 3. Results and discussion The effects of the technological process on quinone reductase induction (II(QR)) by cranberry juices and extracts are presented in Tables 1–3. Also, we evaluated the chemopreventive activity of the cranberry samples according to their CI values which is defined as IC50 value/CD value. The results showed that no cranberry samples induced a growth inhibition in Hepa 1c1c7 cells, with an IC50 value N 200 mg/mL (data not shown). Thus, potential chemopreventive samples showed that high CI values resulted from strong QR induction without cytotoxicity in Hepa 1c1c7 cells. Also, the results showed that the II(QR) and CI for all samples varied with the concentration and that there was an optimal concentration for which the QR induction and the cancer chemopreventive effect were maximal. When data were reported to the quantity of dry matter of each sample, the results showed that the anthocyanin-rich extract (E3) at 25 mg/mL was the most effective among the three fruit extracts with a maximum QR induction of 19.04 II(QR)/mg of dry matter. Among all extracts, the extract rich in apolar phenolic compounds (E2) of depectinized mash presented between 3.12 and 25 mg/mL the highest QR induction with values ranging from 20.34 to 24.45 II(QR)/mg of dry matter. In contrast, extracts of pomace have shown a weak QR induction and the highest concentrations (between 6.25 and 200 mg/mL) of anthocyanin-rich extract (E3) of pomace inhibited the QR activity. The extraction conditions of the extract rich in water-soluble phenolic compounds (E1) (water/ methanol (85:15, v/v)) were similar to those employed for juice (water). Also, the data obtained reveal that the technological process to manufacture cranberry juice has influenced the QR induction. The fruit E1 at 25 mg/mL presented a maximum QR induction with a value of 15.15 II(QR)/mg of dry matter, whereas maximum II(QR) obtained with the depectinized mash E1 at 3.12 mg/mL was 1.33 times higher than that obtained with fruit E1. In addition, the raw and clarified juices presented a maximum QR induction (27.60 and 33.87 II(QR)/mg of dry matter, respectively) that was twice that obtained with fruit E1. However, the maximum QR induction by the juice concentrate (i.e. 3.08 II(QR)/mg of dry matter) was divided by 10 compared to that obtained with the clarified juice. It appears that conditions of the evaporation to obtain a juice concentrate exerted a significant effect (P ≤ 0.05) on inducer potencies of bioactive molecules. Also, the maximum QR induction by pomace E1 at 200 mg/mL was divided by almost 7 compared to that obtained with the depectinized mash which tends to prove that extraction has led to the recovery of most bioactive molecules. Moreover, it is important to note that the maximum QR induction by the depectinized mash E2 was double that obtained with fruit E2, whereas for E3, the maximum QR induction was higher with the fruit compared to depectinized mash. Among all samples, phenolic compounds of fruit E1 showed the highest maximum QR induction (488.70 II(QR)/mg phenol) when results were expressed in II(QR)/mg phenol. The phenolic compounds of fruit E1 showed a maximum QR induction which was 2.5 and 6 times higher than those of phenols of fruit E3 and fruit E2, respectively. Also, the ability of phenols to stimulate the QR activity has been reduced continuously and significantly (P ≤ 0.05) during the technological process. The maximum QR induction by phenols has been divided by almost 5 between fruit E1 and depectinized mash E1, then by 2.5 between depectinized mash E1 and raw juice, and again by 10 at the end of the technological process. Thus, the phenolic compounds of fruit E1 presented a maximum QR induction 97 times higher than that of phenols of juice concentrate. Also, the maximum QR induction by E3 phenols has been divided by 3.33 between fruit and depectinized mash. However, the maximum QR induction by the

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Table 1 Quinone reductase induction by cranberry extract rich in polar phenolic compounds (E1) obtained from samples collected at each step of the industrial juice process. Samplesa

Concentration in mg/mL (extracts) or % (v/v) (juices)

II(QR)/mg of dry matterb,c

II(QR)/mg of phenolb,c

CDc,d (mg/mL)

CIe

Fruit

200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 100.00 50.00 25.00 6.25 3.12 1.56 100.00 50.00 25.00 6.25 3.12 1.56 100.00 50.00 25.00 6.25 3.12 1.56

4.78 ± 0.41f 5.10 ± 0.10f 5.45 ± 0.38f 15.15 ± 0.10n 14.39 ± 0.27m 12.87 ± 0.33l 11.41 ± 0.73k 11.36 ± 0.21k 10.15 ± 0.28j 9.39 ± 0.21i 5.18 ± 0.20f 5.22 ± 0.17f 6.96 ± 0.20g 11.22 ± 0.12k 12.46 ± 1.22kl 10.39 ± 1.19ijk 14.03 ± 0.10m 10.25 ± 0.41j 9.51 ± 0.99hij 7.90 ± 1.03gh 6.90 ± 0.34g 7.99 ± 1.07ghi 8.16 ± 1.11ghi 17.49 ± 0.69o 19.18 ± 0.15p 19.37 ± 0.42p 20.61 ± 1.41p 19.14 ± 0.15p 15.19 ± 0.14n 17.03 ± 0.27o 3.86 ± 0.43e 3.65 ± 0.37e 2.82 ± 0.65cde 2.57 ± 0.45cd 2.31 ± 0.25c 2.28 ± 0.37bc 2.34 ± 0.15c 2.37 ± 0.11c 2.25 ± 0.36bc 2.58 ± 0.51cd 6.87 ± 0.52g 7.49 ± 0.38g 26.83 ± 2.23q 27.60 ± 2.27q 18.26 ± 1.17op 17.75 ± 0.17o 14.56 ± 0.63mn 15.92 ± 0.68n 33.87 ± 1.46r 33.36 ± 1.09r 29.89 ± 1.00q 28.45 ± 0.93q 1.25 ± 0.15a 1.25 ± 0.14a 1.26 ± 0.16a 3.08 ± 0.36de 2.47 ± 0.15c 2.51 ± 0.20c

154.19 ± 15.54p 164.51 ± 3.22p 175.80 ± 12.87p 488.70 ± 25.29s 464.19 ± 21.48s 415.16 ± 24.29r 368.06 ± 23.54r 366.45 ± 25.77r 327.42 ± 11.48q 302.90 ± 16.45q 38.37 ± 3.80hi 38.66 ± 4.02hi 51.55 ± 1.48j 83.11 ± 2.85m 92.29 ± 10.81mno 76.96 ± 5.28lm 103.92 ± 8.18no 75.92 ± 4.10l 70.44 ± 2.33l 58.52 ± 3.62k 38.33 ± 4.15hi 44.38 ± 4.85i 45.33 ± 5.01ij 97.16 ± 3.16n 106.55 ± 4.79o 107.61 ± 5.31o 114.50 ± 11.65o 106.33 ± 4.82o 84.38 ± 4.11m 94.61 ± 1.47n 12.47 ± 0.74f 11.78 ± 0.69f 9.09 ± 0.90e 8.29 ± 1.09de 7.45 ± 0.84d 7.35 ± 0.43d 7.54 ± 0.48d 7.64 ± 0.36d 7.25 ± 0.49d 8.32 ± 0.62de 11.64 ± 0.88f 12.69 ± 0.62f 45.47 ± 5.06ij 46.77 ± 6.46ij 30.94 ± 3.91gh 30.08 ± 2.28g 26.96 ± 1.07g 29.48 ± 2.08g 62.72 ± 3.70k 61.77 ± 2.02k 55.35 ± 5.86jk 52.68 ± 1.72j 2.05 ± 0.25a 2.04 ± 0.23a 2.06 ± 0.18a 5.04 ± 0.17c 4.04 ± 0.23b 4.11 ± 0.30b

1.36 ± 0.07jk 1.29 ± 0.01jk 1.15 ± 0.06hi 0.43 ± 0.03a 0.46 ± 0.02a 0.49 ± 0.03a 0.84 ± 0.05e 1.00 ± 0.02g 1.01 ± 0.02g 1.03 ± 0.05g 1.37 ± 0.07k 1.33 ± 0.06jk 1.26 ± 0.06ijk 0.85 ± 0.07ef 0.71 ± 0.06bcd 0.92 ± 0.02f 0.62 ± 0.04b 0.93 ± 0.03f 1.00 ± 0.03g 1.12 ± 0.03h 1.32 ± 0.05jk 1.23 ± 0.04ij 1.24 ± 0.04ij 0.98 ± 0.08fg 0.74 ± 0.05de 0.73 ± 0.04d 0.63 ± 0.05bc 0.74 ± 0.05de 1.10 ± 0.01h 1.04 ± 0.05gh 1.69 ± 0.05l 1.72 ± 0.06l 1.85 ± 0.06m 1.87 ± 0.07m 1.90 ± 0.06m 1.91 ± 0.08m 1.89 ± 0.07m 1.89 ± 0.07m 1.92 ± 0.08m 1.87 ± 0.07m 1.35 ± 0.05k 1.22 ± 0.03i 0.72 ± 0.05cd 0.71 ± 0.04cd 0.95 ± 0.04fg 0.97 ± 0.05fg 0.94 ± 0.05fg 0.92 ± 0.02f 0.67 ± 0.03bcd 0.67 ± 0.03bcd 0.74 ± 0.04de 0.75 ± 0.05de 3.14 ± 0.25o 3.14 ± 0.23o 3.19 ± 0.22o 1.99 ± 0.14m 2.58 ± 0.18n 2.46 ± 0.19n

N 147.05 N 155.03 N 173.91 N 465.11 N 434.78 N 408.16 N 238.09 N 200.00 N 198.01 N 194.17 N 145.98 N 150.37 N 158.73 N 235.29 N 281.69 N 219.78 N 322.58 N 215.05 N 200.00 N 178.57 N 151.51 N 162.60 N 161.12 N 204.08 N 270.27 N 273.97 N 317.46 N 270.27 N 181.81 N 192.30 N 118.34 N 116.27 N 108.10 N 106.95 N 105.26 N 104.71 N 105.82 N 105.82 N 104.16 N 106.95 N 148.14 N 163.93 N 277.77 N 281.69 N 210.52 N 206.18 N 212.76 N 217.39 N 298.50 N 298.50 N 270.27 N 266.66 N 63.69 N 63.69 N 62.69 N 100.50 N 77.51 N 81.30

Mash

Depectinized mash

Pomace

Raw juice

Clarified juice

Juice concentrate

a E1 is the extract from cranberry fruits and cranberry solids containing the most water-soluble phenolic compounds extracted with water/methanol (85/15, v/v). The extraction conditions used to extract E1 (water/methanol (85/15, v/v)) were similar to those employed for juice (water). b II(QR): Induction index of quinone reductase. c Values are means ± standard deviations. Within each column, means bearing the same lowercase letter are not significantly different (P N 0.05). d Concentration of the cranberry samples to double QR activity. e Chemoprevention index (CI) was calculated by dividing IC50 value with CD value. IC50, concentration to inhibit Hepa 1c1c7 cells growth by 50%, was evaluated by using Cell Proliferation Reagent WST-1 after a 24 h treatment in Hepa 1c1c7 cells: IC50 value N 200 mg/mL for all samples.

phenolic compounds of E2 increased significantly (P ≤ 0.05) between fruit and depectinized mash. The phenolic compounds of three extracts of pomace induced a weak QR induction. Although the three cranberry extracts are good QR inducers, our results indicate that the phenols present in aqueous extract show QR

inductions which are more important than those obtained with phenols present in solvent extracts. The reason is certainly the variation of solubility of compounds extracted in water or solvents, which is connected to their hydrophilic or hydrophobic character, although the three cranberry extracts contain phenolic substances

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Table 2 Quinone reductase induction by cranberry extract rich in apolar phenolic compounds (E2) obtained from samples collected at each step of the industrial juice process. Samplesa

Concentration in mg/mL (extracts) or % (v/v) (juices)

II(QR)/mg of dry matterb,c

II(QR)/mg of phenolb,c

CDc,d (mg/mL)

CIe

Fruit

200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39

1.33 ± 0.17a 2.51 ± 0.31cd 1.75 ± 0.21b 12.41 ± 1.09k 12.19 ± 1.13k 11.90 ± 0.78k 11.93 ± 0.85k 10.87 ± 1.07jk 11.50 ± 0.75k 9.50 ± 1.03hij 3.05 ± 0.48de 3.03 ± 0.47de 3.07 ± 0.34de 3.77 ± 0.47e 5.58 ± 0.44f 9.38 ± 0.71hi 8.38 ± 0.33h 6.78 ± 0.26g 6.96 ± 0.25g 5.54 ± 0.78f 4.77 ± 0.40f 9.36 ± 1.20hij 14.20 ± 0.25l 22.06 ± 1.07p 24.45 ± 2.90p 22.72 ± 1.63p 20.34 ± 1.22op 18.52 ± 1.32no 17.60 ± 0.45n 15.13 ± 0.12m 3.23 ± 0.35de 3.13 ± 0.30de 2.61 ± 0.29cd 2.53 ± 0.41cd 2.27 ± 0.34bc 2.51 ± 0.28cd 2.64 ± 0.47cd 2.51 ± 0.35cd 2.30 ± 0.32c 2.13 ± 0.18bc

8.47 ± 1.26d 15.98 ± 1.97f 11.14 ± 1.13e 79.04 ± 9.61lm 77.64 ± 7.47lm 75.79 ± 5.70lm 75.98 ± 4.58lm 69.23 ± 9.56kl 73.25 ± 4.80l 60.51 ± 3.58k 12.55 ± 0.56e 12.46 ± 0.62e 12.63 ± 0.66e 15.51 ± 1.52f 22.96 ± 1.40g 38.60 ± 4.11ij 34.48 ± 1.35i 27.90 ± 1.09h 28.64 ± 1.02h 22.79 ± 2.23g 21.01 ± 2.08g 41.01 ± 1.28j 62.55 ± 4.60k 97.18 ± 3.47n 107.70 ± 5.50o 100.08 ± 6.06no 89.60 ± 8.01mn 81.58 ± 1.40m 77.53 ± 7.34lm 66.65 ± 5.67kl 5.17 ± 0.42c 5.03 ± 0.15c 4.18 ± 0.24b 4.06 ± 0.27b 3.64 ± 0.33ab 4.02 ± 0.25b 4.23 ± 0.26b 4.02 ± 0.07b 3.69 ± 0.31ab 3.41 ± 0.22a

1.71 ± 0.09lm 1.44 ± 0.07j 1.62 ± 0.08klm 0.64 ± 0.05ab 0.66 ± 0.04b 0.70 ± 0.05bc 0.70 ± 0.05bc 0.77 ± 0.05cd 0.71 ± 0.05bc 0.85 ± 0.06de 1.42 ± 0.07j 1.42 ± 0.07j 1.41 ± 0.07j 1.38 ± 0.05j 1.19 ± 0.06hi 0.85 ± 0.07de 0.94 ± 0.06ef 1.10 ± 0.06gh 1.06 ± 0.06fg 1.18 ± 0.06hi 1.48 ± 0.09jk 1.24 ± 0.06i 1.02 ± 0.05ff 0.62 ± 0.05ab 0.54 ± 0.05a 0.62 ± 0.05ab 0.67 ± 0.03b 0.72 ± 0.05bc 0.79 ± 0.06cd 0.90 ± 0.04e 1.58 ± 0.06kl 1.60 ± 0.06kl 1.69 ± 0,07lm 1.75 ± 0.07mn 1.92 ± 0.08o 1.76 ± 0.08mn 1.67 ± 0.07lm 1.76 ± 0.07mn 1.87 ± 0.08no 2.03 ± 0.12o

N 116.95 N 138.88 N 123.45 N 312.50 N 303.03 N 285.70 N 285.70 N 259.74 N 281.69 N 235.29 N 140.84 N 140.84 N 141.84 N 144.92 N 168.06 N 235.29 N 212.76 N 181.81 N 188.67 N 169.49 N 135.13 N 161.29 N 196.07 N 322.58 N 370.37 N 322.58 N 298.50 N 277.77 N 253.16 N 222.22 N 126.58 N 125.00 N 118.34 N 114.28 N 104.16 N 113.63 N 119.76 N 113.63 N 106.95 N 98.52

Mash

Depectinized mash

Pomace

a

E2 is the extract from cranberry fruits and cranberry solids containing the most apolar phenolic compounds extracted with acetone/methanol/water (40/40/20, v/v). II(QR): Induction index of quinone reductase. c Values are means ± standard deviations. Within each column, means bearing the same lowercase letter are not significantly different (P N 0.05). d Concentration of the cranberry samples to double QR activity. e Chemoprevention index (CI) was calculated by dividing IC50 value with CD value. IC50, concentration to inhibit Hepa 1c1c7 cell growth by 50%, was evaluated by using Cell Proliferation Reagent WST-1 after a 24 h treatment in Hepa 1c1c7 cells: IC50 value N 200 mg/mL for all samples. b

which embrace many classes of compounds, ranging from phenolic acids, colored anthocyanins and simple flavonoids to complex flavonoids (Côté et al., 2010; Neto et al., 2006; Wu & Prior, 2005). Several studies have demonstrated the anticarcinogenic properties of phenolic phytochemicals such as gallic acid, caffeic acid, ferulic acid, catechin and quercetin (Mitscher, Telikepalli, McGhee, & Shankel, 1996; Yamada & Tomita, 1996). Cranberry extracts have been shown to have proapoptotic effects in human cancer cells by inhibiting ornithine decarboxylase (ODC) expression and inducing the xenobiotic detoxification enzyme quinone reductase in vitro (Ramos, Alia, Bravo, & Goya, 2005). However, unlike our study which showed a strong chemopreventive activity without cytotoxicity against Hepa 1c1c7 cells, literature has reported an antiproliferative activity of the cranberry phenolic compounds against many tumor cells. In vitro studies have revealed encouraging potential for cranberry products, extracts, and individual components to inhibit cancer (Neto, 2007). Seeram et al. (2004) reported that cranberry extract and its purified phenolics, including its proanthocyanidins, anthocyanins, and other flavonoids, inhibited the proliferation of human oral, colon and prostate tumor cells in vitro. According to He and Lui (2006), quercetin and 3,5,7,3′,4′-pentahydroxyflavonol-3-O-β-D-glucopyranoside,

compounds isolated from cranberry extract, showed potent inhibitory activity toward the proliferation of MCF-7 cells, with EC50 values of 137.5 ± 2.6, and 23.9 ± 3.9 μM, respectively. Other studies reporting in vitro antiproliferative activity of flavonoid-rich extracts from cranberry have implicated proanthocyanidins as contributing to these activities (Neto, 2007). Proanthocyanidin-rich extracts proved cytotoxic to ovarian cancer cells at concentrations as low as 79 mg/mL and significantly increased the chemotherapeutic activity of paraplatin (Singh et al., 2007). A proanthocyanidin fraction obtained from whole cranberry fruit was observed to selectively inhibit the growth of H460 human large cell lung carcinoma, HT-29 colon adenocarcinoma, and K562 chronic myelogenous leukemia cells in their panel of 8 tumor cell lines. Other in vitro evidence suggests that cranberry phenolics could decrease cell invasion and metastasis by inhibiting MMPs activity, and inhibiting the inflammatory processes including cyclooxygenase (COX) activity (Neto, 2007). Overall, the tumor inhibition by cranberry is likely to involve synergistic activities between the cranberry major phytochemicals, anthocyanins, flavonols and proanthocyanidins. Also, the data obtained reveal that the technological process to manufacture cranberry juice influenced the QR inducer potencies of

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S. Caillet et al. / Food Research International 44 (2011) 902–910

Table 3 Quinone reductase induction by cranberry extract rich in anthocyanins (E3) obtained from samples collected at each step of the industrial juice process. Samplesa

Concentration in mg/mL (extracts) or % (v/v) (juices)

II(QR)/mg of dry matterb,c

II(QR)/mg of phenolb,c

CDc,d (mg/mL)

CIe

Fruit

200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39 200.00 100.00 50.00 25.00 12.50 6.25 3.12 1.56 0.78 0.39

5.57 ± 0.82f 6.02 ± 1.09fg 13.27 ± 0.62l 19.04 ± 1.90o 17.66 ± 0.90o 15.02 ± 1.06mn 13.63 ± 0.95lm 13.27 ± 0.60l 14.83 ± 1.29lmn 11.39 ± 0.10k 4.94 ± 0.35f 5.12 ± 0.15f 5.07 ± 0.17f 8.41 ± 0.45h 11.61 ± 0.45k 13.01 ± 0.47l 13.87 ± 1.20lmn 11.65 ± 0.38k 10.66 ± 0.67jk 9.26 ± 1.01hij 5.74 ± 0.88f 5.89 ± 0.97f 8.72 ± 0.69hi 7.16 ± 0.40g 10.43 ± 0.45j 8.94 ± 0.33hi 13.20 ± 0.24l 9.42 ± 0.24i 9.56 ± 0.12i 8.43 ± 0.46h 0.64 ± 0.12a 0.67 ± 0.07a 0.66 ± 0.09a 0.63 ± 0.19a 0.68 ± 0.04a 0.64 ± 0.23a 2.04 ± 0.32b 3.55 ± 0.25e 2.54 ± 0.26cd 2.98 ± 0.26d

55.70 ± 5.50jk 60.26 ± 4.90k 132.77 ± 6.10m 190.40 ± 19.09o 176.65 ± 16.60no 150.20 ± 13.61mn 136.31 ± 9.01m 132.72 ± 6.01m 148.32 ± 14.90mn 113.90 ± 10.99l 24.21 ± 1.07f 25.09 ± 0.97f 24.85 ± 0.83f 41.22 ± 2.20i 56.91 ± 2.22k 63.77 ± 4.30k 67.99 ± 9.01k 57.10 ± 2.36k 52.25 ± 1.32j 45.39 ± 5.21ij 25.06 ± 0.87f 25.72 ± 1.01f 38.07 ± 3.62hi 31.26 ± 3.23gh 45.54 ± 5.08ij 39.73 ± 1.06i 57.64 ± 2.80k 41.13 ± 2.06i 41.74 ± 1.52i 36.81 ± 3.72hi 1.11 ± 0.23a 1.16 ± 0.14a 1.15 ± 0.15a 1.10 ± 0.32a 1.18 ± 0.07a 1.12 ± 0.40a 3.56 ± 0.19b 6.20 ± 0.43e 4.44 ± 0.41c 5.20 ± 0.32d

2.00 ± 0.17lm 1.85 ± 0.12kl 0.84 ± 0.05cd 0.58 ± 0.03a 0.63 ± 0.05a 0.74 ± 0.05bc 0.82 ± 0.06cd 0.84 ± 0.04d 0.75 ± 0.05bcd 0.98 ± 0.07e 2.95 ± 0.32n 2.36 ± 0.24m 2.22 ± 0.25m 1.33 ± 0.11gh 0.99 ± 0.08e 0.85 ± 0.05d 0.80 ± 0.07bcd 0.96 ± 0.04e 1.05 ± 0.09ef 1.20 ± 0.10fg 3.35 ± 0.32n 3.02 ± 0.30n 1.78 ± 0.15jk 2.17 ± 0.19lm 1.49 ± 0.11hi 1.72 ± 0.12jk 0.96 ± 0.07e 1.63 ± 0.14ij 1.61 ± 0.13ij 1.85 ± 0.16kl 27.14 ± 1.73st 22.39 ± 1.56qr 23.53 ± 1.92qrs 28.96 ± .2.18t 21.09 ± 1.64q 26.45 ± 2.55rst 4.88 ± 0.36p 2.30 ± 0.21m 4.01 ± 0.37o 3.51 ± 0.28no

N 100.00 N 108.11 N 238.09 N 344.82 N 317.46 N 270.27 N 243.90 N 238.09 N 266.66 N 204.08 N 67.79 N 84.74 N 90.09 N 150.37 N 202.02 N 235.29 N 250.00 N 208.33 N 190.47 N 166.66 N 59.70 N 66.22 N 112.35 N 92.16 N 134.22 N 116.27 N 208.33 N 122.69 N 124.22 N 108.10 N 7.36 N 8.93 N 8.49 N 6.90 N 9.48 N 7.56 N 40.98 N 86.95 N 49.87 N 56.98

Mash

Depectinized mash

Pomace

a

E3 is the extract from cranberry fruits and cranberry solids containing anthocyanins extracted with methanol/water/acetic acid (85/14.5/0.5, v/v/v). II(QR): Induction index of quinone reductase. c Values are means ± standard deviations. Within each column, means bearing the same lowercase letter are not significantly different (P N 0.05). d Concentration of the cranberry samples to double QR activity. e Chemoprevention index (CI) was calculated by dividing IC50 value with CD value. IC50, concentration to inhibit Hepa 1c1c7 cell growth by 50%, was evaluated by using Cell Proliferation Reagent WST-1 after a 24 h treatment in Hepa 1c1c7 cells: IC50 value N 200 mg/mL for all samples. b

cranberry bioactive compounds, since juice concentrate showed that QR induction was much lower than those observed with cranberry fruit extracts, particularly with E1 in which the extraction conditions were similar to those used to obtain the juice. Food processing involves changes in structural integrity of the plant material and this produces both negative and positive effects. Juice processing which involves juice extraction, heating steps and juice clarification treatment has an impact on the putative phenolic composition as well as the properties of berries (Pappas & Schaich, 2009). The cellular decompartmentation generally associated with cranberry juice processing may provoke reactions that lead to the formation of new compounds through the transformation and degradation of the anthocyanins, and other phenolics (Skrede, Wrolstad, & Durst, 2000). Occurrence of these reactions appears mainly governed by factors such as pH, storage temperature, light and oxygen, and type and concentration of anthocyanins. But enzymatic degradation and interactions with other food components (fibers, co-pigments with other phenolics) are no less important. In whole fresh cranberries anthocyanins are present at much higher levels than flavonols, but the reverse is true in cranberry juice (Pappas & Schaich, 2009). Anthocyanins are chemically less stable than flavonols, and are

particularly sensitive to oxidation and light degradation during handling and processing. Anthocyanin interactions with ascorbic acid radicals may account for some degradation, but while general types of reactions that cause anthocyanin loss are recognized, specific reactions leading to loss of color and biological properties have not been elucidated (Côté et al., 2010). While flavonol retention during cranberry juice extraction and processing appears to be markedly superior to that of anthocyanins, some flavonol degradation does occur (Pappas & Schaich, 2009). When juices were made, considerable reductions in flavonol contents were observed since only 15% of quercetin and 30% of myricetin (compared to 2–10% for anthocyanins) present in unprocessed berries were retained in juices made by common juice extraction methods (Häkkinen, Kärenlampi, Mykkänen, & Törrönen, 2000). This was due to the fact that the skins of the berries were removed by filtering, and certain flavonols and anthocyanins are known to be concentrated mainly in the skin of fruits (Côté et al., 2010). Proanthocyanidins, monomers, dimers, and trimers account for a higher proportion in commercial pressed cranberry juice than in fresh berries (Prior, Lazarus, Cao, Muccitelli, & Hammerstone, 2001), while higher polymers decrease (Gu et al., 2003). Comparing commercially available juices to whole berries,

S. Caillet et al. / Food Research International 44 (2011) 902–910

benzoic and phenolic acids show variable processing stability (Zhang & Zuo, 2004; Zuo, Wang, & Zhan, 2002). For example, salicylic acid and its isomer p-hydroxybenzoic acid are present at comparable levels in whole fresh cranberries (23.2 μg/g and 21.6 μg/g respectively), but in commercially processed juice, salicylic acid was about 50 times more prevalent than its isomer (3.11 μg/mL and 0.07 μg/mL respectively). Conversions also occur: caffeic acid conjugates of quinic acid are found in processed cranberry juice, but have not been isolated from fresh juice or whole berries (Chen, Zuo, & Deng, 2001; Harnly et al., 2006). The different classes of phenolic compounds had varying susceptibilities to degradation with different processing operations — the highest losses occurred with milling and depectinization, which tend to aggravate native polyphenol oxidase (PPO) activity. Also, Meyer, Let, and Landbo (2003) reported how industrial clarification treatment of blackcurrant juice to remove cloud and sediments decreased the contents of four major anthocyanins by 19–29%. When juice was concentrated, polyphenols and anthocyanin losses were relatively low, except for the procyanidins, which showed marked reduction (Skrede et al., 2000). The amounts of procyanidins in the initial pressed juice was about 40% of that in the fruit, and considerable degradation occurred with an approximate additional 20% loss, when juice was concentrated (Sapers, Jones, & Maher, 1983). Also, pomace, which mainly consists of fruit skins and seeds, is also a rich source of phenolic compounds (Lu & Foo, 1999). However, several phenolics that are found in pomace and other plant products exist in conjugated forms either with sugars (primarily glucose), as glycosides, or as other moieties. This conjugation occurs via the hydroxyl groups of the phenolics, which reduces their ability to function as good antioxidants or antimutagens, because availability of the hydroxyl groups on the phenolic rings is important for resonance stabilization of free radicals (Lu & Foo, 2000). 4. Conclusion This study has shown that bioactive compounds of cranberry can activate significantly (P ≤ 0.05) detoxification enzymatic systems (Phase II). The QR induction for all samples varied with the concentration and there was an optimal concentration for which the QR induction was maximal. However, the polarity of the phenolic compounds has influenced the QR induction. Phenols obtained in aqueous extract stimulated more the QR activity than phenols obtained in solvent extracts. Also, the technological process to manufacture cranberry juice influenced the QR inducer potencies of cranberry bioactive compounds. Thus, the ability of phenols to stimulate the QR activity has been reduced continuously and significantly (P ≤ 0.05) during the technological process; the cancer chemopreventive effect decreased in this order: fruitN mash= depectinized mashN clarified juice N raw juiceN pomaceN juice concentrate. Especially, it appears that conditions of the evaporation to obtain a juice concentrate exerted a significant effect (P ≤ 0.05) on inducer potencies of bioactive molecules. At the present time, observations of cancer chemopreventive effect differences between whole berries and processed juice can mostly serve to generate interest to undertake detailed studies to characterize the cranberry phenolic profile in the juice processing method in order to better understand the significant reduction (P ≤ 0.05) of cancer chemopreventive properties; the data are inadequate to provide explanations, although some speculative explanations may be offered. An understanding of how factors interact, and the putative degradation mechanisms involved, can help improve the occurrence of bioactive phenolics in cranberry juice. Such knowledge could lead to more judicious processing conditions and the production of a more nutritious cranberry juice with improved health benefits. As the health effects from cranberry consumption become more concretely linked to specific chemical components, it will become increasingly important to understand in detail how processing and storage alter key phytochemicals and to develop new processing approaches that optimize their

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conservation and maximize health benefits for consumers. The critical role of cranberry phenolic compounds in health effects argues for the development of new approaches to retain and stabilize these phytonutrients during the processing of cranberry products.

Acknowledgments This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and by Atoka Cranberries Inc. (Manseau, QC, Canada).

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