Process Biochemistry 40 (2005) 22252238 www.elsevier.

com/locate/procbio

Cranberry phenolics-mediated antioxidant enzyme response in oxidatively stressed porcine muscle

D.A. Vattem1, R. Randhir, K. Shetty*
Laboratory of Food Biotechnology, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA Received 26 July 2004; accepted 21 September 2004

Abstract The antioxidant response mechanism by which phenolic phytochemicals show their positive benets in animal systems is not very well understood. The ability of cranberry juice powder (CP), ellagic acid (EA), rosmarinic acid (RA) and their synergies to mediate a cellular antioxidant response in oxidatively stressed porcine muscle tissue was investigated. Results indicated that treatment with CP, EA, RA and their synergies reduced or helped counter oxidative stress as indicated by the formation of malondialdehyde (MDA). It was also observed that CP, EA, RA and their synergies stimulated the pentose phosphate pathway (PPP) linked to the accumulation of free proline suggesting a possible coupling of proline biosynthesis with PPP. This coupling of proline-linked pentose phosphate pathway could be involved in the stimulation of cellular antioxidant enzymic response by replenishing the cellular needs for NADPH2. As a consequence these exogenous phenolic treatments resulted in the stimulation of cellular antioxidant enzyme systems involving superoxide dismutase (SOD), catalase (CAT) and peroxidase, which correlated well with the decreased MDA formation. This suggested that exogenously treated phenolic phytochemicals could be reducing the oxidative stress in porcine muscle by stimulating the PPP linked to proline biosynthesis and by the activation of the cellular antioxidant enzyme system. The results also suggest that pure exogenous phenolics, EA and RA appeared to be effective when they were present in a cranberry phenolic background, suggesting a possible synergistic mode of action between EA, RA and cranberry phenolics in mediating a cellular antioxidant enzyme response. # 2004 Elsevier Ltd. All rights reserved.
Keywords: Phenolic phytochemicals; Antioxidants; Cranberry; Ellagic acid; Rosmarinic acid; Cellular antioxidant enzyme response; Pentose phosphate pathway; Proline biosynthesis

1. Introduction Recent epidemiological studies have indicated that diets rich in fruits and vegetables are associated with lower incidences of oxidation-linked diseases such as cancer, cardiovascular disease and diabetes [1,2]. These protective effects of fruits and vegetables are now linked to the presence of antioxidant vitamins and phenolic phytochemicals having antioxidant activity [3,4]. The ability of dietary antioxidants in managing diseases manifested by oxidative stress is not clearly understood. Most phenolic phytochemicals that have
* Corresponding author. Tel.: +1 413 545 1022; fax: +1 413 545 1262. E-mail address: kalidas@foodsci.umass.edu (K. Shetty). 1 Present address: Nutritional Biomedicine and Biotechnology, Texas State University, San Marcos, TX 78666, USA. 0032-9592/$ see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.09.001

positive effect on health are believed to be functioning by countering the effects of reactive oxygen species (ROS) species generated during cellular metabolism [5] (Fig. 1). Phenolic phytochemicals due to their phenolic ring and hydroxyl substituents can function as effective antioxidants due to their ability to quench free electrons. It is therefore believed that dietary phenolic antioxidants can scavenge harmful free radicals and thus inhibit their oxidative reactions with vital biological molecules [5] and prevent development of many physiological conditions, which can manifest into disease [69]. Recently it has been proposed that the other mechanism by which phenolic phytochemicals function in countering the oxidative stress could be by stimulating the synthesis and/or replenishment of cellular antioxidant status or by inducing and improving host cellular antioxidant enzyme response through superoxide dismutase and catalase

2226

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

Fig. 1. Biological formation of reactive oxygen species. SOD: superoxide dismutase; HOBr/HOCl: hypo(bromide/chloride; EPO: eosinophil peroxidase and NO: nitric oxide.

systems [10,11] (Fig. 2). The synthesis and reduction of cellular antioxidants such as glutathione, as well as the efcient operation of cellular antioxidant enzyme response pathways depend on the availability of reducing equivalents such as FADH2 and NADPH2 [1214]. The cellular needs for NADPH2 can be met by stimulating the pentose phosphate pathway, which commit glucose towards making sugar phosphates for anabolic reactions and in the process regenerate NADPH2 [1517]. The stimulation of pentose phosphate pathway could further be coupled to the biosynthesis of proline that is made from glutamic acid

[11,18] and also requires NADPH2 [1921]. It has been postulated that dietary phenolic phytochemicals can stimulate the biosynthesis of proline in eukaryotic model systems by channeling TCA cycle intermediates such as a-ketoglutarate towards glutamic acid and then to proline biosynthesis, which requires NADPH2 (Fig. 3; [10]). It is hypothesized that the induction of proline biosynthesis can further stimulate the pentose phosphate pathway [10,11] to make more NADPH2, which can be used for replenishing the cellular pool of antioxidants and for efcient functioning of the cellular antioxidant enzyme cascades [10,11].

Fig. 2. The antioxidant defense response of the cell carried out by enzymatic as well as the non enzymatic antioxidants. SOD: superoxide dismutase; CAT: catalase; PER: peroxidase; AP: ascorbate peroxidase; GR: glutathione reductase; GSSG: oxidized glutathione; GSH: reduced glutathione; TP: tocophenrol; DHAR: dehydroascorbate reductase; ASA: reduced ascorbate; DHA: dehydroascorbate; MDA: monodehydroascorbate and MDHA: monodehydroascorbate reductase.

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

2227

Fig. 3. Proline-linked pentose phosphate pathway in eukaryotes for regulating antioxidant response. P5C: pyrroline-5-carboxylate; SOD: superoxide dismutase; CAT: catalase and PER: peroxidase.

The mechanism by which these fruit phenolics carry out their functions is a topic of growing interest as they have recently been linked to a number of health benets [3,22 24]. Cranberry and their products have long been known to have benecial effects on human health and have been used in managing infections of the urinary and digestive tracts. Recent research has also shown that cranberry and its extracts have anti-cancer properties [25] and were able to reduce the risk factors responsible for the development of cardiovascular diseases [26,27]. Although it is now believed that these benecial functional properties of cranberry are linked to specic phenolic phytochemicals, their exact mechanism of functionality is not very well understood. Recent research has also suggested that the phytochemical prole in which a specic functional phenolic is present plays an important role in determining its functionality [28,29]. This is believed to occur due to the synergistic interaction between phenolic phytochemicals in the mixture, which mutually enhance their functionality [11]. Therefore, the aim of this research was to investigate the effect of cranberry phenolics and their synergies with functional biphenyls ellagic acid and rosmarinic acid on modulating cellular antioxidant enzyme response to maintain redox homeostasis in oxidatively stressed porcine muscle tissue. The changes in the cellular antioxidant

enzyme response pathway mediated through the SOD/CAT system was used as a marker for redox status of the tissue. The stimulation of PPP in supporting the activation cellular antioxidant enzyme response and the possible link to proline biosynthesis in driving the PPP was also investigated.

2. Material and methods Freshly harvested porcine muscle (fatless, sirloin) was obtained from Big-Y Supermarkets (Hadley, MA). The tissue was homogenized mildly to disintegrate the tissue and 1 g of the tissue was transferred into a treatment vial. Potassium phosphate buffer (2.5 ml of 0.1 M) of pH 7.5 containing the treatments described in Table 1 were added to the vial. The vials were then incubated at 4 8C and sampled after every 10 h for 40 h. Cranberry powder (CP): Cranberry powder (Decas Cranberry Products Inc., Carver, MA) was added to 1 g of porcine muscle tissue homogenate to give a nal total phenolic concentration of 1 mg/ml. Ellagic acid (EA) and rosmarinic acid (RA): Ellagic acid and rosmarinic acid (Sigma Chemicals, St. Louis, MO) were

2228

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

Table 1 Different treatments used in the porcine muscle study Treatment Phytochemical (phenolic basis, 1 mg/ml) H2 O2 (mM) 0 0 0 0 0 0 100 100 100 100 100 100

Unstressed porcine muscle Control None CP Cranberry powder EA Ellagic acid RA Rosmarinic acid CPEA Cranberry powder + ellagic acid CPRA Cranberry Powder + rosmarinic acid Oxidatively stressed porcine muscle None H2O2 CP + H2O2 Cranberry powder EA + H2O2 Ellagic acid Rosmarinic acid RA + H2O2 CPEA + H2O2 Cranberry powder + ellagic acid CPRA + H2O2 Cranberry powder + rosmarinic acid

Na2CO3 was added to the reaction mixture and allowed to stand for 60 min. The absorbance was read at 725 nm. The absorbance values were converted to total phenolics and were expressed in milligrams equivalents of gallic acid per grams fresh weight (FW) of the sample. Standard curves were established using various concentrations of gallic acid in 95% ethanol. 2.3. Antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) inhibition assay [32] To 3 ml of 60 mM DPPH in ethanol, 500 ml of porcine muscle extract was added, the decrease in absorbance was monitored at 517 nm until a constant reading was obtained. The readings were compared with the controls, which contained 500 ml of 95% ethanol instead of the extract. The % inhibition was calculated by: % inhibition Acontrol Aextract 517 517 100 Acontrol 517

added to 1 g of porcine muscle tissue homogenate to give a nal concentration of 1 mg/ml. Cranberry powder synergies: Based on previous studies, CP synergies with EA and RA were prepared by replacing 30% of phenolics in cranberry powder with equivalent concentration of ellagic acid (CPEA) or rosmarinic acid (CPRA) [30]. These synergy mixtures were added to 1 g of porcine muscle tissue homogenate to give a nal total phenolic concentration of 1 mg/ml. 2.1. Sample extraction Vials containing 1 g of porcine tissue homogenate with the treatments was further homogenized thoroughly at 2000 rpm for 2 min using a tissue homogenizer (Biospec products, OK). The sample was centrifuged at 13,000 rpm for 15 min at 25 8C and stored on ice. The supernatant was used for further analysis. The total phenolic content and antioxidant activity was measured in the porcine tissue homogenate by rst centrifuging the muscle tissue out of the treatment buffer at 13,000 rpm for 15 min at 25 8C. The pellet was resuspended in 2.0 ml of 0.1 M potassium phosphate buffer of pH 7.5. This was then homogenized thoroughly at 2000 rpm for 2 min using a tissue homogenizer (Biospec products, OK). The sample was again centrifuged at 13,000 rpm for 15 min at 25 8C and stored on ice. The supernatant was used for estimating phenolics and antioxidant activity. 2.2. Total phenolics assay Total phenolics were determined by an assay modied from Shetty et al. [31] and was used to determine the amount of phenolic metabolites absorbed by the porcine tissue. Briey, 1 ml of supernatant was transferred into a test tube and mixed with 1 ml of 95% ethanol and 5 ml of distilled water. To each sample 0.5 ml of 50% (v/v) FolinCiocalteu reagent was added and mixed. After 5 min, 1 ml of 5%

2.4. Protein assay Protein content was measured by the method of Bradford [33]. The dye reagent concentrate (Bio-Rad protein assay kit II, Bio-Rad Laboratory, Hercules, CA) was diluted 1:4 with distilled water. Five milliliter of diluted dye reagent was added to 100 ml porcine muscle extract. After vortexing and incubating for 5 min, the absorbance was measured at 595 nm against 5 ml reagent blank and 100 ml buffer using a UVvis Genesys spectrophotometer (Spectronic Instruments Inc., Rochester, NY). 2.5. Proline assay Proline content was determined according to the modied method of Bates et al. [34]. To 750 ml of muscle tissue homogenate 1.25 ml of 3% sulphosalicylic acid was added and vigorously stirred on a vortex mixture. The mixture was then centrifuged at 13,000 rpm for 10 min. One milliliter of the supernatant was then added into a test tube to which 1 ml of glacial acetic acid and 1 ml of freshly prepared acid ninhydrin solution were added (1.25 g ninhydrin dissolved in 30 ml of glacial acetic acid and 20 ml of 6 M orthophosphoric acid). Tubes were incubated in a water bath for 1 h at 100 8C and then allowed to cool to room temperature. Two milliliter of toluene was added and mixed on a vortex mixture for 20 s in a fume hood. The test tubes were allowed to stand at least for 10 min to allow the separation of toluene and aqueous phase. The toluene phase was carefully pipetted out into a glass test tube and the absorbance was measured at 520 nm in a spectrophotometer (Spectronic Instruments Inc., Rochester, NY). The concentration of proline was calculated from a proline standard curve. The concentration of proline was expressed as mmol/g FW.

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

2229

2.6. Malondialdehyde (MDA) assay Malondialdehyde was measured by modifying the method discussed by Tamagnone et al. [35]. Briey, in a test tube 200 ml of the tissue homogenate was mixed with 800 ml of water, 500 ml of 20% (w/v) trichloroacetic acid and 1 ml of 10 mM thiobarbutyric acid. The test tubes were incubated for 30 min at 100 8C and then centrifuged at 13,000 rpm for 10 min. The absorbance of the supernatant was measured at 532 nm and the concentration of MDA was calculated from its molar extinction coefcient (e) 156 mmol1 cm1 and expressed as mmol/g FW. 2.7. Glucose-6-phosphate dehydrogenase (G6PDH) assay A modied version of the assay described by Deutsch [36] was followed. The enzyme reaction mixture containing 5.88 mmol b-NADP, 88.5 mmol MgCl2, 53.7 mmol glucose6-phosphate, and 0.77 mmol maelamide was prepared. This mixture was used to obtain baseline (zero) of the spectrophotometer reading at 339 nm wavelength. To 1 ml of this mixture, 50 ml of the sample was added. The rate of change in absorbance per minute was used to quantify the enzyme in the mixture using the extinction coefcient of NADPH2 (6.22 mM1 cm1). 2.8. Total peroxidase (TPX) activity A modied version of the assay developed by Laloue et al. [37] was used. Briey, the enzyme reaction mixture contained 0.1 M potassium phosphate buffer (pH 6.8), 50 mM guaiacol solution and 0.2 mM hydrogen peroxidase. To 1 ml of this reaction mixture, 50 ml of enzyme extract was added. The absorbance was noted at zero time and then after 5 min. The rate of change in absorbance per minute was used to quantify the enzyme in the mixture using the extinction coefcient of the oxidized product tetraguaiacol (26.6 mM1 cm1). 2.9. Superoxide dismutase (SOD) assay A competitive inhibition assay was performed that used xanthinexanthine oxidase-generated superoxide to reduce nitroblue tetrazolium (NBT) to blue formazan. A spectrophotometric assay of SOD activity was carried out by monitoring the reduction of NBT at 560 nm [38]. The reaction mixture contained 13.8 ml of 50 mM potassium phosphate buffer (pH 7.8) containing 1.33 mM DETAPAC; 0.5 ml of 2.45 mM NBT; 1.7 ml of 1.8 mM xanthine and 40 IU/ml catalase. To 0.8 ml of reagent mixture 100 ml of phosphate buffer and 100 ml of xanthine oxidase was added. The change in absorbance at 560 nm was measured every 20 s for 2 min and the concentration of xanthine oxidase was adjusted to obtain a linear curve with a slope of 0.025 absorbance per minute. The phosphate buffer was then replaced by the enzyme extract and the change in absorbance

was monitored every 20 s for 2 min. One unit of SOD was dened as the amount of protein that inhibits NBT reduction to 50% of the maximum. 2.10. Catalase (CAT) assay A method originally described by Beers and Sizer [39] was used to assay the activity of catalase. Briey, to 1.9 ml of distilled water 1 ml of 0.059 M hydrogen peroxide (H2O2) (Merck Superoxol or equivalent grade) in 0.05 M potassium phosphate, pH 7.0 was added. This mixture was incubated in a spectrophotometer for 45 min to achieve temperature equilibration and to establish a control rate. To this mixture 0.1 ml of diluted enzyme was added and the disappearance of peroxide was followed spectrophotometrically by recording the decrease in absorbance at 240 nm for 2 3 min. The change in absorbance DA240/min from the initial (45 s) linear portion of the curve was calculated. One unit of catalase activity was dened as amount that decomposes 1 mmol of H2O2 Units=mg DA240 =min 1000 43:6 mg enzyme=ml of reaction mixture

2.11. Statistical analysis All experiments were performed at least in duplicates. Analysis at each time point from each experiment was carried out in duplicate or triplicate. Means, standard errors and standard deviations were calculated from replicates within the experiments and analyses using Microsoft Excel XP.

3. Results 3.1. Total absorbed phenolics and antioxidant activity After the treatments with cranberry powder, ellagic acid, rosmarinic acid and their synergies (CPEA and CPRA) the amount of phenolics absorbed into the porcine tissue was assayed using the FolinCiocalteu assay. It was observed that the basal phenolic content in the porcine muscle was around 0.8 mg/g FW (Fig. 4). In the control sample, which did not have any phytochemical treatment and in the H2O2 alone treated tissue sample the value of phenolics did not change over the course of incubation. In the other samples that were incubated with the phenolic treatments it was observed that there was a rapid increase in the total amount of phenolics in the porcine tissue after 2 h, which then remained constant at this higher level for the remaining period of incubation. Phenolics were also absorbed in the porcine muscle tissue that was stressed with H2O2. Higher amounts of total phenolics were absorbed from the biphenyls-containing treatment buffer when the porcine muscle tissue was stressed with H2O2 (Fig. 4).

2230

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

Fig. 4. Total soluble phenolics absorbed by (A) unstressed and (B) stressed porcine muscle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid and their synergies.

The antioxidant properties of the phenolic muscle extract was measured as a function of its DPPH radical inhibition capacity. The DPPH radical inhibition (DRI) of the muscle tissue extracts followed a similar trend to the total phenolics absorbed for both H2O2 stressed and unstressed treatments. The DRI of the muscle extracts rapidly increased within the rst 2 h of the treatment and remained constant for the remaining course of incubation at the higher level (Fig. 5). The DRI of the tissue extracts that were treated with the phenolic phytochemicals alone was much higher than the tissue that was incubated with phenolic phytochemicals and stressed with H2O2 (Fig. 5). The DRI of the control and the H2O2 treatments, which did not contain any phenolic treatment remained constantly low for the course of the incubation. 3.2. Malonaldehyde content The malondialdehyde content of the porcine muscle samples was measured to study the extent of membrane degradation as a result of oxidative stress. In general, it was observed that the MDA content of the tissues increased over the course of incubation and reached a maximum after 40 h (Fig. 6). Control tissue samples, which were not phenolic

treated showed the highest MDA formation. The amount of MDA formed when only the CP, EA, RA and their synergies was used as treatments did not show any signicant difference between each other but were lower than the control (Fig. 6). The amount of MDA formed when the tissues were stressed with H2O2 was much higher than compared to the non-H2O2 stressed tissues. In the tissue samples, which were stressed with H2O2 but did not contain any phytochemical treatment, the amount of MDA formed was highest. The MDA content increased gradually until 10 h after which the rate of increase of MDA was almost exponential (Fig. 6). The porcine tissue, which was stressed with H2O2 but also contained phenolic extracts showed a much different trend. It was observed that for all the tissue samples that were stressed with H2O2 and given a phenolic treatment, the amount of MDA increased gradually until about 1012 h after which the rate of increase in MDA formation was much higher until about 2022 h. After 20 h the rate of increase of MDA formation was lower (Fig. 6). This decrease in the rate of MDA formation was lowest for CPEA and CPRA treatment followed by the porcine tissue stressed with H2O2 in the presence of CP. Among the tissue samples that were stressed with H2O2 in the presence of phenolic treatments, highest MDA was formed when EA was

Fig. 5. Antioxidant activity of (A) unstressed and (B) stressed porcine muscle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid and their synergies.

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

2231

Fig. 6. Changes in the malonaldehyde content of (A) unstressed and (B) stressed porcine muscle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid and their synergies.

used. This increase was however, much lower than the porcine tissue, which was stressed with H2O2 alone and did not have any phenolic treatment (Fig. 6). 3.3. Proline content The changes in proline content in all the different porcine muscle tissue samples were monitored during the course of incubation. The proline content in all the different treatments increased over the course of incubation. The control sample, which was not incubated with a phenolic treatment or stressed with H2O2 showed a linear increase in the amount of proline (Fig. 7). The proline content increased gradually from 0 to 2 h reaching a maximum value after 40 42 h of incubation. The proline content in the control unstressed porcine tissue samples showed the lowest increase. The porcine muscle tissues incubated with only CP, EA, RA and their synergies showed a different trend. Here, the rate of increase in proline content was rapid when the tissue was incubated for 1012 h after which the rate of increase in proline content gradually decreased for the remaining incubation time (Fig. 7). This rate of change in the

formation of proline during the course of incubation followed almost hyperbolic rate kinetics. The rate of increase in the formation of proline was lowest when tissues were incubated with EA alone. There was no signicant change in the amount of proline formed when the porcine tissue was incubated with CP, RA and CPRA alone (Fig. 7). When the porcine tissue was stressed with H2O2 it was observed that the rate of increase in the proline content was signicantly different compared to the tissues, which were not stressed with H2O2 (Fig. 7). When the porcine tissue was stressed with H2O2 only without any phenolic treatment, the proline content increased rapidly until 1012 h after which it remained constant for the remaining incubation time. The nal late stage increase in proline content was lowest among all the stressed and unstressed porcine tissues without phytochemical treatment (Fig. 7). For the porcine tissue samples that were stressed with H2O2 along with a phenolic phytochemical treatment the rate of change of proline content was similar to the unstressed tissue samples with phenolic phytochemicals. The amount of proline formed rst increased rapidly for 1012 h after which the rate of increase slowly decreased for the remaining time of

Fig. 7. Changes in the proline content of (A) unstressed and (B) stressed porcine muscle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid and their synergies.

2232

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

Fig. 8. Changes in the G6PDH activity in (A) unstressed and (B) stressed porcine muscle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid and their synergies.

incubation (Fig. 7). Highest amounts of proline were formed when CPEA and CPRA were used as phenolic treatments in the porcine tissues stressed with H2O2. For all the treatments the maximum proline content was reached after 3032 h incubation after which the proline content remained constant (Fig. 7). Only, when CP alone was present with the H2O2 stressed tissue the amount of proline continued to increase until the end of incubation period. The proline content in H2O2 stressed porcine muscle incubated with EA and RA was lower than other phytochemical treatments (Fig. 7). 3.4. Glucose-6-phosphate dehydrogenase (G6PDH) activity The G6PDH activity of the porcine muscle was assayed in order to measure the stimulation of pentose phosphate pathway in response to phenolic treatments. In the control samples, which were not incubated with phenolic extracts, the G6PDH activity increased slightly until 2022 h after which it started to decline (Fig. 8). In the porcine tissue samples, which were given phytochemical treatment but not stressed with H2O2 the G6PDH activity gradually increased until about 1012 h after which they showed a sharp increase in the activity. This increase was highest for CPEA and CP RA treated porcine muscle extracts. The next highest increase in the G6PDH activity was obtained in the porcine muscle samples that were incubated with CP extracts, which had also reached its maximum value after 2022 h of incubation (Fig. 8). The G6PDH activity gradually started to decline for the remaining duration of incubation until 40 42 h. The porcine muscle tissue incubated with pure EA behaved similarly, however, the rate of increase of the activity in this case was much lower than the porcine muscle tissues incubated with cranberry treatments (Fig. 8). Among all the treatments, the changes in the G6PDH activity in porcine muscle tissue incubated with RA showed a different trend. The G6PDH activity in this sample continued to

increase gradually until about 3032 h at which the activity reached its maximum value. The G6PDH activity then declined for the remaining period of incubation. The G6PDH activity was assayed in the porcine muscle tissues stressed with H2O2 alone behaved similar to the control tissue. The G6PDH activity after increasing slightly until 2022 h declined for the remaining period of incubation. However, the rate of decline in the G6PDH activity was more rapid in the H2O2 stressed porcine muscle compared to the control (Fig. 8). The trends for the changes in the G6PDH activity when the porcine muscle tissue was stressed with H2O2 and incubated with pure EA, RA and CP were similar. It was observed that the G6PDH activity in these samples increased gradually from the basal values for 1012 h. The rate of increase in G6PDH activity after 1012 h of incubation was higher and peaked after 3032 h of incubation before declining for the subsequent period of incubation (Fig. 8). The G6PDH activity in the stressed porcine tissues in the presence of CPEA and CPRA increased gradually before reaching a maximum value after 2022 h of incubation (Fig. 8). Further incubation resulted in a gradual decrease in the activity of this enzyme. This was different compared to the trends obtained in the unstressed tissues with the same treatments where the increase in G6PDH activity showed two different rates of increase (Fig. 8). 3.5. Superoxide dismutase (SOD) activity The SOD activity in the control sample gradually increased over the course of incubation and reached its maximum after 3032 h of incubation, after which it did not increase any further (Fig. 9). For the porcine muscle tissues that were treated with CP, EA and RA the SOD activity increased rapidly after 1012 h of incubation, beyond which the SOD activity gradually decreased slightly over the remaining course of incubation (Fig. 9). The porcine muscle tissue that was incubated with CPEA and CPRA treatments behaved differently. The SOD activity in these

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

2233

Fig. 9. Changes in the SOD activity in (A) unstressed and (B) stressed porcine muscle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid and their synergies.

tissues gradually increased to reach their maximum value after 3032 h of incubation before declining slightly by the end of the incubation. In general, the SOD activity of the CPEA and CPRA treated porcine muscle extracts was higher than the other treatments, this was especially true during the later stages of incubation (Fig. 9). The SOD activity in the porcine muscle tissue stressed with H2O2 was higher for all the treatments compared to the SOD activity in the unstressed muscles. The SOD activity in all the treatments at the beginning of the of the incubation (02 h) was signicantly different from each other (Fig. 9). It was observed that at the beginning of the incubation (0 2 h) SOD activity of the porcine muscle tissue stressed with H2O2 alone was the lowest compared to the H2O2 stressed porcine muscle tissues incubated with phenolic treatments. The highest SOD activity at 02 h was obtained when the porcine muscle tissue was stressed with H2O2 in the presence of CPRA treatment (Fig. 9). For all the tissue samples the SOD activity slightly decreased for the next 10 h of incubation before increasing again and reaching their maximum value after 3032 h of incubation. The SOD activity for all the H2O2 stressed tissues decreased during the

last 1012 h of incubation. For all the time points the SOD activity of the CPEA extract was highest compared to the SOD activity obtained when other phenolic treatments were used (Fig. 9). 3.6. Catalase (CAT) activity The catalase activity in the porcine muscle tissue was monitored over the course of incubation for all the different treatments. It was observed that for the unstressed muscle tissue the catalase activity increased gradually over the course of incubation (Fig. 10). All the porcine muscle tissues incubated with phenolic treatments had higher CAT activity compared to the control. Differences in the CAT activity among the phenolic treatments were not signicantly different (Fig. 10). The CAT activities observed in the stressed tissues were much higher than obtained in the unstressed porcine muscle tissues for all the treatments (Fig. 10). The CAT activity in the porcine muscle tissue stressed with H2O2 was much higher than the control at the beginning of the incubation (0 2 h). The CAT activity continued to increase for the

Fig. 10. Changes in the CAT activity in (A) unstressed and (B) stressed porcine muscle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid and their synergies.

2234

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

Fig. 11. Changes in the total peroxidase activity in (A) unstressed and (B) stressed porcine muscle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid and their synergies.

remaining period of incubation before slightly decreasing towards the end of the incubation time (Fig. 10). In the porcine muscle tissue samples that were stressed with H2O2 in the presence of phenolic treatments the CAT activities were signicantly higher. When CAT activity was measured immediately at the beginning of incubation (02 h), the samples from the CPRA, CPEA and RA treatments were higher than the CAT activity obtained in the H2O2 stressed porcine muscle treated with EA and CP (Fig. 10). The CAT activity continued to increase rapidly after 1012 h of incubation when it reached its maximum value for all the treatments. The CAT activity then decreased rapidly for the next 1012 h of incubation for all the treatments in the H2O2 stressed porcine muscle tissues. After this decrease, the CAT activity for all the treatments did not change signicantly for the remaining course of incubation (Fig. 10). 3.7. Total peroxidase (TPX) activity The TPX activity in the control tissue, which was not incubated with any phenolic treatment did not change signicantly (Fig. 11). In the unstressed porcine muscle tissues incubated with the different phenolic treatments the enzyme activity then increased gradually until 1012 h after which the enzyme activity rapidly increased reaching a maximum value after 2022 h of incubation. The enzyme activity decreased gradually for the remaining period of incubation (Fig. 11). There was no signicant difference between the TPX activities among the different treatments (Fig. 11). In the porcine muscle tissues that were stressed with H2O2 the changes in the TPX activity were different compared to the unstressed tissue samples (Fig. 11). The TPX activity in the porcine muscle sample stressed with H2O2 alone increased gradually until 2022 h of incubation after which it slightly declined. The increase in the TPX activity when the stressed porcine muscle tissues were incubated with the phenolic treatments was much higher (Fig. 11). The TPX activity for all the phenolic treated H2O2 stressed porcine muscle tissue extracts increased

gradually up to 1012 h of incubation. Subsequently TPX activity for all the treatments rapidly increased to a maximum value after 2022 h of incubation (Fig. 11). The rate of increase in the TPX activity for CP and CPEA treated porcine muscle tissue extracts was signicantly higher than the TPX activity obtained with other phenolic treatments in H2O2 stressed porcine muscle tissues (Fig. 11). For the H2O2 stressed porcine muscle tissues that were incubated with EA, RA and CPRA treatments the TPX activity was maintained at their highest level even after 30 32 h of incubation after which the TPX activity declined slightly (Fig. 11).

4. Discussion The results suggest that the phenolic treatments had a protective effect on maintaining the cellular redox homeostasis through the stimulation of cellular antioxidant enzyme response in the porcine muscle tissue. In general, the pure phenolic treatments EA and RA were less effective than the treatments with CP, CPEA and CPRA. These results suggest that the functionality of these biphenyls was enhanced when they were present in a CP background indicating a possible synergistic interaction between CP phenolics and the biphenyls. The total phenolics in the porcine muscle tissue increased upon treatment with the cranberry powder, ellagic acid, rosmarinic acid and their synergies CPEA and CPRA showing that the phenolic phytochemicals were readily absorbed by the porcine muscle tissues. The total phenolic content was slightly higher in oxidatively stressed porcine muscle that was treated with EA and RA compared to the unstressed tissue. Oxidative stress in the porcine muscle was induced with the help of hydrogen peroxide as a source of reactive oxygen species. H2O2 like other reactive oxygen species is known to interact with the membrane lipids and carry out their oxidation. Oxidation of membrane lipids had been shown to change the membrane plasticity and exibility, which can cause an increase in the membrane

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

2235

permeability. This increase in membrane permeability could have resulted in increased uptake of partially hydrophobic biphenyls in the presence of H2O2. The changes in the antioxidant activity measured by the increase in the DPPH radical scavenging activity was enhanced with the total soluble phenolics absorbed. The antioxidant activity in the oxdatively stressed porcine muscle tissues with phytochemicals was lower than the DRI of the unstressed tissue. This could be possibly due to the involvement of the phenolic antioxidants in quenching the peroxide radical from H2O2 resulting in lower net antioxidant activity. Whether or not the absorption of phenolic antioxidants by the porcine muscle had any effect on the redox homeostasis was investigated by measuring the amount of MDA formed in the porcine muscle tissue. Oxidation of lipids in biological systems by reactive oxygen species results in the formation of malondialdehyde, which is a metabolite of lipid hydroperoxides [40]. It is a secondary oxidation product of lipids and serves as a good marker for lipid oxidation and cell membrane injury [41]. MDA is naturally formed in all living cells as a result of lipid oxidation from endogenously produced ROS. In an actively metabolizing tissue this ROS is quickly removed with the help of several cellular antioxidants and cellular antioxidant enzymes such as SOD and CAT [12,13]. The MDA content in the unstressed porcine muscle samples increased with incubation time and highest MDA was formed in the control tissue sample, which was signicantly higher than the other treatments. For the constant removal of ROS from the system it is essential for the cells to replenish cellular antioxidant pools either by reducing oxidized antioxidants or by inducing synthesis of cellular antioxidants and antioxidant enzymes. Both these processes require reducing equivalents from NADPH2, which probably were exhausted in the control tissue after 20 h of incubation. This could probably have resulted in a rapid increase in the formation of MDA due to the cascading oxidant activity of ROS [9]. Phenolic phytochemicals are often linked to free radical scavenging antioxidant activity due to their ability to delocalize electrons [5]. Lower amounts of MDA were formed in the porcine muscle tissue that were incubated with different phenolic treatments. This could probably be due to the free radical scavenging antioxidant activity of CP, EA and RA, which could have helped the cell to manage the removal of endogenous ROS. The MDA content in the H2O2 stressed muscle tissues was much higher than the unstressed tissue samples. This could be due to the rapid progression of the secondary oxidation of the lipids induced by the external H2O2, which could have exceeded the capacity of the limited reserves of cellular antioxidants and reduced cellular antioxidant enzyme response. The MDA content in the oxidatively stressed porcine muscle in the presence of CP and their synergies was still lower than the other treatments, which could again indicate a possible involvement of the phenolic antioxidants from CP and their synergies in removing the ROS. However, since the stoichiometric concentration of the

H2O2 was signicantly higher than the antioxidant capacity of the phenolic phytochemicals supplied by the CP and their synergies, it could possibly suggest that the phenolic treatments were able to replenish the cellular antioxidants by possibly inducing cellular antioxidant enzyme systems, which were able to manage the H2O2 induced oxidative stress. The replenishment of cellular antioxidant systems by reducing the oxidized forms of GSH, ascorbate and tocopherols and efcient functioning of the cellular antioxidant enzyme systems need a constant supply of reducing equivalents in the form of NADPH2 [10,11]. We therefore investigated the effect of phenolic treatments on G6PDH, which is the rst committed enzyme in the PPP involved in the generation of NADPH2. The G6PDH activity in the unstressed porcine muscle tissue showed that when CP, EA, RA and their synergies were used, the enzyme activity was higher than the control. Interestingly, this was also true when the porcine muscle tissue was oxidatively stressed with H2O2 and then incubated with these phytochemical treatments. These results suggest a possible stimulation of PPP by phenolic phytochemicals, which led to the increase in NADPH2 in porcine muscle tissues. Recent empirical evidence has now shown that some phenolic phytochemicals can mimic the functions of biological signaling molecules and trigger the signal transduction pathways [4244]. Phenolics from cranberry, biphenyls and phenolic acids can create conditions suitable for activating signaling pathways responsible for the stimulation of PPP [4244]. The acidic nature of the phenolic acids from cranberry as well as the strong chelating ability of larger phenolic phytochemicals such as ellagic acid, rosmarinic acid and avanoids from cranberry can alter the ionic as well as proton gradients across the cell membrane [11]. An apparent modulation in the concentrations of these ions and protons can activate these cellular signaling cascades, which could have resulted in the changes in many physiological pathways including the stimulation of the PPP [15,4547]. The partially hydrophobic nature of certain larger phenolic phytochemicals permits them to directly interact with membranes, ion channels, and pumps causing changes in the membrane permeability and function of these channels and pumps [4850]. These changes can alter the electrochemical gradient across the cell membrane causing rapid inux of protons and ions into the cytosol and may activate many signal cascades leading to the dehydrogenase-linked stimulation of PPP [51]. This stimulation of PPP resulting in the formation of NADPH2 could possibly help the regeneration of the cellular antioxidants such as glutathione and ascorbic acid. Proline synthesis in biological systems is a NADPH2 intensive process and it has been previously proposed that phenolic phytochemicals are able to induce proline synthesis, thereby creating a higher demand for the NADPH2, which can therefore further stimulate PPP [10,11]. The biosynthesis of proline could create a demand

2236

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

for the TCA cycle intermediates such as a-ketoglutarate to be channeled to glutamic acid and then to NADPH2requiring proline biosynthesis [19,20] (Fig. 3). We investigated a possible link between the stimulation of PPP and concomitant stimulation in proline biosynthesis by measuring the changes in the free proline content during incubation. It was observed that for all the treatments the proline content increased with incubation time. This increase in proline content correlated well with the increase in the G6PDH activity suggesting a possible coupling of the stimulation of G6PDH and proline synthesis. It is therefore likely that the coupling of proline biosynthesis and PPP can generate more NADPH2, which can be used by the proline biosynthesis and cellular antioxidant enzyme response pathways [10,11]. The higher rates of increase in the proline content in the oxidatively stressed porcine muscle tissue compared to the unstressed tissue could possibly indicate that the induction of proline synthesis is an inherent natural response in cellular systems against oxidation stress. This can further be concluded by the higher rate of proline increase in the porcine muscle tissue that was stressed with H2O2 compared to the control. One possible function of phenolic phytochemicals could be in favoring this switch to proline synthesis by stimulating the PPP independently, which could be the reason for higher proline values obtained both in the stressed and unstressed porcine muscle tissues in the presence of phytochemical treatments. The cellular demands for reducing equivalents are coupled to the needs for ATP, which is the source of energy in biological systems. ATP is synthesized by oxidative phosphorylation of ADP by an enzyme ATPase in the mitochondria by reduction of molecular oxygen to water with the help of electrons from reducing equivalents such as NADH and FADH2. Excessive cellular requirement for ATP usually results in incomplete reduction of oxygen to make reactive oxygen species, which have implications in manifestation of various oxidative stress related diseases [52,53]. Proline has been shown to be able to function as a reductant in cellular systems [54,55]. Therefore, proline could be functioning as an alternative reductant (instead of NADH) (Fig. 3) for mitochondrial oxidative phosphorylation to generate ATP [10,11]. This can reduce the cellular need for NADH-linked ATP synthesis, which can reduce excessive mitochondrial oxidative burst to limit the leakage of reactive oxygen species into cytosol during oxidative phosphorylation. This was indicated by the reduced MDA formation in porcine muscle tissues treated with cranberry phenolics, biphenyls and their synergies. To conrm if cranberry and biphenyl treatments were also able to maintain redox homeostasis in the cell by inducing cellular antioxidant enzyme response we investigated the activities of cellular antioxidant enzymes SOD, CAT and TPX. The results indicated that the activity of SOD and CAT increased gradually with incubation time.

The rate of increase in SOD activity was higher with the phenolic treatments than compared to the control suggesting that CP, EA, RA and their synergies were able to induce SOD. Higher SOD and CAT activity obtained at zero time in the peroxide stressed muscle could indicate a natural biological response against oxidation stress in eukaryotic systems. Rapid increases in the activity of SOD and other cellular antioxidant enzyme systems including CAT were probably required to quickly remove the ROS to prevent oxidative damage to the cell. It possible that the activity of SOD and CAT was further stimulated by an antioxidant response element (ARE)-mediated induction in enzyme expression similar to the induction of NAD(P)H:quinone reductase and glutathione S-transferase-Y genes [42,56]. Peroxidases such as glutathione peroxidases are expressed in eukaryotic systems to reduce the reactive peroxide species and protect them against oxidative stress [9]. TPX activity was measured to investigate if any peroxidases were induced as a result of phenolic treatments. The TPX activity measures the total peroxidase activity of glutathione peroxidase and phenolic-dependent peroxidases that have been previously reported to be induced in plant model systems by cranberry phenolics and in porcine muscle systems in response to oregano phenolics [57,58]. In plants these peroxidases protect the tissues from oxidation stress by removing the ROS and using them to oxidatively couple phenolic phytochemicals to make lignin and other crosslinked phenolics [59,60]. The TPX activity in the CP, EA and RA treated porcine muscle tissue was higher than control, suggesting that this peroxidase was a phenolic dependent peroxidase similar to the ones seen in plant systems [59,60]. We suspect that these phenolic-dependent peroxidases could be involved in reducing oxidative stress by removing reactive oxygen species by oxidatively polymerizing phenolics from cranberry and their synergies without affecting the cellular pools of glutathione, ascorbate and other antioxidants [10]. The total peroxidase activity was found to be higher in all the phenolic phytochemical treated porcine muscle tissue samples indicating that peroxidases were induced in response to phytochemical treatments. TPX activity in the stressed porcine muscle was however, higher than the unstressed muscle, which could possibly be due to the forward stimulation in the activity of the TPX activity by H2O2, which is one of the substrate for peroxidase.

5. Conclusion The mechanism of cranberry phenolics and their synergies with functional biphenyls ellagic acid and rosmarinic acid on modulating the cellular antioxidant enzyme response in oxidatively stressed porcine muscle tissue was investigated. Results suggested that phenolic treatments reduced the oxidative stress on the porcine

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238

2237

muscle as indicated by the reduced MDA formation. It was also observed that treatment with phenolic phytochemicals led to increased activity of the enzyme G6PDH suggesting that these treatments stimulated the pentose phosphate pathway, which could provide NADPH2 for stimulating cellular antioxidant enzyme response. We showed in our earlier work that the stimulation of the pentose phosphate pathway was linked to a concomitant increase in proline biosynthesis both in plant as well as in porcine muscle models [57,58]. The results in this study also indicated that the increased activity of the enzyme G6PDH correlated closely with the increase in the proline formation. This suggests that treatment with phenolic phytochemicals stimulated the NADPH2-dependent proline biosynthetic pathway, which can further stimulate the PPP. Increased proline biosynthesis could potentially reduce oxidative burst from the mitochondria by functioning as an alternate reductant for ATP synthesis without depending on NADH from the complete operation of TCA cycle. The activity of the cellular antioxidant enzymes SOD and CAT was also stimulated by CP, EA, RA and their synergies. The higher activities of these enzymes in response to phenolic treatments correlated well with the lower amounts of MDA that were formed in both the oxidatively stressed and unstressed muscles. This suggests a possible role of phenolic phytochemicals in reducing the oxidative stress by inducing cellular antioxidant enzymes. Another cellular antioxidant enzyme, peroxidase was also found to be induced in the porcine muscle tissue samples, which were incubated with the phenolic phytochemicals. Glutathione peroxidases have been shown to be induced in response to oxidative stress [9]. Phenolic phytochemical dependent peroxidases have previously been reported to be induced by CP, EA, RA and their synergies in plant systems [57]. The above peroxidase could also be involved in reducing oxidative stress by removing reactive oxygen species by oxidatively polymerizing phenolics from cranberry and their synergies without affecting the cellular pools of glutathione, ascorbate and other antioxidants [10]. From this investigation phenolic antioxidants from plants appear to mediate their biological functionality by modulating cellular antioxidant systems in eukaryotes by more than one mechanism. These functions were carried out either by functioning as free radical scavenging antioxidants and more importantly, by inducing cellular antioxidant enzyme responses. The cellular antioxidant enzyme responses could be mediated by the stimulation of the PPP-linked to proline biosynthesis, which can provide the reducing equivalents required for the efcient functioning of these enzymes [10,11]. In most parameters that were evaluated it appeared as though the pure biphenyls functioned more efciently when they were in a cranberry background suggesting that the conditions created by cranberry phenolics in synergistic combinations signicantly improved the functionality of rosmarinic acid and ellagic acid. The results provide an important insight into the

possible mechanism of action of fruit phytochemicals in biological systems and also showed that the functionality can be improved in synergy with specic biphenyls.

References
[1] Block G, Patterson B, Subar A. Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr Cancer 1992;18:129. [2] Serdula MK, Byers MAH, Simoes E, Mendlein JM, Coates RJ. The association between fruit and vegetable intake and chronic disease risk factors. Epidemiology 1996;7:1615. [3] Tapiero H, Tew KD, Ba GN, Mathe G. Polyphenols: do they play a role in the prevention of human pathologies? Biomed Pharmacother 2002;56(4):2007. [4] Duthie GG, Gardner PT, Kyle JA. Plant polyphenols: are they the new magic bullet? Proc Nutr Soc 2003;62(3):599603. [5] Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of avonoids and phenolic acids. Free Radic Biol Med 1996;20:93356. [6] Schwarz KB. Oxidative stress during viral infection: a review. Free Radic Biol Med 1996;21(5):6419. [7] Gotz JM, vanKan CI, Verspaget HW, Biemond I, Lamers CBHW, Veenendaal RA. Gastric mucosal superoxide dismutases in Helicobacter pylori infection. Gut 1996;38:5026. [8] Jakus V. The role of free radicals, oxidative stress and antioxidant systems in diabetic vascular disease. Bratisl Lek Listy 2000;101(10):54151. [9] Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82(1):4795. [10] Shetty K. Role of proline-linked pentose phosphate pathway in biosynthesis of plant phenolics for functional food and environmental applications: a review. Process Biochem 2004;39:789803. [11] Shetty K, Wahlqvist ML. A model for the role of proline-linked pentose phosphate pathway in phenolic phytochemical biosynthesis and mechanism of action for human health and environmental applications. Asia Pac J Clin Nutr 2004;13(1):124. [12] Mates JM, Perez-Gomez C, Nunez de Castro I. Antioxidant enzymes and human diseases. Clin Biochem 1999;32(8):595603. [13] Mates JM, Sanchez-Jimenez F. Antioxidant enzymes and their implications in pathophysiologic processes. Front Biosci 1999;4:33945. [14] Nordberg J, Arner ES. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med 2001;31(11):1287312. [15] Fabregat I, Vitorica J, Satrustegui J, Machado A. The pentose phosphate cycle is regulated by NADPH/NADP ratio in rat liver. Arch Biochem Biophys 1985;236(1):1108. [16] Pfeifer R, Karl G, Scholz R. Does the pentose cycle play a major role for NADPH supply in the heart? Biol Chem Hoppe Seyler 1986;367(10):10618. [17] Cabezas H, Raposo RR, Melendez-Hevia E. Activity and metabolic roles of the pentose phosphate cycle in several rat tissues. Mol Cell Biochem 1999;201(12):5763. [18] Shetty K. Biotechnology to harness the benets of dietary phenolics; Focus on Lamiaceae. Asia Pac J Clin Nutr 1997;6:16271. [19] Wu G. Intestinal mucosal amino acid catabolism. J Nutr 1998;128(8):124952. [20] Brosnan JT. Glutamate, at the interface between amino acid and carbohydrate metabolism. J Nutr 2000;130(Suppl 4S):988S90S. [21] Newsholme P, Procopio J, Lima MM, Pithon-Curi TC, Curi R. Glutamine and glutamate-their central role in cell metabolism and function. Cell Biochem Funct 2003;21(1):19. [22] Steinmetz KA, Potter JD. Vegetables, fruit, and cancer prevention: a review. J Am Diet Assoc 1996;96(10):102739.

2238

D.A. Vattem et al. / Process Biochemistry 40 (2005) 22252238 [42] Barch DH, Rundhaugen LM. Ellagic acid induces NAD(P)H:quinone reductase through activation of the antioxidant responsive element of the rat NAD(P)H:quinone reductase gene. Carcinogenesis 1994;15(9):20658. [43] Chen C, Shen G, Hebbar V, Hu R, Owuor ED, Kong AN. Epigallocatechin-3-gallate-induced stress signals in HT-29 human colon adenocarcinoma cells. Carcinogenesis 2003;24(8):136978. [44] Chen C, Yu R, Owuor ED, Kong AN. Activation of antioxidantresponse element (ARE), mitogen-activated protein kinases (MAPKs) and caspases by major green tea polyphenol components during cell survival and death. Arch Pharm Res 2000;23(6):60512. [45] Bellomo G, Thor H, Orrenius S. Increase in cytosolic Ca2+ concentration during t-butyl hydroperoxide metabolism by isolated hepatocytes involves NADPH oxidation and mobilization of intracellular Ca2+ stores. FEBS Lett 1984;168(1):3842. [46] Stout C, Charles A. Modulation of intercellular calcium signaling in astrocytes by extracellular calcium and magnesium. Glia 2003;43(3):26573. [47] Cohen JE, Fields RD. Extracellular calcium depletion in synaptic transmission. Neuroscientist 2004;10(1):127. [48] Hagerman AE, Butler LG. The specicity of proanthocyanidin-protein interactions. J Biol Chem 1981;256(9):44947. [49] Tsuchiya H, Sato M, Kameyama Y, Takagi N, Namikawa I. Effect of lidocaine on phospholipid and fatty acid composition of bacterial membranes. Lett Appl Microbiol 1987;4(6):1414. [50] Tsuchiya H. Biphasic membrane effects of capsaicin, an active component in Capsicum species. J Ethnopharmacol 2001;75(2 3):2959. [51] Pan CY, Kao YH, Fox AP. Enhancement of inward Ca2+ currents in bovine chromafn cells by green tea polyphenol extracts. Neurochem Int 2002;40(2):1317. [52] Sarkela TM, Berthiaume J, Elfering S, Gybina AA, Giulivi C. The modulation of oxygen radical production by nitric oxide in mitochondria. J Biol Chem 2001;276(10):69459. [53] Cadenas E. Mitochondrial free radical production and cell signaling. Mol Aspects Med 2004;25(12):1726. [54] Hagedorn CH, Phang JM. Transfer of reducing equivalents into mitochondria by the interconversions of proline and a-pyrroline-5carboxylate. Arch Biochem Biophys 1983;225:95101. [55] Phang JM. The regulatory functions of proline and pyrroline-5-carboxylic acid. Curr Top Cell Regul 1985;25:91132. [56] Barch DH, Rundhaugen LM, Pillay NS. Ellagic acid induces transcription of the rat glutathione S-transferase-Ya gene. Carcinogenesis 1995;16(3):6658. [57] Vattem DA, Randhir R, Shetty K. Cranberry phenolics-mediated elicitation of antioxidant enzyme response in fava bean (Vicia faba) sprouts. J Food Biochem 2004 [in press]. [58] Randhir R, Vattem DA, Shetty K. Antioxidant response studies on the effect of oregano phenolics on H2O2 stressed porcine muscle. Process Biochem 2004 [in press]. [59] Moorales M, Barcelo ARA. Basic peroxidase isoenzyme from vacuoles and cell walls of Vitis vinifera. Phytochemistry 1997;45: 22932. [60] Barcelo AR, Pomar F. Oxidation of cinnamyl alcohols and aldehydes by a basic peroxidase from lignifying Zinnia elegans hypocotyls. Phytochemistry 2001;57(7):110513.

[23] Barbaste M, Berke B, Dumas M, Soulet S, Delaunay JC, Castagnino C, et al. Dietary antioxidants, peroxidation and cardiovascular risks. J Nutr Health Aging 2002;6(3):20923. [24] Riboli E, Norat T. Epidemiologic evidence of the protective effect of fruit and vegetables on cancer risk. Am J Clin Nutr 2003;78(Suppl 3):559S69S. [25] Bomser J, Madhavi DL, Singletary K, Smith MA. In vitro anticancer activity of fruit extracts from Vaccinium species. Planta Med 1996;62(3):2126. [26] Reed JD, Krueger CG, Porter ML. Cranberry juice powder decreases low density lipoprotein cholesterol in hypercholesterolemic swine. FASEB J 2001;15(45):54. [27] Reed J. Cranberry avonoids, atherosclerosis and cardiovascular health. Crit Rev Food Sci Nutr 2002;42(Suppl):30116. [28] Abraham SK. Anti-genotoxic effects in mice after the interaction between coffee and dietary constituents. Food Chem Toxicol 1996;34:1520. [29] Chan MM, Mattiacci JA, Hwang HS, Shah A, Fong D. Synergy between ethanol and grape polyphenols, quercetin, and resveratrol, in the inhibition of the inducible nitric oxide synthase pathway. Biochem Pharmacol 2000;60(10):153948. [30] Vattem DA, Jang H-D, Levin R, Shetty K. Synergism of cranberry phenolics with ellagic acid and rosmarinic acid for antimutagenic and DNA-protection functions. J. Food Biochem 2004 [submitted for publication]. [31] Shetty K, Curtis OF, Levin RE, Witkowsky R, Ang W. Prevention of vitrication associated with in vitro shoot culture of oregano (Origanum vulgare) by Pseudomonas spp. J Plant Physiol 1995;147:44751. [32] Cervato G, Carabelli M, Gervasio S, Cittera A, Cazzola R, Cestaro B. Antioxidant properties of oregano (Origanum vulgare) leaf extracts. J Food Biochem 2000;24:45365. [33] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:24854. [34] Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant Soil 1973;39(1):2057. [35] Tamagnone L, Merida A, Stacey N, Plaskitt K, Parr A, Chang CF, et al. Inhibition of phenolic acid metabolism results in precocious cell death and altered cell morphology in leaves of transgenic tobacco plants. Plant Cell 1998;10(11):180116. [36] Deutsch J. Glucose-6-phosphate dehydrogenase. In: Methods of enzymatic analysis. 3rd ed. Verlag Chemie; 1983. p. 191197. [37] Laloue H, Weber-Lofti F, Lucau-Danila A, Gullemat P. Identication of ascorbate and guaiacol peroxidase in needle chloroplasts of spruce trees. Plant Physiol Biochem 1997;35:3416. [38] Oberley LW, Spitz DR. Assay of SOD activity in tumor tissue. In: Methods in enzymology, vol. 105. Academic Press; 1984. p. 457461. [39] Beers R, Sizer IA. Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 1952;195:13340. [40] Moore K, Roberts LJ. Measurement of lipid peroxidation. Free Radic Res 1998;28(6):65971. [41] Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR, Grandjean P. Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors. Clin Chem 1997;43(7):1209 14.