Shetty Chamberry Antioxidants

www.elsevier.com/locate/procbio

Process Biochemistry 40 (2005) 2225–2238

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 benefits 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: [email protected] (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 [6–9]. 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

D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–22382226

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

efficient operation of cellular antioxidant enzyme response

pathways depend on the availability of reducing equivalents

such as FADH2 and NADPH2 [12–14]. 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 [15–17]. The stimulation of pentose

phosphate pathway could further be coupled to the

biosynthesis of proline that is made from glutamic acid

Fig. 2. The antioxidant defense response of the cell carried out by enzymatic as

catalase; PER: peroxidase; AP: ascorbate peroxidase; GR: glutathione reductase;

DHAR: dehydroascorbate reductase; ASA: reduced ascorbate; DHA: dehydroasc

reductase.

[11,18] and also requires NADPH2 [19–21]. 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 efficient functioning of the cellular

antioxidant enzyme cascades [10,11].

well as the non enzymatic antioxidants. SOD: superoxide dismutase; CAT:

GSSG: oxidized glutathione; GSH: reduced glutathione; TP: tocophenrol;

orbate; MDA: monodehydroascorbate and MDHA: monodehydroascorbate

D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–2238 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 benefits [3,22–

24]. Cranberry and their products have long been known to

have beneficial 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 beneficial functional properties of cranberry are

linked to specific phenolic phytochemicals, their exact

mechanism of functionality is not very well understood.

Recent research has also suggested that the phytochemical

profile in which a specific 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 cell-

ular 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 Cran-

berry Products Inc., Carver, MA) was added to 1 g of porcine

muscle tissue homogenate to give a final 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


Table 1

Different treatments used in the porcine muscle study

Treatment Phytochemical

(phenolic basis, 1 mg/ml)

H2O2

(mM)

Unstressed porcine muscle

Control None 0

CP Cranberry powder 0

EA Ellagic acid 0

RA Rosmarinic acid 0

CP–EA Cranberry powder + ellagic acid 0

CP–RA Cranberry Powder + rosmarinic acid 0

Oxidatively stressed porcine muscle

H2O2 None 100

CP + H2O2 Cranberry powder 100

EA + H2O2 Ellagic acid 100

RA + H2O2 Rosmarinic acid 100

CP–EA + H2O2 Cranberry powder + ellagic acid 100

CP–RA + H2O2 Cranberry powder + rosmarinic acid 100

added to 1 g of porcine muscle tissue homogenate to give a

final 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 concen-

tration of ellagic acid (CP–EA) or rosmarinic acid (CP–RA)

[30]. These synergy mixtures were added to 1 g of porcine

muscle tissue homogenate to give a final 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 2–5 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 first cen-

trifuging the muscle tissue out of the treatment buffer at

13,000 rpm for 15 min at 2–5 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

2–5 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 modified

from Shetty et al. [31] and was used to determine the amount

of phenolic metabolites absorbed by the porcine tissue.

Briefly, 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) Folin–Ciocalteu

reagent was added and mixed. After 5 min, 1 ml of 5%

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 ¼ Acontrol517 � Aextract

517

Acontrol517

�100

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

UV–vis Genesys spectrophotometer (Spectronic Instru-

ments Inc., Rochester, NY).

2.5. Proline assay

Proline content was determined according to the modified

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 orthopho-

sphoric 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.


2.6. Malondialdehyde (MDA) assay

Malondialdehyde was measured by modifying the

method discussed by Tamagnone et al. [35]. Briefly, 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 coefficient (e)156 mmol�1 cm�1 and expressed as mmol/g FW.

2.7. Glucose-6-phosphate dehydrogenase (G6PDH) assay

A modified 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 glucose-

6-phosphate, and 0.77 mmol maelamide was prepared. This

mixture was used to obtain baseline (zero) of the spectro-

photometer 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 coefficient of NADPH2

(6.22 mM�1 cm�1).

2.8. Total peroxidase (TPX) activity

A modified version of the assay developed by Laloue et

al. [37] was used. Briefly, 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 coefficient of the oxidized product tetraguaiacol

(26.6 mM�1 cm�1).

2.9. Superoxide dismutase (SOD) assay

A competitive inhibition assay was performed that used

xanthine–xanthine oxidase-generated superoxide to reduce

nitroblue tetrazolium (NBT) to blue formazan. A spectro-

photometric 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

defined 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. Briefly, 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 4–5 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 defined as amount that decomposes

1 mmol of H2O2

Units=mg ¼ ðDA240=minÞ � 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 (CP–EA and CP–RA)

the amount of phenolics absorbed into the porcine tissue was

assayed using the Folin–Ciocalteu 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).


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

first 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

Fig. 5. Antioxidant activity of (A) unstressed and (B) stressed porcine muscle tiss

synergies.

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 significant

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 10–12 h after which the rate of increase in MDA

formation was much higher until about 20–22 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

CP–EA and CP–RA 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 MDAwas formed when EAwas

ue incubated with cranberry powder, ellagic acid, rosmarinic acid and their


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 10–12 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

Fig. 7. Changes in the proline content of (A) unstressed and (B) stressed porcine m


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 significant

change in the amount of proline formed when the porcine

tissue was incubated with CP, RA and CP–RA alone (Fig. 7).

When the porcine tissue was stressed with H2O2 it was

observed that the rate of increase in the proline content was

significantly 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 10–12 h after which it

remained constant for the remaining incubation time. The

final 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

first increased rapidly for 10–12 h after which the rate of

increase slowly decreased for the remaining time of

uscle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid


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


incubation (Fig. 7). Highest amounts of proline were formed

when CP–EA and CP–RA were used as phenolic treatments

in the porcine tissues stressed with H2O2. For all the

treatments the maximum proline content was reached after

30–32 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 20–22 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 10–12 h after which they showed a sharp increase

in the activity. This increase was highest for CP–EA 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 20–22 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 30–32 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 20–22 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 10–12 h. The rate

of increase in G6PDH activity after 10–12 h of incubation was

higher and peaked after 30–32 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 CP–EA and CP–RA increased gradually before

reaching a maximum value after 20–22 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 30–32 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 10–12 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 CP–EA and CP–RA

treatments behaved differently. The SOD activity in these


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 30–32 h of incubation before declining slightly by the

end of the incubation. In general, the SOD activity of the

CP–EA and CP–RA 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

(0–2 h) was significantly 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 0–2 h was obtained when the

porcine muscle tissue was stressed with H2O2 in the

presence of CP–RA 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 30–32 h of incubation. The SOD

activity for all the H2O2 stressed tissues decreased during the

Fig. 10. Changes in the CATactivity in (A) unstressed and (B) stressed porcine mus

their synergies.

last 10–12 h of incubation. For all the time points the SOD

activity of the CP–EA 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 significantly

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

cle tissue incubated with cranberry powder, ellagic acid, rosmarinic acid and


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 significantly higher. When CAT activity was measured

immediately at the beginning of incubation (0–2 h), the

samples from the CP–RA, CP–EA 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 10–12 h of

incubation when it reached its maximum value for all the

treatments. The CAT activity then decreased rapidly for the

next 10–12 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 significantly 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

significantly (Fig. 11). In the unstressed porcine muscle

tissues incubated with the different phenolic treatments the

enzyme activity then increased gradually until 10–12 h after

which the enzyme activity rapidly increased reaching a

maximum value after 20–22 h of incubation. The enzyme

activity decreased gradually for the remaining period of

incubation (Fig. 11). There was no significant 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 20–22 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 10–12 h of incubation. Subsequently TPX

activity for all the treatments rapidly increased to a

maximum value after 20–22 h of incubation (Fig. 11).

The rate of increase in the TPX activity for CP and CP–EA

treated porcine muscle tissue extracts was significantly

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 CP–RA 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 home-

ostasis 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, CP–EA and CP–RA. 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 CP–EA and CP–RA

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

flexibility, which can cause an increase in the membrane


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 phytochem-

icals 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 MDAwas formed in the control tissue sample, which

was significantly 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 significantly 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 efficient 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 first 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 [42–44]. Phenolics from cranberry,

biphenyls and phenolic acids can create conditions suitable

for activating signaling pathways responsible for the

stimulation of PPP [42–44]. 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 flavanoids from cranberry can alter

the ionic as well as proton gradients across the cell

membrane [11]. An apparent modulation in the concentra-

tions 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 [15,45–47]. 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 [48–50]. These changes can

alter the electrochemical gradient across the cell membrane

causing rapid influx 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


for the TCA cycle intermediates such as a-ketoglutarate to

be channeled to glutamic acid and then to NADPH2-

requiring 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 confirm if cranberry and biphenyl treatments were

also able to maintain redox homeostasis in the cell by

inducing cellular antioxidant enzyme response we inves-

tigated 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 cross-

linked 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


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 efficient functioning of

these enzymes [10,11]. In most parameters that were

evaluated it appeared as though the pure biphenyls

functioned more efficiently when they were in a cranberry

background suggesting that the conditions created by

cranberry phenolics in synergistic combinations signifi-

cantly 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 specific biphenyls.

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