shetty chamberry antioxidants
TRANSCRIPT
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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
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–22382228
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.
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–2238 2229
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).
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–22382230
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
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–2238 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 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
and their synergies.
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
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–22382232
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 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
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–2238 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 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
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–22382234
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
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–2238 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 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
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–22382236
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
D.A. Vattem et al. / Process Biochemistry 40 (2005) 2225–2238 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 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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