IABG 2003Cambridge University, UK Aging, Exercise and Phytochemicals: Promises and Pitfalls

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IABG 2003 Cambridge University, UKAging,
Exercise and PhytochemicalsPromises and
Pitfalls

• Li Li Ji
• Departments of Kinesiology
• Intedisciplinary Nutritional Science
• and Institute on Aging
• University of Wisconsin-Madison, USA

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ROS generation in Skeletal Muscle

• Exercise is an important means to keep fit,
  prevent diseases and improve quality of life, or
  at least to maintain mobility.

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• Mitochondrial respiratory Chain
• increased oxygen consumption produces more O2.-
  and H2O2.
• Xanthine oxidase
• Insufficient blood flow (hypoxia) leads to
  degradation of ATP to hypoxanthine producing O2.-
  and H2O2 .
• Neutrophil (PMN)
• Respiratory burst by NADPH oxidase
• IL-1, IL-6 and TNF-? increases adhesion
  molecules and PMN infiltration
• Lipoxygenase/cycloxygenase
• Activated by cytokines, hormones and toxins

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Source of Free Radicals in Skeletal Muscle

• With 2 mM pyruvate and 2 mM malate as
  mitochondrial respiration substrates
• Replace pyr-
• malate wiith
• 1.7 mM ADP, 0.1 mM NADPH and Fe3
• Ji Bejma J.A.P. (1999)

• An acute bout of exercise in rats increases ROS
  production in skeletal muscle.
• Aged rats generates more ROS at rest and during
  exercise (15 m/min, 0) at the same relative
  workload as young rats (25 m/min, 10).
• Both mitochondria and NADPH oxidase are sources
  of ROS in young muscle during exercise.
• For aged muscle, mitochondria seem to be the
  main source.
• ROS generation is also increased in the heart.

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• The ability of the cell to resist or prevent
  oxidative stress is a key determinant of its
  longevity.
• - Finkel and Holbrook. Science 2000
• Strategies to boost antioxidant defense
• Caloric restriction
• Transgene
• Dietary supplementation
• Antioxidant mimics
• Adaptation (Exercise training)

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Antioxidant Supplementation

• Nature offers rich sources of antioxidants
  contained mainly in plants (fruits, vegetables
  and herbs) known as phytochemical antioxidants.
• Phenolic compounds are important antioxidants
• due to their redox properties in absorbing,
• quenching and decomposing ROS.

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IABG 2003 Cambridge University, UK

• Hypothesis 1
• Ginseng supplementation ameliorates
  age-associated oxidative stress in rats
• Fu Ji, J. Nutr. (in press)

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Ginseng Antioxidant Property

• There are two kinds of ginsengs, Panax C. A.
  Meyer (Asian ginseng) and Panax quinquefolius L.
  (Wisconsin ginseng)
• The primary activate ingredients are a mixture of
  saponin glycosides, known as ginsenosides .
• Higher ginsenoside content is found in Panax
  quinquefolius.

R1, R2, and R3 may vary between glucose,
arabinose and rhamnose and their combination
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Ginseng Antioxidant Property

• Phytopanaxadiols are a group of ginsenosides
  containing two glucose moieties on C-3 position
  while differing between glucose and arabinose on
  C-20 such as Rb1, Rb2,
• etc.
• Phytopanaxadiols appear to not only scavenge free
  radicals and chelate metal ions, but also
  influence gene expression of antioxidant enzymes.

• Rb1 interact with hydroxyl radicals and protect
  ischemic neuron. Rb2 stimulate nuclear protein
  biding to gene regulatory sequences on CuZn SOD
  promoter.

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IABG 2003 Cambridge University, UK

• Experimental Design
• Animals Female Fischer 344 Rats at 8 month
  (young) and 26 month age (old)
• Diet AIN-95 purified diet with 0.5 mg/kg (low
  dose) or 2.5 mg/kg (high dose) Wisconsin
  ginseng for 4 months.
• Ginsenosides (11.95) Rb1, 1.5 Rb2, 0.02, Rc,
  1.67 Rd, 1.86 Re, 3.42 Rg1, 1.09

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Heart
Muscle

• Oxidant production rate (dichlorofluorescin,
  DCFH as probe) in the homogenate of rat heart
  and skeletal muscle (deep portion of vastus
  lateralis). The assay buffer contained 130 mM
  KCl, 5 mM MgCl2, 20 mM NaH2PO4, 20 mM Tris-HCl,
  and 30 mM glucose (pH 7.4) with 5 ?M
  DCFH-diacetate dissolved in 1.25 mM methanol.
  Each bar represents mean ? SEM with number of
  rats in each group specified in previous slide.
  plt0.05, Low-dose or High-dose ginseng vs.
  Control. plt0.05, main age effect plt0.01, 26
  month vs. 8 month old rats.

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Heart
Muscle

• Protein carbonyl content in rat heart and
  skeletal muscle (deep portion of vastus
  lateralis). Each bar represents mean ? SEM.
  plt0.05, Low-dose or High-dose ginseng vs.
  Control. plt0.05 plt0.01, 26 month vs. 8 month
  old rats

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Heart
Muscle

H L C
H L C
80kD
50kD

• Western blot analysis of Reactive carbonyl
  derivative in the heart and skeletal muscle (deep
  portion of vastus lateralis) of old rats with
  control (C), low-dose (L) or high-dose (H)
  ginseng diet.

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Heart
Muscle

MDA content in rat heart and skeletal muscle
(deep portion of vastus lateralis). Each bar
represents mean ? SEM. plt0.05, Low-dose vs. or
High-dose. plt0.05 26 month vs. 8 month old rats
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Heart
Muscle

• Superoxide dismutase (SOD) activity in rat heart
  and skeletal muscle (deep portion of vastus
  lateralis). Each bar represents mean ? SEM.
  plt0.05, Low-dose or High-dose ginseng vs.
  Control. plt0.05 plt0.01, 26 month vs. 8 month
  old rats

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Soleus
Vastus lateralis

• Glutathione peroxidase (GPX) activity in rat
  soleus and deep portion of vastus lateralis (DVL)
  muscle. Each bar represents mean ? SEM. plt0.05,
  Low-dose or High-dose ginseng vs. Control.
  plt0.05 26 month vs. 8 month old rats

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Potential Side-effects

Body wt was lower (Plt0.01) in high-dose compared
to low-dose and control groups of old rats.

• Body wt was not significantly different among
  three dietary groups of young rats

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Heart
Muscle

• Citrate synthase (CS) activity in rat heart and
  skeletal muscle (deep portion of vastus
  lateralis). Each bar represents mean ? SEM.
  plt0.05, Low-dose or High-dose ginseng vs.
  Control. plt0.05 plt0.01, 26 month vs. 8 month
  old rats

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IABG 2003 Cambridge University, UK

• Summary1
• Dietary supplementation of ginseng for 4 months
  in rats decreased oxidant production and
  age-related oxidative damage to protein in the
  heart and skeletal muscle.
• Elevated SOD and GPX activities may partially
  explain these protective effects.
• The effects seem to be dose-dependent. Possible
  side-effects on growth should be examined.

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IABG 2003 Cambridge University, UK

• Hypothesis 2
• Oat antioxidant supplementation attenuates
  exercise-induced oxidative damage in rats
• Ji et al. Nutr. Res. (in press)

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Oat Antioxidants

• Oat (Avena sativa L.) contains several families
  of compounds displaying antioxidant properties.
• Non-flavonoid phenols Flavonoids

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Compositions of the Experimental Diet

• Ingredient Control Flour
  Pearling
• Casein 200.0 125.0 170.2
• L-Cystine 3.0 3.0 3.0
• Oat Flour 0.0 500.0 0.0
• Oat Pearling 0.0 0.0 200.0
• Corn Starch 392.0 46.55 295.7
• Maltodextrin 129.49 129.49 129.49
• Sucrose 58.0 58.0 58.0
• Corn Oil 100.0 64.3 84.1
• Cholesterol 10.0 10.0 10.0
• Cholic Acid 2.0 2.0 2.0
• Cellulose 58.0 14.15 0.0
• Mineral Mix (AIN-93-MX) 35.0 35.0 35.0
• Vitamin Mix (AIN-93-VX) 10.0 10.5 10.0
• Choline bitartrate 2.5 2.5 2.5
• TBHQ (Antioxidant) 0.01 0.01
  0.01
• Total (g) 1000.0 1000.0 1000.0
  
  
  

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• ROS Production in Muscle
• Plt0.05
• Exercise vs. Rest
• Plt0.05
• Oat vs. Control.

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Muscle Glutathione Status

• GSH GSSG GSHGSSG
• Control
• Rested 0.69 0.03 0.02 0.002 35.1
  3.23
• Exercised 0.67 0.04 0.03 0.003
  22.0 1.93
• Oat Flour
•   Rested 0.75 0.02 0.03 0.002 30.1
  2.37
• Exercised 0.75 0.04 0.03 0.004 26.8
  2.91
•    Oat Pearling
•   Rested 0.71 0.08 0.03 0.002 24.5
  3.26
• Exercise 0.67 0.04 0.04 0.003 19.6
  1.54
•    ANOVA, P Diet 0.08 Diet 0.026
• Exer 0.006 Exer
  0.002

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Oat Antioxidants Avenathramides are anionic,
substituted cinnamic acid conjugates found only
in oats that demonstrate high antioxidant potency.

• General structure of avenathramides. Variations
  of R1 and R2 moiety give three classes as Aven.
  A, B, and C.

• HPLC chromatographs of Aven. A, B, and C.

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Avenathramide Supplementation

• Purpose of the Study
• To investigated the efficacy of dietary
  supplementation of avenathramides in rats at rest
  and after an acute bout of oxidative stress
  imposed by heavy exercise
• Hypotheses
• Avenathramide supplementation increases
  endogenous antioxidant defense capacity
• Avenathramide supplementation decreases
  intracellular ROS generation at rest and during
  exercise
• Avenathramide supplementation decreases tissue
  oxidative damage at rest and after exercise

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Study Design

• Animals Female Sprague-Dawley rats (n48, age
  6-7 wk)
• Diet
• AIN-93 based control diet or a diet containing
  0.1 g/kg AVEN Bc
• (N-3,4-dihydroxycinnamoyl)-5-hydroxyanthranilic
  acid) 50 days.
• Exercise
• Treadmill running at 22.5 m/min, 10 grade for 1
  hour.
• Tissue Collection
• Heart, liver, kidney, Deep vastus lateralis
  (DVL) and soleus muscle.

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• Body weight of the rats was not different among
  various treatment groups at the beginning or the
  end of the experiments. Food consumption showed
  no difference between groups of rats.
• Final Body Weight of Rats

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• ROS Production

• Exercise increased ROS in the liver. Aven did
  not affect ROS generation.

• Aven increased ROS generation (Plt0.05) in the
  heart of exercise rats.

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ROS generation

• Aven attenuated exercise induced ROS (Plt0.05) in
  soleus muscle.

• ROS generation in DVL muscle was increased
  (Plt0.1) with exercise. No Aven effect was found.

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SOD Activity

• Aven increased SOD activity in the liver
  (Plt0.01).

• Aven increased SOD activity in the kidney
  (Plt0.01).

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SOD Activity

• Aven decreased SOD activity in the heart (Plt0.05)
  of exercise rats.

• Aven increased SOD activity in the DVL muscle
  (Plt0.01).

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GPX Activity

• Exercise increased GPX activity in the liver
  (Plt0.05). Aven had no effect on GPX activity.

• GPX activity tended to be higher (Plt0.09) in the
  heart of Aven vs. Control rats.

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GPX Activity

• GPX activity was not affected by Aven or exercise
  in Kidney or DVL muscle.

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Lipid Peroxidation

• Exercise increased MDA content in the liver
  (Plt0.05). Aven did not affect exercise-induced
  lipid peroxidation.

• Exercise increased MDA content in the heart
  (Plt0.01). Aven attenuated exercise-induced lipid
  peroxidation (Plt0.05).

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Lipid Peroxidation

• Exercise increased MDA content in DVL muscle
  (Plt0.05). No Aven effect.

• MDA content was not changed in kidney with Aven
  or exercise.

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IABG 2003 Cambridge University, UK

• Summary 2
• Aven appears to be a potential antioxidant
  supplement as it decreased ROS generation in
  oxidative muscle soleus and increased SOD
  activity in most tissues.
• The wide tissue-specific and sometimes adverse
  effects are currently unexplained and require
  further investigation.

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IABG 2003 Cambridge University, UK

• Conclusion
• Phytochemicals offer a wide-spectrum of
  antioxidant properties ranging from scavenging
  ROS to increasing antioxidant enzymes in various
  tissues In vitro and in vivo. They can provide
  protective effects against age- and
  exercise-induced oxidative damage.
• Except for a few well-defined ones, we still know
  little about the bioavailability, tissue
  distribution, dose response and potential side
  effects of most phytochemicals.

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IABG 2003 Cambridge University, UK

• Acknowledgment
• Ying Fu, MS
• David Lay, MS
• Euhee Chung, MS
• Stacey Brickson, Ph.D.
• David Peterson, Ph.D.
• USDA CSREES grant
• UW UIR grant
• Kaiser Farms, Inc. for ginseng supply
• Now Foods Inc. for travel support