1. ARCLI:
Antioxidants
TAMARA SIMPSON & EMMA BARVICH.

2. Antioxidants
• Molecules which prevent the oxidation of other molecules
• Oxidation reactions can lead to free radical damage
• Free radical damage can lead to cell death
• Some also act as free radical scavengers
• Broadly classed as either: 1) Hydrophillic 2) Lipophillic
• Hydrophillic antioxidants react with oxidants in cellular cytosol as
well as blood plasma
• Lipophillic antioxidants protect cell membranes from lipid
peroxidation
Antioxidants can also become pro-oxidant following reduction
of oxidizing molecules (e.g. Vitamin C)

3. Oxidative stress
• Oxidative stress is a condition whereby cellular
antioxidant defences are insufficient to keep the
level of reactive oxygen species (ROS) below a
toxic threshold.
• It can be thought of as a balancing act between
antioxidants and free radicals/ROS
• This is because both free radicals and ROS have
important roles to play.
• ROS: cellular redox signalling, particularly H2O2
• Free radicals: thought to play a role in inducing
an endogenous response to protect against
exogenous radicals (i.e. radiation, smoking, etc)
• Lipid peroxidation, the oxidation of cellular lipids,
is a central feature of oxidative stress.
• Lipid peroxidation is implicated as playing a
causative role in the pathophysiology of a
number of diseases.

4. Pycnogenol
What is it?
◦ French Maritime Tree Bark.
◦ Pro-cyanidins (flavanols) make up 65 – 75 % of the extract
What does it do?
◦ Pro-cyanidins in the bark show high anti-oxidant; free radical scavenging
activity
◦ Anti-inflammatory
• Going back to the broad classes of antioxidants, pro-
cyanidins are moderately water soluble - Pycnogenol’s
antioxidant component is likely to react with oxidants
in the blood plasma and cytosol of the cell

5. Pycnogenol
research findings
Note: PYC = Pycnogenol, RCT = Randomized Controlled Trial, CDR = Cognitive Drug
Research computerized assessment system, F2I = F2 Isoprostanes, BP = Blood
Pressure, GSH = Glutathione, GSSG = glutathione oxidised disulfide
Author/Year Intervention/dosa
ge
Design summary Result
Ryan et al. 2008 PYC – 150 mg daily 3 arm – 3 months –
RCT- sample 60 –
85 Yrs (n = 101)
PYC sign. Improved
working memory
(CDR) – decr. F2I
Enseleit et al. 2011 PYC – 200 mg daily 2 arm – 2 months –
RCT – clinical
sample (n = 23)
F2I decr. – No sign.
Improvements for
BP
Dvorakova et al.
2006
PYC – 1 mg/kg
body weight daily
2 arm – 1 month –
RCT – Sample
ADHD 6 – 14 yrs (n
= 43)
PYC improved GSH
levels as well as
GSH:GSSG ratio

6. Bacopa monnieri
What is it?
• a traditional Ayurvedic herb
• used for memory decline, inflammation, pain, fever, epilepsy and as a sedative
• Steroidal sapponins, Bacosides A and B are the active constituents believed to be
responsible for improving both learning and memory
Antioxidant effect:
• may be due to the phytochemicals (alkaloids, flavanoids, steroids) in the plant
• may act at the initiation or termination level or as a chain breaker in free radical
reaction
• it is believed the metal chelating effect on transition metals (ferrous iron) act to
inhibit the formation of free radicals
• high concentrations of Bacopa have showed a marked enhancement in the rate of
oxidation (GSH), thus there may be a balancing act between dosage and duration

7. Bacopa
randomised controlled trial examples
Author/Year Intervention/
dosage
Design summary Result
Stough et al.
(2001)
BM/300mg
daily
2 arm, 3 months, parallel group RCT.
Sample aged 18-60y (n=46)
BM significantly improved speed of visual
information processing (IT), learning rate and
memory (AVLT) & state anxiety
Roodenrys et al.
(2002)
BM/300mg
daily
2 arm, 3 months, parallel group RCT.
Sample aged 40-65y (n=76)
BM significantly improved retention of new
information in delayed recall of word pairs.
Stough et al.
(2008)
BM/300mg
daily
2 arm, 3 months, parallel group RCT.
Sample aged 18-60y (n=62)
BM significantly improved working memory
(CDR) and RVIP
Calabrese et al.
(2008)
BM/300mg
daily
2 arm, 3months, BM significantly improved learning rate and
memory (AVLT) & state anxiety
Peth-Nui et al.
(2012)
BM/300mg or
600mg daily
3 arm, 3 months, parallel group RCT.
Sample mean age 62.62y, SD 6.46 (n=60)
BM improved WM decrease in N100 &P300
latencies. Reduced plasma AChE occurred.
Benson et al.
(2013)
BM/320mg or
640mg
2 arm, acute, cross over RCT.
Sample aged 18-44y (n=17)
BM improved processing speed/selective
attention (Stroop/Letter Search) 1hr & 2hrs post
consumption and positive mood effects and
reduction in cortisol.
Note: PYC = Pycnogenol, BM = Bacopa, RCT = Randomized Controlled Trial, AVLT= Auditory Verbal Learning Test, IT = Inspection Time, RVIP =
Rapid Visual Information Processing, CDR = Cognitive Drug Research computerized assessment system

8. Active Ingredient Dose Role Action
Folic acid 400µg Essential for lots of bodily functions • Synthesise and repair DNA
• regulate homocysteine levels
• reduce neural tube defects
• required in methylation
Vitamin B12 500µg AKA cobalamin, plays a key role in the normal
functioning of the brain and nervous system
• amino acid, fatty acid metabolism
• DNA synthesis and regulation
• identified to reduce brain atrophy associated with AD and
impaired cognitive function
• required in methylation reactions and regulate homocysteine
levels
Vitamin B 6 25mg Coenzyme involved in metabolic processes • amino acid, glucose and lipid metabolism
• neurotransmitter, histamine synthesis
• haemoglobin synthesis and function
• Gene expression
Phosphatidylserine 50mg Phospholipid for brain cells • responsible for cell cycle signaling, specifically in apoptosis
Lipoic acid 300mg AKA alpha lipoic acid, essential for aerobic
metabolism, antioxidant from red meat, broccoli,
spinach, potatoes, carrots, water and fat soluble
• involved in the Krebs cycle – converting carbs into energy
• In a ‘free’ state, excessive amounts act as an antioxidant
removing heavy metals
• may also assist in regenerating antioxidants Vitamins C and E
Vitamin E 30IU
~20mg
Fat soluble antioxidant • stops the production of ROS when fat undergoes oxidation
• Involved in cell signaling, gene expression (connective tissue
growth factor)
• plays a role in cognition
CoQ10 50mg Found in the mitrochondria, part of electron
transport and aerobic cellular respiration, powerful
antioxidant
• key role in producing energy (ATP) for mitochondria
• believed to increase energy production in heart muscle
• may increase dopamine which is low in PD patients
• may assist with heart related conditions due to improving cell
energy production and preventing blood clot formation
• may assist in lowering glycemic index of diabetic patients
Blackmores® Multivitamin
Proprietary blend of micronutrients
and vitamins, not yet commercially
available

9. Multivitamin
and B vitamin RCT examples
Author/Year Intervention/
dosage
Design summary Result
Cognition
de Jager et al. (2011) 0.8mg FA, 0.5 mg
B12, 20mg B6
(VITACOG-MCI)
2 arm, 2yrs, parallel group RCT. Sample aged
≥ 70y (n=266)
BV improved global cognition (MMSE), episodic memory (p=0.001),
semantic memory (p=0.037)
Pipingas et al.
(2014)
Swisse Men’s or
Women’s Ultivite
2 arm, 4 month, parallel group RCT. Sample
aged 20-50y (n=138)
Strong trend for males’ (p=0.01) improvement in selective
attention/response inhibition(Stroop incongruent). No cognitive
benefits for women. Multivitamins increased blood levels of Vit
B6, B12 and folate for both genders, decreased homocysteine in
men.
Kennedy et al.
(2011)
Berocca®, 1
tablet per ay
2 arm, 1 month, parallel group RCT. Male
sample aged 30-55y (n=198)
No cognitive enhancing effects. Subjective ratings of having
greater ‘physical stamina’ across assessments and weeks.
Subjective ratings of greater ‘concentration’ and mental stamina’
during the wo
Atrophy
Douaud et al. (2013) 0.8mg FA, 0.5 mg
B12, 20mg B6
(VITACOG-MCI)
2 arm, 2yrs, parallel group RCT. Sample aged
≥ 70y (n=156)
Whole brain shrinkage slowed as well as, bilateral hippocampus,
parahippocampal gyrus, retrosplenial precuneus, lingual and
fusiform gyrus cerebellum (p < 0.001 FWE corrected for multiple
comparisons)

10. Antioxidant biomarkers
recruited in ARCLI
Glutathione (GSH) in vivo
(hopefully GSH assay)
and
F2 isoprostanes

11. Glutathione
• Most abundant antioxidant in the human body
• Protects cells against reactive oxygen species - from free radical damage and
peroxides and maintains exogenous antioxidants (Vits C & E) in their active form
• It is represented as a reduced form (GSH) and as an oxidised disulphide (GSSG)
• GSH is the major tissue antioxidant. More than 90% of the total glutathione pool is in
this reduced form in healthy cells
• Glutathione peroxidase (GPx) reduces lipid hydroperoxide to their corresponding
alcohols and hydrogen peroxide to water. Low levels have been correlated with free
radical related disorders
• GPx catalyzes, generating oxidised glutathione (GSSG)
• Glutathione reductase (GR) is coupled to GPx and recycles GSSG to GSH

12. cont…
• Plays an essential role in the synthesis and degradation of proteins and DNA
synthesis and repair
• Conjugates with foreign proteins (drugs) and with compounds formed in metabolism
( eg. estrogens, prostaglandins) participating in their metabolism
• A depletion of GSH is indicative of mitochondrial dysfunction and decreased NAA
• Higher concentrations of GSH has been identified in:
• astrocytes vs neurons,
• grey matter vs white matter and
• females vs males
• GSH levels can be raised by mild stress, possibly as a measure of support to handle
increases in stress levels
• With age, GSH levels are diminished

13. Rae, Neurochem Res (2014)39; 1-36
• GSH is made from:
glutamate (Glu),
cysteine (Cys) and
glycine (Gly)
in a two step pathway requiring
ATP
• GSH converted to GSSG via GPx
redox (oxidation-reduction)
reactions
• GSSG can leave the cell
via a multidrug
resistance pump as:
GSH, GSSG or as a mixed
bonded disulphide
• In astrocytes, the major source
of cysteine for GSH synthesis is
from cystine
• Cystine enters astrocytes via the
cystine/glutamate exchanger
• Cysteine is also made through
transsulfuration using
methionine via
S-adenosylmethionine,
S-adenosylhomocysteine,
homocysteine and cystathione
Glutathione in neurons & astrocytes

14. Measuring GSH endogenously
•blood assay measuring total glutathione (GSH: GSSG) ratio
• Kits incorporate glutathione reductase to enable GSH & GSSG to be
measured, reflecting total glutathione
•blood assay measuring GSH peroxidase (GPx)
• kits incorporate cumene hydroperoxide to measure GPx activity
• GPX reduces cumene hdroperoxide while oxidising GSH to GSSG. GSSG is reduced to GSH with the
consumption of NADPH by GR. The decrease in NADPH (measured at A340 monitored as a function of
time) is proportional to GPx activity
GSSG (oxidised state) accumulates when cells are exposed to increased levels of oxidative stress, thus
the ratio of GSSG to GSH increases
An increased ratio of GSSG to GSH is indicative of oxidative stress
The monitoring of reduced and oxidised GSH in biological samples is essential for evaluating the redox
and detoxification status of the cells and tissues against oxidative and free radicals mediated cell injury

15. Research including blood assays isolating GSH and
the relationship to atherosclerosis
114 healthy adults underwent blood assays (GSH:GSSG, high-sensitivity C-reactive
protein, HDL, LDL, triglycerides). BMI, Age, Framingham risk score and carotid
intimamedia thickness (IMT) measured using ultrasound, were also collected.
GSH:GSSG was identified to be an independent predictor for the presence of
atherosclerosis (narrowing of arteries due to plaques) in an otherwise healthy
cohort.

16. Measuring GSH in vivo
Magnetic Resonance Spectroscopy (MRS)
• a technique that complements standard MRI to examine small molecules
• The signal is obtained from the abundance of hydrogen protons (1H) to determine
metabolite peak areas that reflect concentrations of metabolites in brain tissue
• Each metabolite is associated with a peak that occurs at a known frequency
Brennan et al., Biological Psychiatry,(2013)73:24-31.

17. Research applying the technique of MRS
isolating GSH in response to supplements
Ethyl-eicosapentaenoic Acid (E-EPA- 2g), an omega-3 fatty acid, was administered
for 12 weeks with 24 first episode psychosis patients. GSH increased bilaterally and
glutamate/glutamine increased in the left hemisphere post E-EPA administration.
Improvement in negative symptoms correlated with increased GSH (r=-0.57).

18. Research applying the technique of MRS isolating
GSH in response to intravenous application of N-
acetylcysteine
Infusion of 150mg/kg NAC with 3 PD, 3GD and 3 healthy controls increased blood GSH
redox ratios. This was followed by an increase in GSH neurometabolite concentrations
across the participants identified using MRS.

19. F2 - isoprostanes
•Prostaglandin-like compounds formed from free radical induced peroxidation of
arachadonic acid (ubiquitous omega-6 polyunsaturated acid from food eg. egg,
red meat)
•Lipid peroxidation can be very damaging, it leads to alterations in the
biophysical properties of the cell membrane, impairing normal cellular function
•F2 isoprostanes are a significant and accurate marker of oxidative stress in vivo
in humans and animals
•They are the most studied prostaglandin due to their stability which enables the
most accurate measure of oxidative stress
•Elevated levels have been linked to a myriad of human disorders eg. smoking,
diabetes, Huntington’s disease, AD, Chron’s diesease, atherosclerosis
•Clinically, F2 isoprostanes are useful for monitoring disease and response to
therapy
•F2 isprostanes in ARCLI are measured through blood assays

20. Biological effects
of F2 - isoprostanes
Isoprostanes:
• produce inflammation and
atherogenesis (formation of
plaques in arteries) via Mitogen-
Activated Protein kinases
• Promote platelet activation and
induces mitogenesis (triggering
of mitosis) in vascular smooth
muscle cells, stimulates
responses in fibroblasts, alters
endothelial cell biology (COX
activation-produces
inflammation, PGF2a-induces
labour)
• Asprin and ibuprofen inhibit COX
activation
Kaviarasan et al. J Clin Biochem Nutr (2009)45(1):1-8.
Diabetes:
• Associated with high F2
levels
• Deficiencies in: ascorbate,
GSH, superoxide
dismutase.
• Low levels of GSH found in
diabetic neutrophils (white
blood cell, hallmark of
acute inflam) and
monocytes (white blood
cell, elicit immune
response)
• Low levels of ascorbate
found in both diabetic
plasma and mononuclear
cells (lymphocytes,
monocytes, dendritic cells)

21. Examples of F2 isoprostane research
Weight loss in obese women was associated with a significant reduction in
isoprostane formation.
Based on 3,000 participants in the Framingham Heart study, elevated F2
isoprostane levels in both males and females was strongly associated with
increasing BMI.

22. Possible directions using
baseline data
Baseline correlations of F2 isoprostane levels, BMI, weight and diet
(FFQ)
Baseline correlations of F2 isoprostane levels and GSH concentrations in
vivo to elucidate markers of oxidative stress
Baseline correlations of F2 isoprostane levels, GSH:GSSG bloods,
different age ranges to determine the trajectory of normal age related
cognition
GSH:GSSG ratio and the relationship to F2 isoprostane levels
Baseline assessment of brain atrophy between different age groups and
correlation to cognition or WASI

23. Possible directions using
longitudinal data
Pycnogenol
Cognition: Based on Ryan et al. (2008) – improvements expected for PYC group on CDR
F2 Isoprostanes: Expected decrease in PYC group compared to control (Ryan et al., 2008 & Enseleit et al., 2011)
GSH levels: Based on quite tentative findings, we could see an increase in PYC group (Dvorakova et al., 2006)
Bacopa
Cognition: Based on the body of CHP work and others, improvement is expected at 3mths and more importantly 6mths and 12mths. Any
permutation of the cognitive variables in ARCLI with MRS, F2’s and/ or cardio measures to investigate improvement over time.
GSH levels: If assayed will be interesting to investigate any changes that occur, as low doses for a long duration has been suggested to
possibly be more effective (Tripathi et al., 1996).
Multivitamin
Cognition: Correlation to forestalling of brain atrophy over time (Duoud et al. 2013).
F2 Isoprostanes: A reduction in levels may be mediated through B Vits and antioxidants in the blend - speculative
Imaging
Cognition: correlation to MRS (NAA, Cho, Cre) and supplements, correlation to GSH in vivo and supplements, correlation to MRS, GSH in
vivo and blood flow (ASL), supplements and PWV, correlation to resting state (DMN) and supplements

24. Thank you
Dr Doug Mitchell
Swinburne Alumni Benefactor
Acknowledgements: