GARLIC A DAY KEEPS THE OXIDATIVE

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GARLIC A DAY KEEPS THE OXIDATIVE STRESS AWAY
Dr. Khalid Rahman School of Biomolecular
Sciences Faculty of Science Liverpool John Moores
University Liverpool, UK.
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TRADITIONAL MEDICINES

• Usage over many centuries - Before advent of
  modern medicines
• Fastest growing segment of food health industry
• Market estimated at 6 - 60 billion
• Growing at gt5 pa

• Last 20 years international resurgence of herbs
• Enhanced interest in natural systems
• Questioning of an over dependence on synthetic
  drugs
• More consumer awareness/demand for preventative
  medicines
• Cost effective way of sustaining health

• EU Legislation

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OXIDATIVE STRESS
Free radicals (ROS)
Arrest of ageing/rejuvenation being sought
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NUTRITION PROBLEMS

• Ignorance confusion
• Unwilling or unable to follow dietary guidelines
• Modern day lifestyle

EPIDEDMIOLOGICAL STUDIES

• Diet rich in plant foods (antioxidants) may
  reduce/delay certain chronic diseases eg cancer,
  atherosclerosis

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• Renewed interest in garlic

• Composition of fresh garlicvariable
• Commercial garlic preparations available
• Garlic powder tablets (allicin)
• Oil of steam-distilled garlic
• Oil of oil-macerated garlic
• Ether extracted oil of garlic

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AGED GARLIC EXTRACT (KYOLIC)
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AGED GARLIC EXTRACT (KYOLIC)
Garlic soaked in 15-20 aqueous ethanol for up to
20 months
?
Extract filtered and concentrated under reduced
pressure
?
Marketed in both dry (tablets powder capsules)
liquid form
?
Major sulphur compund is S-Allyl Cysteine
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ANTI-OXIDANT PROPERTIES OF AGED GARLIC EXTRACT IN
RELATION TO CARDIOVASCULAR DISEASE
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REACTIONS OF LIPID PEROXIDATION
LH X ? L XH
INITIATION OXYGEN ADDITION CHAIN
PROPAGATION INHIBITION BY ANTI-OXIDANTS
L O2 ? LOO
LOO LH ? LOOH L
LOO AH ? LOOH A
LH Lipid - polyunsaturated fatty acid X
Initiating free radical LOO Lipid alkoxyl
radical LOOH Lipid hydroperoxide AH
Anti-oxidants
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INITIATION OF LIPID PEROXIDATION
LDL
?
mm - LDL minor amounts of hydroperoxides
LIPID PEROXIDATION CHAIN REACTION
HYDROPEROXY FATTY ACIDS MODIFICATION,
FRAGMENTATION OF apoB
?
Continued
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Stimulates platelet aggregation
Immunogenic
Chemotactic
Stimulates smooth muscle cell proliferation
Induces expression of proinflammatory cytokines
Unregulate uptake by macrophages (foam cell
formation)
Stimulates smooth muscle collagen production
Monocyte ? resident macrophages
Impairs vasodilation
Cytotoxic
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AGE PREPARATIONS
Whole AGE interferes with some spectrophotometric
assays. 1 part of AGE 2 parts of diethyl
ether. ? Diethyl ether extract removed, dried
and residue resuspended in PBS
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Xanthine-xanthine oxidase.. ? Superoxide
ions.. ? Reduce cytochrome C at 550nm ? Xanthine
oxidase activity monitored as uric acid
production at 284nm
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The effect of SOD, ascorbic acid, whole
AGE/extract on superoxide and uric acid
production by xanthine-xanthine oxidase (X-XO).
plt0.05
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Effect of AGE/extract on reduction of cytochrome
C by superoxide ions generated by xanthine
oxidase and xanthine.









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The effect of whole AGE/extract and EDTA on
superoxide and uric acid production by
xanthine-xanthine oxidase (X-XO) in the presence
of 50?m CuSO4.
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15-Lipoxygenase ? Catalyses oxidation of Linoleic
acid ? Forms hydroperoxy lipids ? Reaction
monitored as formation of conjugated dienes at
234nm
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The effect of Trolox? and a diethyl ether extract
of AGE on hydroperoxy lipid formation by
15-lipoxygenase (15-LO).
plt0.05
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Effect of AGE extract on conjugated diene
formation by 15-soybean lipoxygenase and
linoleic acid
plt0.05
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LDL oxidatively modified by Cu2,
Xanthine-Xanthine oxidase and 15-Lipoxygenase ?
Oxidised LDL characterised by assessing for
TBA-RS (malondialdehyde (MDA) equivalents) and
electrophoretic mobility as an indicator of lipid
and protein oxidation respectively
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Effect of AGE extract on oxidative modification
of LDL
80
LDLPBS
70
LDLBorate Buffer

60
LDL Cu2
LDLXO/X
50
LDL15-SLO
MDA (nmoles/mg LDL protein)
40
30
20
10
0
Controls
5 Ex
10 Ex
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plt0.05
Effect of AGE extract on oxidative modification
of LDL
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Time course for the oxidative modification of LDL
80
Cu2
Cu25Ex
60
Cu210Ex
LDL ONLY
MDA (nmoles/mg LDL protein)
40
20
0
0
1
2
3
4
5
Time (hrs)
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Electrophoretic mobility of LDL
CUSO4
CUSO4 AGE
X-XO AGE
X-XO
LDL
CUSO4 AGE extract
lipoxygenase LA AGE extract
X-XO AGE extract
lipoxygenase LA AGE
lipoxygenase LA
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CAN THIS BE SHOWN IN A CLINICAL TRIAL?
Oxidative stress markers in clinical settings are
non-specific, unreliable or inaccurate
Family of novel prostaglandin F2 isomers called
F2-isoprostanes have been described
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Arachidonic acid
F2-isoprostanes (released from membrane
phospholipids)
F2-isoprostanes -plasma? free form or esters in
phospholipid -urine ? free form excreted
F2-isoprostanes - sensetive - specific indicator
of lipid peroxidation (oxidative stress)
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DOES AGED GARLIC EXTRACT REDUCE OXIDATIVE
STRESS IN SMOKERS AND NON SMOKERS?
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PROTOCOL

• 20 apparently healthy subjects
• 10 smokers
• 5 men, 5 women, mean age 41 ? 4.1y
• 10 nonsmokers
• 5 men, 5 women, mean age 41.6 ? 4y
• Age, height, weight, alcohol, cigarettes
• Body mass index determined
• Day zero
• 10mL urine venous blood collected
• 5mL aged garlic extract/day for 14 days
• Day 14
• Urine blood sample collected
• discontinue AGE for 14 days
• Day 28
• Urine blood sample collected

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ANALYSIS

• Biochemical analysis

• plasma urinary creatinine
• creatinine clearance
• plasma protein
• total plasma cholesterol
• total plasma triglycerides

• Enzyme immuno-assay

• plasma total (esterified free)
  8-Iso-Prostaglandin F2?
• urine free 8-Iso-Prostaglandin F2?

• Antioxidant capacity of plasma

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RESULTS
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CLINICAL / BIOCHEMICAL CHARACTERISTICS
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RESULTS

• Subjects
• similar in age, height and weight
• normal lipidaemic
• normal kidney function
• similar alcohol consumption
• no effect on plasma lipid profiles
• no effect on plasma and urinary creatinine
  concentration
• only difference was in smoking habits

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PLASMA 8-iso-PGF2?
Different from non-smokers Different from
day 0
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URINARY 8-iso-PGF2?
Different from non-smokers Different
from day 0 Different from day 14
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PLASMA ANTIOXIDANT CAPACITY
Different from non-smokers Different
from day 0 Different from day 14
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SUMMARY

• Aged garlic extract
• (inhibits platelet aggregation)
• Scavenges superoxide inhibits formation of
  lipid peroxide
• Inhibits in-vitro oxidation of LDL
• Reduces LDL oxidation by chelation of Cu2
• Reduces oxidative stress in smokers non-smokers

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CONCLUSIONS
Dietary supplementation with Aged Garlic Extract
may protect against an increase in oxidative
stress and related diseases
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GARLIC A DAY KEEPS THE OXIDATIVE STRESS AWAY
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GARLIC A DAY KEEPS THE OXIDATIVE STRESS AWAY
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ACKNOWLEDGEMENTS
Stephanie Dillon Gillian Allison Dr. Gordon
Lowe Quest Vitamins Ltd. Wakunaga of America
Ltd.