Lipids

Class Presentations Will be

Discussed at the End of Class
Exams back next Monday
No class this Wednesday !!!!!

Lipids
Main functions of lipids in foods
Energy and maintain human health
Influence on food flavor
Fatty

acids impart flavor

Lipids carry flavors/nutrients
Influence
Solids

on food texture

or liquids at room temperature

Change with changing temperature
Participation in emulsions

Lipids
Lipids

are soluble in many organic solvents

Ethers

(n-alkanes)
Alcohols
Benzene
DMSO (dimethyl sulfoxide)
They

are generally NOT soluble in water

C, H, O and sometimes P, N, S

Lipids

Neutral Lipids

Waxes

Long-chain alcohols (20+ carbons in length)

Cholesterol esters
Vitamin A esters
Vitamin D esters

Conjugated Lipids

Triacylglycerols

Phospholipids, glycolipids, sulfolipids

Derived Lipids

Fatty acids, fatty alcohols/aldehydes, hydrocarbons

Fat-soluble vitamins

Lipids
Structure
Triglycerides or triacylglycerols
Glycerol + 3 fatty acids
>20 different fatty acids

Lipids 101
Fatty

acids- the building block of fats

A fat with no double bonds in its structure is said to
be saturated (with hydrogen)
Fats with double bonds are referred to as mono-, di-,
or tri- Unsaturated, referring to the number of
double bonds. Some fish oils may have 4 or 5
double bonds (polyunsat).
Fats are named based on carbon number and number
of double bonds (16:0, 16:1, 18:2 etc)

Lipids
Oil-

liquid triacylglycerides Oleins

Fat- solid or semi-solid mixtures of crystalline
and liquid TAGs Stearins
Lipid content, physical properties, and
preservation are all highly important areas for
food research, analysis, and product
development.
Many preservation and packaging schemes are
aimed at prevention of lipid oxidation.

Nomenclature
The

first letter C represents Carbon

The number after C and before the colon
indicates the Number of Carbons
The letter after the colon shows the Number of
Double Bonds
The letter n (or w) and the last number indicate
the Position of the Double Bonds

Saturated Fatty Acids

Saturated Fatty Acids

8
7
CH3 CH 2

5
3
4
6
CH 2 CH 2 CH 2 CH 2

Octanoic Acid

O
2
1
CH 2 C OH

Mono-Unsaturated Fatty Acids

Poly-Unsaturated Fatty Acids

Fatty Acids
Melting Points and Solubility in Water

Response

Melting Point

Solubility in H2O
2

Fatty acid chain length

Unsaturated Fatty Acids

8
CH 3

7
CH2

5
6
CH 2 CH 2

4
CH 2

3
2
CH 2 CH 2

O
1
C OH

3 - Octenoic Acid

8
7
CH3 CH 2

5
3
4
6
CH 2 CH 2 CH 2 CH 2
3, 6 - Octadienoic Acid

O
2
1
CH 2 C OH

Lipids
Properties depend on structure
Length of fatty acids (# of carbons)
Position of fatty acids (1st, 2nd, 3rd)
Degree of unsaturation:

Double bonds tend to make them a liquid oil

Significantly lowers the melting point

Hydrogenation: tends to make a solid fat

Significantly increases the melting point

Unsaturated fats oxidize faster

Preventing lipid oxidation is a constant battle in the food
industry

Fatty Acids
Melting Points and Solubility in Water

Response

Melting Point

Solubility in H2O
2

Fatty acid chain length

Characteristics of Fatty Acids

Fatty Acids

M.P.(C)

mg/100 ml
in H2O

C4

-8

C6

-4

970

C8

16

75

C10

31

C12

44

0.55

C14

54

0.18

C16

63

0.08

C18

70

0.04

Fatty Acids
O
R C OH
#1 Carbon
O
R C OH

Acid Group
Polar End - Hydrophilic End

Non-polar End - Hydrophobic End

(Fat-soluble tail)

Lipids 101
Fatty

acid profile- quantitative determination of the

amount and type of fatty acids present following
hydrolysis.
To help orient ourselves, we start counting the
number of carbons starting with 1 at the
carboxylic acid end.
O
C C C C C C C C C C C C C C C C C C
18
1

OH

Lipids 101
For

the 18-series (18:0, 18:1, 18:2, 18:3) the

double bonds are usually located between carbons
9=10 12=13
15=16.
O
C C C C C C C C C C C C C C C C C C
18
16 15 13 12 10 9
1

OH

Lipids 101
The

biomedical field started using the OMEGA (w)

system (or n fatty acids).
With this system, you count just the opposite.
Begin counting with the methyl end
Now the 15=16 double bond is a 3=4 double bond
or as the medical folks call it.an w-3 fatty acid
C
C C C C C C C C C C C C C C C C C C
1
6 7
3 4
18
10
9

OH

Tuning Fork Analogy-TAGs

Envision a Triacylglyceride as a loosely-jointed E
Now, pick up the compound by the middle chain,
allowing the bottom chain to hang downward in a
straight line.

The top chain will then curve forward and form an h

Thus the tuning fork shape
Fats will tilt and twist to the lowest free energy level

Lipids

Lipids are categorized into two broad classes.

The first, simple lipids, upon hydrolysis, yield up to two types

of primary products, i.e., a glycerol molecule and fatty acid(s).

The other, complex lipids, yields three or more primary

hydrolysis products.

Most complex lipids are either glycerophospholipids, or

simply phospholipids

contain a polar phosphorus moiety and a glycerol backbone

or glycolipids, which contain a polar carbohydrate moiety

instead of phosphorus.

Lipids

Other types of lipids

Phospholipids
Structure similar to triacylglycerol
High in vegetable oil
Egg yolks
Act as emulsifiers

Where Do We Get Fats and Oils?

Crude fats and oils are derived from plant and animal sources
Several commercial processes exist to extract food grade oils
Most can not be used without first refining before they reach
consumers
During oil refining, water, carbohydrates, proteins, pigments,
phospholipids, and free fatty acids are removed.
Crude fats and oils can therefore be converted into high quality
edible oils
In general, fat and oil undergo four processing steps:

Extraction
Neutralization
Bleaching
Deodorization

Oilseeds, nuts, olives, beef tallow, fish skins, etc.

Rendering, mechanical pressing, and solvent extraction.

Fats and Oils: Processing

Extraction
Rendering
Pressing oilseeds
Solvent extraction
Soybean

Peanut

Rape Seed

Safflower

Sesame

Fats and Oils

Further Processing

Degumming

Refining

Remove free fatty acids (alkali +

water)

Bleaching

Remove phospholipids with water

Remove pigments (charcoal filters)

Deodorization

Remove off-odors (steam, vacuum)

Where Do We Get Fats and Oils?

Rendering
Primarily for extracting oils from animal tissues.
Oil-bearing tissues are chopped into small pieces and
boiled in water.
The oil floats to the surface of the water and skimmed.
Water, carbohydrates, proteins, and phospholipids
remain in the aqueous phase and are removed from the
oil.
Degumming may be performed to remove excess
phospholipids.
Remaining proteins are often used as animal feeds or
fertilizers.

Where Do We Get Fats and Oils?

Mechanical Pressing
Mechanical pressing is often used to extract oil from
seeds and nuts with oil >50%.
Prior to pressing, seed kernels or meats are ground into
small sized to rupture cellular structures.
The coarse meal is then heated (optional) and pressed in
hydraulic or screw presses to extract the oil.
Olive oils is commonly cold pressed to get extra virgin
or virgin olive oil. It contains the least amount of
impurities and is often edible without further processing.
Some oilseeds are first pressed or placed into a screwpress to remove a large proportion of the oil before
solvent extraction.

Where Do We Get Fats and Oils?

Solvent Extraction
Organic solvents such as petroleum ether, hexane, and 2-propanol can be added to
ground or flaked oilseeds to recover oil.
The solvent is separated from the meal, and evaporated from the oil.
Neutralization
Free fatty acids, phospholipids, pigments, and waxes exist in the crude oil
These promote lipid oxidation and off-flavors (in due time)
Removed by heating fats and adding caustic soda (sodium hydroxide) or soda ash
(sodium carbonate).
Impurities settle to the bottom and are drawn off.
The refined oils are lighter in color, less viscous, and more susceptible to oxidation
(without protection).
Bleaching
The removal of colored materials in the oil.
Heated oil can be treated with diatomaceous earth, activated carbon, or activated
clays.
Colored impurities include chlorophyll and carotenoids
Bleaching can promote lipid oxidation since some natural antioxidants are
removed.

Where Do We Get Fats and Oils?

Deodorization
The final step in the refining of oils.
Steam distillation under reduced pressure (vacuum).
Conducted at high temperatures of 235 - 250C.
Volatile compounds with undesirable odors and tastes
can be removed.
The resultant oil is referred to as "refined" and is ready
to be consumed.
About 0.01% citric acid may be added to inactivate prooxidant metals.

Fats and Oils

Further Processing
Hydrogenation
Add hydrogen to an oil to saturate the fatty acid
double bonds

Conducted with heated oil

Often under pressure
In the presence of a catalyst (usually nickel)

Converts liquid oils to solid fats

Raises melting point

Hydrogenating Vegetable oils can

produce trans-fats
H H
C C
Cis-

H
C C
Trans-

The cis- and trans- forms of a fatty acid

Fats and Oils in Foods

SOLID FATS are made up of microscopic fat crystals. Many fats

are considered semi-solid, or plastic.
PLASTICITY is a term to describe a fats softness or the
temperature range over which it remains a solid.
Even a fat that appears liquid at room temperature contains a small number of
microscopic solid fat crystals suspended in the oil..and vice versa

PLASTIC FATS are a 2 phase system:

Plasticity is a result of the ratio of solid to liquid components.

Solid phase (the fat crystals)

Liquid phase (the oil surrounding the crystals).

Plasticity ratio = volume of crystals / volume of oil

Measured by a solid fat index or amount of solid fat or liquid oil in a lipid

As the temperature of a plastic fat increases the fat crystals melt

and the fat will soften and eventually turn to a liquid.

Fat and Oil: Further Processing

Winterizing

(oil)

Cooling

a lipid to precipitate solid fat crystals

DIFFERENT from hydrogenation
Plasticizing
Modifying

(cooling)

Tempering

(fat)
fats by melting (heating) and solidifying

(fat)

Holding

the fat at a low temperature for several

hours to several days to alter fat crystal properties

(Fat will hold more air, emulsify better, and have

a more consistent melting point)

Lipid Oxidation

Oleic acid

Radical Damage,
Hydrogen
Abstraction

Formation of a
Peroxyl Radical

Simplified scheme of lipoxidation

H H H H

H H H H

H H H H

R C C C C R

R C C C C R

R C C C C R

+ Catalyst

+ Oxygen

O
O

Primary Drivers
Temperature-basic

rxn kinetics

Water Activity
Both

high and low Aw

At low Aw, peroxides decompose faster and metal ions
are better catalysts in a dry environment
Metal

Ions-catalysts
Light-energy source
Singlet Oxygen- ROS, highly electrophilic
Reacts

1,500 times faster at C=C than ground state O2

Enzymes

ie. Lipoxygenase (LOX)

Initiation of Lipid Oxidation

There must be a catalytic event that causes the initiation of

the oxidative process

Enzyme catalyzed
Auto-oxidation

Excited oxygen states (i.e singlet oxygen): 1O2

Triplet oxygen (ground state) has 2 unpaired electrons in the same spin in
different orbitals.
Singlet oxygen (excited state) has 2 unpaired electrons of opposite spin in the
same orbital.

Metal ion induced (iron, copper, etc)

Light
Heat
Free radicals
Pro-oxidants
Chlorophyll
Water activity

Considerations for Lipid Oxidation

Which

hydrogen will be lost from an unsaturated

fatty acid?
The longer the chain and the more double
bonds.the lower the energy needed.

Bond Strength
85 kCal

103 kCal

65 kCal

65 kCal

Fatty acid
OH or other
Free radicals
.

..

Propagation Reactions
Initiation

Peroxyl radical

Ground state oxygen

Hydroperoxide
decomposition

Hydroperoxide

Alkoxyl radical
Start all over again

New
Radical

Hydroxyl radical!!

Mechanism of Photooxidation

Chlorophyll

O2

3
1

or

Singlet Oxygen Oxidation

1

O2

HOOC

OOH
HOOC

OOH
HOOC

OOH
HOOC

OOH
HOOC

Autoxidation
9

HOOC

10

12

13

11

14

H
9

10

12

13

11
8

10

12

14

13

10

11

12

13

11
14

14

O2
OO
10
9
8

12

13

11
14

Singlet Oxygen Oxidation

1

O2

HOOC

HOOC
OOH

OH

Nonanal

Secondary Products: Aldehydes

Volatile Aldehydes from Oxidation

Lipid Oxidation

Peroxide decomposition can generate aldehydes, ketones, alcohols, various

hydrocarbons, and epoxides. Many are volatile, and many are unappealing.

Relative Oxidation Rates

How

fast is lipid oxidation?

Fatty Acid
Relative Rate
Steric acid (18:0) 1
Oleic acid (18:1 n9)100
Linoleic acid (18:2 n6) 1,200
Linolenic acid (18:3 n3) 2,500

Termination of Lipid Oxidation

Although radicals can meet and terminate propagation
by sharing electrons.
The presence or addition of antioxidants is the best way in
a food system.
Antioxidants can donate an electron without becoming a
free radical itself.

Antioxidants and Lipid Oxidation

BHT butylated hydroxytoluene
BHA butylated hydroxyanisole
TBHQ tertiary butylhydroquinone
Propyl gallate
Tocopherol vitamin E
NDGA nordihydroguaiaretic acid
Carotenoids

Polar Antioxidants

Most effective in
nonpolar or less polar
environment

Bulk oils

Located at the oil-air

interface or in reverse
micelles

High amount of
oxidants present here

Yellow Oil
Blue water
Phospholipids

Non-polar Antioxidants

Most effective in polar

environment

Oil-in-water emulsions

Located at the water-oil

interface

Dissolved in oil droplets

of the emulsion
Allows access to
oxidizing agents located
in the water phase

Peroxides
Oxidizing metals

Chemical Tests for

Lipid Characterizations

Iodine Value
Measure

of the degree of unsaturation in an oil

or the number of double bonds in relation to
the amount of lipid present
Defined as the grams of iodine absorbed per
100-g of sample.
The

higher the amount of unsaturation, the

more iodine is absorbed.
Therefore the higher the iodine value, the
greater the degree of unsaturation.

Iodine Value
A known

solution of KI is used to reduce excess ICl

(or IBr) to free iodine
R-C-C = C-C-R + ICl R-C-CI - CCl-C-R + ICl
[Excess]

Reaction

(remaining)

scheme: ICl + 2KI KCl + KI + I2

The

liberated iodine is then titrated with a

standardized solution of sodium thiosulfate using a
starch indicator
I2 + Starch + thiosulfate = colorless endpoint
(Blue colored)

Iodine Value
Used to characterize oils:
Following hydrogenation
During oil refining (edible oils)
Degree of oxidation (unsaturation decreases
during oxidation)
Comparison of oils
Quality control

Iodine value: g absorbed I2/ 100 g fat

Highly saturated
High in 18:1
High in 18:1 and 18:2)
18:1, 18:2, 18:3
18:1, 18:2, 18:3
(longer chains)

What can we conclude about the COMPOSITION

or STRUCTURE of each of these oil types?

Automated
Iodine Value
Determination

Standard Iodine Value

A = 23
B = 44
C = 67
D = 89
E = 111
Measures IBr or ICl
Consumption (neg. peak)

Consumption over
time

Chemical Tests
Saponification Value

Saponification Value
Saponification is the process of breaking down or
degrading a neutral fat into glycerol and fatty acids
by treating the sample with alkali.
Heat

Triacylglyceride ---> Fatty acids + Glycerol

KOH

Definition: mg KOH required to titrate 1g fat

(amount of alkali needed to saponify a given amount of fat)

Typical values: Peanut = 190, Butterfat = 220

Saponification Value
The mg KOH required to saponify
triacylglycerides into glycerol plus fatty acids
is related to:
average fatty acid chain length or
average fatty acid molecular weight
Divide molecular weight by 3 to get average of
the fatty acids present

Chemical Tests for Oxidation

Lipid Oxidation
Hydrolysis
Peroxide Value
Oxidation Tests

LIPID OXIDATION
Lipid System Under
Oxidizing Conditions

Free Fatty Acids (FFAs)

Degree of hydrolysis (hydrolytic rancidity)

High level of FFA means a poorly refined fat
or fat breakdown after storage or use.

Measures of Oxidation
Oxidation is a very complex reaction - no one test

will measure all of the reactants or products.

Some assays measure intermediates while others

measure end products.

Peroxide Value
Measures peroxides and hydroperoxides in an

oil which are the primary oxidation products

(usually the first things formed).
The peroxide value measures the present

status of the oil. Since peroxides are

destroyed by heat and other oxidative
reactions, a seriously degraded oil could have
a low PV.

Peroxide Value
KI + peroxyl radical yields free Iodine (I2)
The iodine released from the reaction is measured in the
same way as an iodine value.
I2 in the presence of amylose is blue.
I2 is reduced to KI and the endpoint determined by loss of
blue color.

4I + O2 + 4H 2I2 + 2H2O

Thiobarbituric acid (reactive substances) TBA OR TBARS

Tests for end products of oxidation aldehydes, Malonaldehyde
(primary compound), alkenals, and 2,4-dienals
A pink pigment is formed and measured at ~530 nm.

TBARS is firmly entrenched in meat oxidation research and is a

method of choice.
TBARS measure compounds that are volatile and may react further
with proteins or related compounds.
High TBA = High Oxidative Rancidity

HEXANAL Determination
Good indictor of the end products of oxidation

(if there are any).

Standard method in many industries.
Aldehyde formation from lipid oxidation.
Nonenal is also a common end-product

Conjugated Fatty Acids

During oxidation, double bond migration
occurs and conjugated fatty acids are
formed.
They absorb light efficiently and can be
monitored in a spectrophotometer.

R C C C C C C C C R

TECHNIQUES OF MEASURING OXIDATIVE

STABILITY
Induction Period: is defined as the length of time
before detectable rancidity or time before rapid
acceleration of lipid oxidation

MEASURING OXIDATIVE STABILITY

Active

Oxygen Method - Air is bubbled through oil or fat at

97.8C. Time required to reach peroxide value of 100 meq/kg
fat determined. (method replaced by OSI)
Oil

Stability Index automated Rancimat (instrumental

method). Air bubbled through sample (110C). Oil degrades to
many acidic volatiles (e.g. formic acid) which are carried by
the air into a water trap. Conductivity of the water can then be
assessed.

Free Radicals

 What

are free radicals?

Where are free radicals from?
How damaging are free radicals?
How do we control free radicals?

What are free radicals?

Any molecular species capable of independent

existence, which contains one or more
unpaired valence electrons not contributing to
intramolecular bonding.is a free radical.

The most frequent radicals are oxygen-derived free radicals, also

known as reactive oxygen species (ROS):
Superoxide (O2-)
Peroxyl (ROO)
Alkoxyl (RO)
Hydroxyl (HO)
Nitric oxide (NO)
Other ROS are non-radicals such as singlet oxygen (O2), hydrogen
peroxide (H2O2), and hypochlorous acid (HClO).

Where do they come from?

Free radicals are produced by oxidation/reduction

reactions in which there is a transfer of only one electron
at a time, or when a covalent bond is broken and one
electron from each pair remains with each atom.

1) Normal ongoing metabolism, especially from the

electron transport system in the mitochondria and
from a number of normally functioning enzymes

2) Environmental factors such as pollution, radiation,

cigarette smoke and toxins can also spawn
biologically-derived free radicals.

How damaging are free radicals?

ROS may be very damaging, since they can

attack:

Lipids in cell membranes

Proteins in tissues or enzymes
Carbohydrates
DNA

These cause cell membrane damage, protein

modification, and DNA damage.
Thought to play a role in aging and several
degenerative diseases (heart disease, cataracts,
cognitive dysfunction, and cancer).
Oxidative damage can accumulate with age.

Our Body vs. Our Food

Biological

radicals
Food-based radicals
Where

do these 2 areas cross?

Functional Foods Concept

Certain

food ingredients have health benefits

beyond basic nutrition
Recent development only: since ~1975
The concept that non-nutrients were beneficial
has taken off since then
First idea in scientific community: antioxidant
compounds may protect against chronic
diseases

Free Radicals
Early

1950s: cell damage is due to reactive

oxygen species called free radicals
Unstable, damaged molecule that is missing an
electron
Highly reactive; reacting to some measurable
extent with any molecule they come in contact
with
In living systems, cell injury or disease
In foods, quality-degrading impact

Reactive Oxygen Species (ROS)

Primary

target list: protein, lipid, DNA, and

carbohydrates
End results: cancer, CHD, stroke, arterial
disease, rheumatoid arthritis,
Parkinsons/Alzheimer disease, cataracts,
macular degeneration.many more
Aging by slow oxidation?

The Defense
Minimize

contact between free radicals and

important systems (like cellular components)
Cell membranes are one of our best barriers
Metal chelation system in-place
Protease enzymes are in place to remove
damaged proteins for replacement by new
Repair enzymes help to restore DNA
Antioxidant enzymes-superoxide dismutase,
catalase, glutathione peroxidase

Best DefenseA Good Offense

Nutrients

that cant be synthesized in vivo:

vitamin C, vitamin E, (pro)vitamin A
Non-nutrients: polyphenolics/carotenoids
Diet

What

is only source.are they essential?

about conditions of oxidative stress?

This

is a condition when pro-oxidants

outnumber antioxidants (I.e. decreased immune
response, environmental factors, hypertension,
poor diet).

Foods and the Antioxidant Link

Soy- isoflavones, polyphenolics
Tea- polyphenolics, flavans
Coffee- polyphenolics
Wine- polyphenolics
Rosemary- carnosic acid, rosmaric acid
Citrus- flavonoids
Onions- sulfur cpds, flavonoids
Berries- flavonoids, polyphenolics
Vegetables- carotenoids, polyphenolics

Antioxidants in Food Systems

Oxidative Stress-the food remedy

Diet:
Inflammation-

tocopherol
Smoke- ascorbic acid
Physical stress- carotenoids
Pollution- carotenoids
Environment:
Radiation- glutathione
Carcinogens- antioxidant enzymes, diet
modification

Oxidative Stress and Foods

Tocopherol-

vegetable oil, whole grains,

vegetables, fish/poultry
Ascorbic acid- citrus, berries, tomato, leafy
veggies, brassicas (broccoli, cauliflower)
Carotenoids- yellow/orange fruits and
veggies, tomatoes, green leafy veggies.
Polyphenolics- coffee/tea, grains, all fruits
and vegetables

Magic Bulletsfor our body?

Most likely not
Will increasing the intake of antioxidants
modulate disease prevention? Will we live longer
with no health problems?
Lung cancer and -carotene: Whoa
Antioxidant compounds have demonstrated
benefits (both acute and long-term) of preventing
or postponing the onset of many degenerative
diseases, but clinical trials are full of holes, and
conclusive evidence of the bullet is still not with
us.

Magic Bulletsfor our foods?

Will increasing the use of antioxidants in foods
modulate all oxidative damage?
Will food products live longer with no quality
problems?
Pro-oxidant nature of ascorbic acid: Whoa
Ascorbic acid does not always act linearly in food
systems
In the presence of metal ions (ie. Fe/Cu) it can
generate reactive oxygen species (peroxides) or free
radicals (hydroxyl radicals)

Causes and Effects

-carotene and lung cancer: small, but significant
increase with smokers
Tocopherols and CHD: protect lipoproteins or inhibit
blood clotting (which initiates heart attacks)
Tocopherols and Alzheimers: reducing oxidative
stress by supplementation
Cataracts and vitamins (A,C,E): inverse association
Macular degeneration and carotenoids: inverse
Vitamin C and the Common Cold: shorter, milder
colds

Structure-Based AOX
Polyphenolics

Structure of flavonoids
OH
3

HO

7
6

2
3

OH O
Flavonols: X=OH
Flavones: X=H
Flavanones: No 2-3 db

4
5

OH

B-Ring Substitutions
HO

A C

OH

Kaempferol

OH

OH O

OH

Quercetin
OH

HO

Myricetin

OH

OH

OH O

OH
HO

OH

OH
OH O

Quercetin
4 OH groups

OH
OH

HO

O
2-3 db

OH
OH O

3-OH
4-oxo function

Catechin
4 OH groups

OH
OH

HO

O
OH
OH

3-OH

Cyanidin
4 OH groups

OH
OH

+
HO

O
OH
OH

Structurally Similar Compounds

OH

OH

OH

OH
HO

HO

O
OH

OH
OH

OH O
Quercetin
AOX = 4.7

OH
+
O

HO

OH

OH

Cyanidin
OH AOX = 4.4

Catechin
AOX = 2.4

OH

Importance of the
3-OH group

OH
HO

O
O

OH

Quercetin-3-glucoside
AOX ~ 2.5

O
OH

OH

OH O

Quercetin
AOX = 4.7

OH O

OH
HO

OH
HO

OH O

Luteolin
AOX = 2.1

Importance of the 4-Oxo Function

Works

with the 2-3 double bond in the C-ring and

is responsible for electron delocalization from the
B-ring.
3-OH and 5-OH substitutions with the 4-oxo
function are best for maximum AOX properties
OH
OH
HO

Structurally, quercetin has

all the right components to
make for the perfect antioxidant.

O
OH
OH O

Importance of the 2-3 db

OH

OH

OH

OH
HO

HO

O
OH

OH
OH O

Quercetin
AOX = 4.7

OH O

Taxifolin
AOX = 1.9

More on the Phenolic Acids

COOH

COOH

COOH

1
2 Ortho
3
4
Para

Meta

Hydroxybenzoic
Acid
(HBA)

1
2

Hydroxyphenylacetic
Acid
(HPA)

Cinnamic
Acid
(CA)

COOH

HO

0.08
COOH

HO

1.19

p-OH-benzoic
p-coumaric

Protocatechuic
Caffeic

HO

COOH

2.22
HO

1.26

COOH

HO

MeO

MeO
HO

HO

COOH

1.43

Vanillic
Ferulic

HO

1.90

COOH

Antioxidants in Food Systems

What Makes a Good Antioxidant?

Polyphenolics- Radical scavengers
Number

of hydroxy groups (-OH)

Location of hydroxy groups (on benzene ring)
Presence of a 2-3 double bond (flavylium ring)
4-oxo function (flavylium ring)
Synergistic/antagonistic

reactions with other

antioxidant compounds
OH
OH
HO

HO
COOH

COOH

HO

OH
OH O

What Makes a Good Antioxidant?

Carotenoids
The number of conjugated double bonds (9+ is best)
Substitutions on -ionone group (on the end)

Radical

scavengers

Beta-Carotene

R* + CAR => R- + CAR*+

Chain

breakers

ROO*+ CAR => ROO-CAR*

ROO-CAR* + ROO* => ROO-CAR-ROO
Singlet

oxygen quenchers

O2* + 1CAR =>

O2 + 3CAR*

Tocopherol

Alpha-tocopherol = Vitamin E

beta and gamma forms also

Synergist with carotenoids and selenium and is

regenerated by vitamin C
Efficiency determined by the bond dissociation energy
of the phenolic -OH bond
The heterocyclic chromanol ring is optimized for
resonance stabilization of an unpaired electron.

HO
O

Antagonism-Synergism-Metals
Many antioxidant work for and against each other
An antioxidant in a biological system my be
regenerated
In mixed ROSinefficiency of one antioxidant to
quench all the different radicals.
No way of knowing if the better antioxidant for a
particular radical is doing all the work or not.
Will a better antioxidant for a given food system beat
out a lesser antioxidant (antagonistic response) in
order to quench the radicals.

Example: Factors Affecting

AOX of Bell Peppers
Chemical interactions
In vitro models
Find synergistic/antagonistic effects
Free metal ions
Diluted isolates
Add metal chelator

Flavonoid Ascorbic

AOX ?

AOX with Quercetin Interactions

(-Carotene Bleaching)

OH
OH

450 ppm Caffeic = 47 %

880 ppm Ascorbic = 15%

HO

O
OH
OH O

AOX with Luteolin Interactions

(-Carotene Bleaching)

OH
OH

450 ppm Caffeic = 47 %

880 ppm Ascorbic = 15%

HO

OH O

Theoretical Quercetin
Regeneration Scheme

Quercetin

Delocalization
of C-ring

Reduced resonation in A and B rings

Minor regeneration by ascorbic acid
Minor regeneration by caffeic acid

Theoretical Luteolin
Regeneration Scheme

Luteolin

Electron donation
by C-ring

Excellent resonance stability in A-ring

Highly regenerated by ascorbic acid
No regeneration by caffeic acid

% Inhibition of Carotene Bleaching

Antioxidant Activity after Dilution

% Inhibition of Bleaching

Antioxidant Activity with Chelator

Antioxidant Methods

HAT and SET Reactions

Hydrogen Atom

Transfer (HAT) vs. Single

Electron Transfer (SET)
Antioxidants can work in one of two ways (HAT
or SET).
End result is the same for both, differing in
kinetics and side rxns.
HAT and SET rxns may occur in parallel
Determined

by antioxidant structure and properties

Solubility and partition coefficient
System solvent, system pH

HAT
HAT-based

methods measure the classical

ability of an antioxidant to quench free radicals
by hydrogen donation (AH = any H donor)

SET
SET-based

methods detect the ability of a

potential antioxidant to transfer one electron to
reduce any compound, including metals,
carbonyls, and radicals.
Also based on deprotonation, so pH dependent

HAT vs SET
HAT
Selectivity in HAT rxs are determined by the bond dissociation
energy of the H-donating group in the antioxidant
Antioxidant reactivity or capacity measurements are therefore
based on competition kinetics.
Reactions are solvent and pH independent and are very fast
Common reducing agents (Vitamin C) are an interference
SET
Usually slow and can require long times to reach completion
Antioxidant reactivity is based on a percent decrease, rather than
kinetics
Very sensitive to ascorbic acid and other reducing agents.
Trace amounts of metal ions will interfere, and cause overestimation and inconsistent results.

Antioxidants and Radicals

Four sources of antioxidants:

Enzymes

Large molecules

estrogen, angiotensin, melatonin

Multiple free radical and oxidant sources

ascorbic acid, glutathione, uric acid, tocopherol, carotenoids, phenols

Hormones

albumin, ferritin, other proteins

Small molecules

Superoxide dismutase, glutathione peroxidase, and catalase

O2, O2-, HO, NO, ONOO-, HOCl, RO(O), LO(O)

Oxidants and antioxidants have different chemical and

physical characteristics.

Complex Systems: Singlet Oxygen

Carotenoids are not good peroxyl radical quenchers compared to

polyphenolics
Carotenoids are exceptional singlet oxygen quenchers compared
to polyphenolics
However, singlet oxygen is not a radical and does not react via
radical mechanisms
Singlet oxygen reacts by its addition to fatty acid double bonds,
forming endoperoxides, that can be reduced to alkoxyl radicals,
that initiate radical chain reactions.
Now we have multiple reaction characteristics and multiple
mechanisms
No single assay will accurately reflect all of the radical sources
or test all the antioxidants in such a complex system.

Method Selections for Antioxidants

Controversy exists over standard methods for antioxidant

determination
Historical use and peer-review acceptance is critical
Use my multiple labs to highlight strength, weakness, and
effectivness
New methods take time to adopt and accept
An ideal method:

Measures chemistry actually occurring in potential application

Utilizes a biologically relevant radical source
Simple to run
Uses a defined endpoint and chemical mechanism
Instrumentation is readily available
Good within-run and between-day reproducibility
Adaptable for both hydrophilic and lipophilic antioxidants
Adaptable for multiple radical sources
Adaptable for high-through-put analysis
Understanding of the range of use and recognition of interfering agents

HAT assays
ORAC
Oxygen

Radical Absorbance Capacity

Measures inhibition of peroxyl radical induced
oxidations in chain breaking activity by H atom
transfer
TRAP
Total

Radical-Trapping Antioxidant Parameter

Measures the ability to interfere with peroxyl
radicals or stable free radicals

SET assays
FRAP
Ferric

Reducing Antioxidant Power

The reaction measures the reduction capacity of a
ferric compound to a color end-product
CUPRAC
Copper

Reduction Assay
Variant of FRAP assay using Cu instead of Fe
Folin-Ciocalteu
Reduction

assay

of oxidized iron and molybdenum