25 Natural Phenolic Antioxidants

and Their Impact on Health

Edwin N. Frankel
Department of Food Science and Technology
University of California
Davis, California 95616

INTRODUCTION

Flavonoids are an important part of the diet because they can modulate
lipid peroxidation involved in atherogenesis, thrombosis, and carcinogene-
sis (Kinsella et al., 1993). Known properties of flavonoids include free
radical scavenging, strong antioxidant activities in preventing the oxida-
tion of low-density lipoproteins (LDL), inhibition of hydrolytic and ox-
idative enzymes (phospholipase A2, cyclooxygenase, lipoxygenase), and
anti-inflammatory actions.
Diets rich in fruits and vegetables are associated with lower risks of
coronary heart disease and cancer (Block et al., 1992). Fruits and vegeta-
bles are considered to be the main source of dietary antioxidants. Although
vitamin E, vitamin C, and carotenoids have received the most attention as
anticarcinogens and as defenses against degenerative diseases of aging
(Ames et al., 1993), the nutritional role of flavonoid antioxidants in fruits
and vegetables has generally been overlooked. These flavonoids are found
in many fruits and vegetables in a great variety of structures and specifici-
ties. As active antioxidants, flavonoids may be especially important in pro-
tecting against human degenerative diseases. However, little is known about
their absorption and excretion in humans and very little is known about
their bioavailability.
This chapter reviews some of the chemical and biological properties of
plant phenolic antioxidants in foods and possible mechanisms for their pro-

Antioxidant Food Supplements in Human Health

Copyright 9 1999 by Academic Press. All rights of reproduction in any form reserved. 385
386 Edwin N. Frankel

tective effects in biological systems. The aim is to relate the extensive in vitro
studies on flavonoids to their potential health effects in foods and beverages.

CHEMICAL AND BIOLOGICAL PROPERTIES

Two important properties of flavonoids include their antioxidant activ-

ity and their metal chelation properties. They behave as antioxidants by do-
nating electrons to radicals and breaking the radical chains. Several antiox-
idant mechanisms may be considered, including scavenging radicals (ROO',
RO') and activated oxygen species (HO', 02", 102), inactivating metal ions
by chelation, complexing with proteins (enzymes, apoprotein-B of LDL)
and metal-binding sites of enzymes, exhibiting synergism by reducing radi-
cals from oxidized c~-tocopherol and ascorbic acid, and partitioning ac-
cording to their polarity to become distributed at different oxidation sites.
Flavonoids can also bind one or two copper atoms by chelation, thus in-
hibiting copper-activating enzymes.
Oxidation of LDL plays a key role in the initial phases of atherosclero-
sis (Steinberg et al., 1989; Esterbauer et al., 1992). When polyunsaturated
lipids (LH) in LDL or tissue membranes become oxidized in the presence of
metals and hydroperoxides, an alkyl radical L" is formed (Fig. 1). The alkyl
radical reacts with oxygen to form peroxyl radicals (LOO'), which react
again with LH to produce hydroperoxides (LOOH). These hydroperoxides
decompose readily in the presence of metals to produce aldehydes. These
aldehydes react with the apoprotein-B of LDL to produce oxidized LDL.
Phenolic antioxidants (AH) can inhibit two important steps in the free rad-
ical chain reaction by reacting with (1) peroxyl radicals and inhibiting hy-
droperoxide formation and (2) alkoxyl radicals and inhibiting aldehyde for-
mation. To properly evaluate the effectiveness of phenolic antioxidants, it is
not only necessary to measure the formation of hydroperoxides, but also
the formation of aldehydes, which cause biological damage. The inhibition
of aldehyde formation is an important function of phenolic antioxidants
that has been poorly appreciated in the literature.

M+/M2+ 02 AH
LH ~ L~ ; LOO'+ L H ~ /
LOOH

LOOH
AH M+/M2+~)
Aldehydes ~ I LO"

Figure I Oxidation of polyunsaturated lipids in LDL and mechanism of antioxidant action.

 25. Natural Phenolic Antioxidants 387

Many plant flavonoids inhibit enzymatic reactions, especially those that

produce and respond to oxidized lipids. These enzymes are highly sensitive
to the presence of oxidants and antioxidants. Phenolic antioxidants may re-
duce the peroxidation of polyunsaturated lipids and LDL and decrease
macrophage foam cell formation.

PROTECTIVE ROLE OF PLANT FLAVONOIDS

Flavonoids are known to inhibit cyclooxygenase and lipoxygenase

(Moroney et al., 1988; Kinsella et al., 1993). These enzymes play a key role
in platelet aggregation and in the formation of macrophages,
prostaglandins, and leukotrienes. By downregulating these enzymes, phe-
nolic compounds may reduce thrombosis and chronic inflammatory reac-
tions involved in immune suppressions by reducing hydroperoxides, down-
regulating the arachidonic acid cascade, and reducing platelet aggregation
and thrombotic tendencies. These physiological events tend to increase
chronic diseases such as heart diseases and cancer. Three possible mecha-
nisms for the protective role of plant flavonoids include (a) inhibiting LDL
oxidation, protecting cholesterol from forming cytotoxic compounds and
preventing atherosclerosis, (b) interfering with the i m m u n e response by re-
ducing monocytes from vessel walls, and (c) interfering with platelet aggre-
gation involved in the clotting process and preventing thrombosis.
The susceptibility of lipoprotein particles to oxidation in vitro was
shown to increase with prolonged time spent in circulation, or aging, in
vivo in an avian model (Walzem et al., 1995). These results imply that
plasma LDL from hypercholesterimic individuals is older than LDL from
individuals with normal plasma cholesterol. Accordingly, LDL particles
from hypercholesterimic individuals are subjected to elevated oxidant stress
by prolonging their exposure to intravascular oxidation (German et al.,
1997). Therefore, individuals with high LDL cholesterol may require a
higher antioxidant intake for protection against the damaging effects of
higher circulating levels of oxidatively susceptible LDL.

NATURAL PHENOLIC ANTIOXIDANTS

Wines
Considerable research has been published on the flavonoid compounds
of grapes and wine. Grapes are the world's largest fruit crop, and about
80% of the total crop is used in wine making (Mazza, 1995). Dealcoholized
red wine and red grapes were found to be important sources of flavonoid
388 Edwin N. Frankel

antioxidants that inhibit the oxidation of LDL in vitro (Frankel et al.,

1993). The antioxidant activity of phenolics in wines was evaluated in a
number of in vitro systems using either copper or biological catalysts (myo-
globin, cytochrome c, and iron ascorbate) (Kanner et al., 1994). Lipid per-
oxidation inhibition of 50% (Iso) by wine phenolics was achieved at con-
centrations of 0.2 to 0.9/,g/ml phenolics. The relative antioxidant activity
of 20 commercial California wines in inhibiting LDL oxidation in vitro var-
ied from 46 to 100% with red wines and from 3 to 6% with white wines
(Frankel et al., 1995). The activity of wines to protect LDL from oxidation
was distributed widely among the principal phenolic components, including
catechin, gallic acid, epicatechin, malvidin 3-glucoside, rutin, myricetin,
quercetin, caffeic acid, and cyanidin.

Grapes and Grape Juice

The antioxidant activities of extracts of different table and wine grapes
were tested in lecithin liposomes and in human LDL in vitro. In lecithin li-
posomes oxidized with copper acetate, grape extracts inhibited conjugated
diene formation by 25-68% and hexanal formation by 49-98% (Yi et al.,
1997). Anthocyanins were the most abundant phenolic compounds in ex-
tracts of red grapes, and flavonols were most abundant in extracts of white
grapes. In LDL oxidized with copper sulfate, hexanal formation was inhib-
ited by 62-91% (Meyer et al., 1997). The relative antioxidant activity of
grape extracts toward LDL oxidation correlated with the concentration of
total phenols, anthocyanins, and flavonols. Extracts of different grape vari-
eties showed significantly different trends in antioxidant activity between li-
posome and LDL systems. Commercial white and red grape juices also sig-
nificantly inhibited the oxidation of LDL (66-73%) (Frankel et al., 1997).
Therefore, grape extracts and juices, especially red grape juices, contribute
a significant source of phenolic antioxidants that, like wine, may have po-
tential beneficial health effects in protecting against atherosclerosis.

Pure Phenolic Compounds

The more active fractions purified from a Petite Syrah wine containing
components of the catechin family were evaluated for their activity in in-
hibiting human LDL oxidation by copper (Teissedre et al., 1996). The high-
est activity was found with the procyanidin dimers, the trimer, and the
monomers catechin, epicatechin and myricetin, and lower activity for the
gallic acid, quercetin, caffeic acid, and rutin. The relative antioxidant activ-
ity of pure phenolic compounds tested at two concentrations decreased in
the following order: catechin > myricetin = epicatechin - rutin > gallic
 25. Natural Phenolic Antioxidants 389

acid > quercetin > cyanidin, a-Tocopherol had relatively weak activity.
Some dimers and trimers separated from red grape seeds had the same ac-
tivity as catechin, but other dimers and trimers were less active. These
oligomers may have different protein-binding capacity than the monomeric
catechin. Thus, the numerous phenolic compounds in wines, grapes, and
plant flavonoids found in the diet are potent antioxidants in various physi-
ologically relevant systems. Several anthocyanins were tested in vitro on hu-
man LDL and on lecithin liposomes (Satu~-Gracia et al., 1997). In LDL ox-
idized with 10 ktM copper, malvidin was the best antioxidant, followed by
delphinidin, cyanidin, and pelargonin. In the liposome system oxidized with
10/~M copper, pelargonin followed by malvidin in antioxidant activity, but
cyanidin and delphinidin were prooxidants.
The properties of various phenolic compounds are thus very system de-
pendent. Caution is required in interpreting the antioxidant activities de-
termined with nonspecific assays in the absence of suitable substrate targets.

Rosemary Extracts
Rosemary extracts provide a major source of natural antioxidants used
commercially in foods. Carnosic acid and carnosol are the most important
active components of rosemary extracts and rosmarinic acid is a minor con-
stituent. Rosemary extracts, carnosic acid, and rosmarinic acid were more
active in bulk corn oil than carnosol (Frankel et al., 1996). However, rose-
mary compounds were less active in corn oil emulsions than in bulk corn
oil, and rosemary extracts, carnosic acid, and carnosol were more active
than rosmarinic acid. The polar hydrophilic components of rosemary ex-
tracts were less active in the emulsion system because they partitioned into
the water phase and became less protective than in the bulk oil system.

Green Tea Catechins

Green tea catechins have attracted much attention because of their
physiological effects, including antimutagenic and antitumorigenic activi-
ties. Studies showed that the hydrophilic catechol-type flavonoids of green
tea were more effective in inhibiting the oxidation of liposomes composed
of egg phosphatidylcholine than the lipophilic ce-tocopherol (Terao et al.,
1994). However, kinetic studies showed that flavonoids had much smaller
rate constants for the inhibition of oxidation of methyl linoleate than c~-
tocopherol. The marked differences in activity between hydrophilic cate-
chins and lipohilic ce-tocopherol may be due to their relative partition
between the water phase and the surface environment of the phospholipid
bilayers. This interracial phenomenon was invoked to explain the contrast-
390 Edwin N. Frankel

ing properties of polar and nonpolar antioxidants in bulk oil versus oil-in-
water emulsion systems (Frankel et al., 1994).
The antioxidant activities of pure components of green tea and differ-
ent commercial green teas were evaluated in different lipid systems (Huang
and Frankel, 1997; Frankel et al., 1997). Green teas were active antioxi-
dants in bulk corn oil but were prooxidant in the corresponding oil-in-
water emulsions oxidized. Green tea catechins were also active antioxidants
in lecithin liposomes oxidized in the presence of cupric acetate as the cata-
lyst. The marked variation in activity of green teas may be attributed in part
to differences in their relative partition between phases in various lipid sys-
tems. The improved antioxidant activity observed for green teas in lecithin
liposomes compared to corn oil emulsions can be explained by the greater
affinity of the polar catechin gallates for the polar surface environment of
the lecithin bilayers, thus affording better protection against oxidation.

CONCLUSIONS

Dealcoholized red wine and grapes are important sources of flavonoid

antioxidants that are potent inhibitors of in vitro oxidation of LDL. The ac-
tivity of commercial wines to protect LDL from oxidation was compared
with the activity of pure phenolic compounds found in wine. The antioxi-
dant activities of grape extracts and commercial grape juices were compa-
rable to that of commercial wines. The extracts of various grape varieties
showed different trends in antioxidant activity between liposome and LDL
systems. Rosemary extracts and components, tea catechins, and commercial
green teas had strong antioxidant activities in corn oil and lecithin lipo-
somes, but not in corn oil emulsions. Therefore, the relative activity of nat-
ural phenolic antioxidants is affected greatly by the test system used and the
biological target to be protected.
Natural phenolic compounds are found in many fruits and vegetables
in a great variety of structures and specificities. A 140-ml glass of red wine
or tea is estimated to contain approximately 0.3 g of phenolic materials
consisting largely of flavonoids. As potent antioxidants, flavonoids may be
especially important in protecting against human disease and represent a
positive potential in our diet that requires further research to improve our
understanding of their mechanism of action.
Natural phenolic antioxidants inhibit biological oxidation by a com-
plex multistep mechanism (Fig. 1). Unfortunately, many contemporary
studies tend to use nonspecific indicators of oxidation and antioxidation to
determine "antioxidant capacity" or "antioxidant status." Phenolic com-
pounds can participate in several antioxidant defenses, including preventing
 25. Natural Phenolic Antioxidants 391

oxidant formation, scavenging activated oxidants, reducing reactive inter-

mediates, and inducing repair systems. To improve our understanding of
these complex interactions in different systems, the use of nonspecific assays
for antioxidant capacity would be risky because they do not provide infor-
mation on the biological target(s) protected. The better approach is to mea-
sure specific products of oxidation in both relevant in vitro and in vivo bi-
ological systems.

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