Terpenoids as Plant Antioxidants

15

Vitamins and Hormones, Volume 72

Copyright 2005, Elsevier Inc. All rights reserve

Terpenoids as Plant

Antioxidants

J. Graßmann

Institute of Vegetable Science—Quality of Vegetal FoodstuV

Life Science Center Weihenstephan, Durnast 2, 85350 Freising, Germany

I.
I ntroduction
0083-d. DOI: 10.1016/S0083-67505

II.
P lant Antioxidants
I
II. T erpenoids
A.
S ynthesis
B.
M onoterpenes, Sesquiterpenes, and Diterpenes
C.
T etraterpenes—Carotenoids
R
eferences
oxidants are composed of a broad variety of diVe
Plant anti rent subs-
tances like ascorbic acid and tocopherols, polyphenolic compounds, or

terpenoids. They perform several important functions in plants and

humans (e.g., carotenoids function as accessory pigments for light

harvesting and provide photoprotection and pigmentation in plants).

Monoterpenes and diterpenes, which are the main components of

essential oils, act as allelopathic agents, attractants in plant–plant or

plant–pathogen/herbivore interactions or repellants.

For humans, carotenoids play an important role for health, carotenoids

with provitamin A activity are important for vision; other carote-

noids influence the human immune function and gap‐junctional com-

munication (GJC). Additionally, their antioxidative capacity is believed

to be responsible for the health promoting properties of fruits and

vegetables. Three main ways of antioxidant action of carotenoids have

6729/05 $35.0029(05)72015-X

506 Graßmann

been detected until now (i.e., quenching of singlet oxygen, hydrogen

transfer, or electron transfer). These mechanisms and investigation of

antioxidant activity in vitro are discussed in detail. The monoterpenes

limonene and perillyl alcohol may be promising substances in cancer

therapy. Several investigations have studied the antioxidant activity of

monoterpenes and diterpenes or essential oils in vitro. Results as well as

the action of a newly discovered, very eVective antioxidant (i.e., g‐terpinene) are discussed.

An important point when assessing the antioxidant activity of plant

antioxidants is to consider their interaction with other antioxidants.

Especially combinations of hydrophilic and lipophilic antioxidants may

exert synergistic eVects, as has been shown for rutin in combination with

g‐terpinene, lutein, or lycopene. # 2005 Elsevier Inc.

I. INTRODUCTION

Bioactive compounds are defined as nonnutritive constituents of food,

which usually occur in very small quantities. They are composed of thousands

of substances, which can be divided by virtue of their structure in nine classes:

glucosinolates, organo‐sulfur compounds, phytosterols, saponins, protease‐inhibitors, phytoestrogenes, terpenoids, and polyphenolic compounds. Some

of them were shown to lower total cholesterol, LDL‐cholesterol, or triglycer-ides as well as blood pressure. Glucosinolates and organo‐sulfur compounds

are believed to protect from cancer by inducing phase‐II enzymes, and some

phytosterines seem to be helpful in lowering cholesterol in humans, there-

by protecting human health (Goldberg, 2003; Kris‐Etherton et al., 2002).

Another important property of bioactive compounds is to protect from

oxidative stress (i.e., they possess antioxidative capacity [AC]).

This AC may help to prevent cardiovascular disease (CVD) or cancer, as

the involvement of reactive oxygen species (ROS) in these is probable

(Halliwell, 1996; Stanner et al., 2004; Vendemiale et al., 1999). Oxidative

stress has been postulated to be involved in the development of several

chronic diseases. The reaction of ROS with biomolecules like lipids, pro-

teins, and DNA may lead to increased risk of chronic diseases, such as

cancer, CVD, atherosclerosis, age‐related macular degeneration (AMD), or

cataract. Therefore, the inactivation of ROS by (dietary) antioxidants may

be a promising preventive strategy. However, there is increasing evidence

that the most prudent public health advice is to increase the consumption of

plant foods (and in this way increasing the antioxidant intake) instead of

using single supplements (Hercberg et al., 1998; Kaur and Kapoor, 2001;

Stanner et al., 2004; Tapiero et al., 2004). The following contribution will

give an overview of terpenoids as plant antioxidants.

Terpenoids as Plant Antioxidants 507

II. PLANT ANTIOXIDANTS

The imbalance between oxidants and antioxidants in favor of the oxi-

dants, potentially leading to damage, forms the core of the definition of

‘‘oxidative stress.’’ Oxidative stress may occur in plants as well as in humans.

In plants, one major source of oxidative stress is the photosystem since

chlorophyll may act as photosensitizer forming singlet oxygen. But also

in other compartments like mitochondria, microsomes, peroxisomes, and

others the formation of ROS may occur (Schempp et al., 2005).

In humans, about 1–3% of the O2 consumed by the body is converted into

superoxide and other ROS under physiological conditions (Fridovich, 1986;

Halliwell, 1996). They perform many important functions in physiological

processes (e.g., microbial killing, cell signaling, or gene transcription [Droge,

2002]). However, besides these desirable eVects they may also damage DNA,

proteins, or lipids. These deleterious eVects are found to be responsible for

the development of diseases like CVD, cancer, or AMD (Halliwell, 1996;

Stanner et al., 2004; Vendemiale et al., 1999). To cope up with this threat

of ROS‐induced damage, the body has developed an antioxidant defense

system, which consists mainly of antioxidant enzymes like superoxide dis-

mutase or catalase, and chain‐breaking antioxidants (radical scavengers) likeascorbic acid, tocopherols (vitamin E), and uric acid. During exercise and

certain disorders, this antioxidant system is enhanced (Clarkson and

Thompson, 2000), which may reflect an attempt to keep the balance between

prooxidants and antioxidants. Another possibility to improve antioxidant

defense is to increase the dietary intake of antioxidants. This can be achieved

by enhancing the intake of fruits and vegetables, since they contain a broad

spectrum of antioxidants, the most important of them being ascorbic acid

and vitamin E, polyphenolic compounds and terpenoids. Since antioxidant

actions and biological functions of ascorbic acid and vitamin E as well as

polyphenolic compounds have gained great interest and therefore have been

addressed in numerous comprehensive reviews (Asard et al., 2004; Bramley

et al., 2000; Davey et al., 2000; Harborne and Williams, 2000; Kim and Lee,

2004; Parr and Bowell, 2000; Pietta, 2000), the main focus of this study will

be on terpenoids.

III. TERPENOIDS

A. SYNTHESIS

Terpenoids are substances that are built up from isoprene; therefore, they

are also called isoprenoids. They are divided on the basis of their C‐skeleton;Table I gives an overview.

TABLE I. Classes of Terpenoids

Terpenoid

Number of

C‐atoms

Number of

isoprene subunits

Monoterpene 10 2

Sesquiterpene 15 3

Diterpene 20 4

Triterpene 30 6

Tetraterpene 40 8

Polyterpene >40 >8

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The generation of terpenoids comprises three steps:

1. Formation of the C5‐subunit2. Condensation of these subunits form the skeleton of the diVerent

terpenoids

3. Conversion of the resulting prenyldiphosphates to end products

Synthesis is accomplished either by the mevalonate or the methylerythri-

tol‐4‐phosphate (MEP) pathway (which was originally named nonmevalo-

nate pathway and in the meantime also DXP‐ or DOXP‐pathway). Theformer has been known for a long time and is located in the cytoplasm.

By this pathway, sesquiterpenes, triterpenes, and polyterpenes are synthe-

sized (Bruneton, 1999; Loza‐Tavera, 1999). The latter was discovered in the

early 1990s and produces monoterpenes, diterpenes, sesterterpenes, and

tetraterpenes (Lichtenthaler, 1999; Rodrıguez‐Concepcion and Boronat,

2002). Their common intermediate is isopentenylpyrophosphate (IPP;

‘‘activated isoprene’’) from which all terpenoids are formed. Catalyzed by

prenyltransferases, IPP polymerizes to prenylpyrophosphates. In the third

phase of synthesis prenylpyrophosphates are finally converted to terpenes.

These reactions are carried out by the large group of terpene synthases

(Kreuzwieser et al., 1999). Figure 1 shows the basic reactions of both the

pathways.

B. MONOTERPENES, SESQUITERPENES,

AND DITERPENES

1. Significance for Plants and Men

Monoterpenes and sesquiterpenes are the main constituents of essential

oils (e.g., those from citrus fruits, herbs, and spices). Essential oils have numer-

ous ecological functions in the plant kingdom, such as acting as allelopathic

agents, repellants, or attractants in plant–plant or plant–pathogen/herbivore

FIGURE 1. Basic reactions during terpenoids synthesis. Abbreviations: GA‐3‐P, D‐glyceraldehyde‐3‐phosphate; DOXP, 1‐deoxy‐D‐xylulose‐5‐phosphate; MEP, methylerythritol‐4‐phosphate; IPP, isopentenyl diphosphate; GPP, geranyl diphosphate; GGPP, geranylgeranyl

diphosphate; HMG, hydroxymethylglutaryl; DMAPP, dimethylallyl diphosphate; FPP,

farnesyl diphosphate.


interactions (Dudareve et al., 2004; Pare and Tumlinson, 1999; Wink and

Latz‐Bruning, 1995). Another function is seen in defense and wound healing

in pine tree species or in increasing thermotolerance in plants (Singsaas

et al., 1997).

Most investigations regarding their role in human health have been

carried out with limonene, perillyl alcohol, carvone, and carveol due to their

chemotherapeutic activities. A number of dietary monoterpenes possess

antitumor activity in animal models or diVerent cell lines, although human

clinical trials are under way. The monoterpenes inhibit carcinogenesis both

in the initiation and promotion/progression stages, and are eVective in

treating early and advanced cancers (Crowell, 1997, 1999; Gould, 1997;

Wagner and Elmadfa, 2003). Antioxidant properties of monoterpenes are

discussed later, some monoterpenes are shown in Fig. 2

Sesquiterpenes are the most diverse group of isoprenoids. In plants, they

function as pheromones and juvenile hormones. The acyclic representatives

are also called farnesans, the term, which is derived from the basic structure,

farnesol. Examples for bicyclic sesquiterpenes are a‐caryophyllene and

b‐caryophyllene. Some sesquiterpenes are shown in Fig. 3

FIGURE 2. Structures of some important monoterpenes.

510 Graßmann

FIGURE 3. Structures of some important sesquiterpenes.


The most important diterpenes for human health are those with vitamin A

activity, which plays a fundamental role in the process of viewing (Wagner

and Elmadfa, 2003). Besides this, retinoids regulate the growth and diVer-entiation of normal, premalignant, and malignant cells. These functions are

achieved by changes in gene expression mainly through interaction with the

retinoic acid receptors and retinoid X receptors (Evans and Kaye, 1999;

Okuno et al., 2004). Figure 4 shows the structures of the most important

diterpenes.

2. Antioxidative Properties of Monoterpenes, Sesquiterpenes,

and Diterpenes

a. AC in DiVerent Test Systems In Vitro

Monoterpenes and sesquiterpenes as well as diterpenes show antioxidant

activity. Most investigations, however, were conducted with essential oils

and it was shown that they exhibit AC in diVerent in vitro model systems.

FIGURE 4. Structures of some important diterpenes.

512 Graßmann

Besides chemical composition of an essential oil, extraction methods and the

system used to determine AC influence the results (Dapkevicius et al., 1998;

Fadel et al., 1999; Hopia et al., 1996; Mantle et al., 1998). Many investiga-

tions were carried out by examining the influence of essential oils on oxida-

tion of fats like sunflower oil, lard, primrose oil, or others (Abdalla and

Roozen, 1999; Dang et al., 2001; Schwarz et al., 1992; Youdim et al., 1999).

This may be useful in identifying those essential oils, monoterpenes or

diterpenes, which might be used for food preservation; however, it has no

physiological relevance.


Baratta et al. (1998a,b) investigated a broad range of essential oils for

their AC using egg yolk and rat liver as oxidizable substrates and 2,20‐azobis(2‐amidinopropane) dihydrochloride (AAPH) as a radical inducer. The oils

showed diVerent eVectiveness, majoran and oregano oil being the most

eVective. Other examinations used the 2,2‐diphenyl‐1‐picrylhydrazyl(DPPH) assay or diVerent lipid peroxidation assays for quantifying AC, or

tested essential oils for their ability for hydroxyl or superoxide radical

scavenging. The results confirmed the hydrogen‐donating properties of

essential oils but gave inconsistent results regarding the activity towards

hydroxyl radical or the superoxide radical anion.

A major problem in reviewing the literature regarding AC of essential oils

is the broad variation of the used test systems. Furthermore, results, which

were obtained in the same test system (e.g., the DPPH assay), are given in

quite diVerent units like percent remaining activity, trolox equivalents, IC50

values, and others. Because of this reason, a comparison of results from

diVerent groups is extremely complex or even impossible. Table II gives an

overview of the most frequently used model systems and the tested oils.

Some investigations confirmed good AC for rosemary and thyme oil and

in most studies phenolic substances like the phenolic diterpenes carnosic acid

and carnosol from rosemary extracts or the phenolic monoterpenes carva-

crol and thymol were held responsible for the observed antioxidative eVects(Aeschbach et al., 1994; Hopia et al., 1996; Richheimer et al., 1996; Schwarz

et al., 1996). Figure 5 shows the structure of these terpenoids.

The phenolic diterpenes from rosemary were shown to act as ‘‘primary

antioxidants’’ by donating hydrogen to lipid radicals and thereby slowing

down lipid peroxidation.

Citrus oils have also been shown to be quite eVective antioxidants in the

DPPH assay (Choi et al., 2000) and g‐terpinene was shown to be an important

nonphenolic antioxidant.

All the used test systems provide some conclusions about hydrogen‐donating or radical‐scavenging activities of monoterpenes and diterpenes

as well as their eVectiveness in inhibiting lipid peroxidation (LPO). This may

be useful when one is looking for antioxidant to enhance oxidative stability

of edible lipids but mostly lacks significance regarding human pathologies.

Investigations using activated neutrophils in whole blood as a source for

ROS showed a strong inhibition by essential oils, which may be due to

interactions of the lipophilic essential oils with the membrane of neutro-

phils. This may also be an explanation for the anti‐inflammatory eVects ofessential oils (Graßmann et al., 2000).

b. AC Regarding Oxidation of LDL

Another physiological model system in this context is oxidation of low‐density lipoproteins (LDL). Low‐density lipoproteins oxidation is believed

to be implicated in atherogenesis (Chisolm and Steinberg, 2000). Therefore,

TABLE II. AC of Essential Oils and Their Components in Various In Vitro Test Systems

Test system

Tested oils and

components (selection) References

Lipid peroxidation

(Rancimat

method, Schaal

test and other)

Rosemary, ginger,

cinnamon, lemongrass

Dang et al. (2001)

Thyme Youdim et al. (1999)

Thyme Schwarz et al. (1996)

Rosemary, sage Schwarz et al. (1992)

Catnip, hyssop,

lemon balm,

oregano, sage, thyme

Abdalla and Roozen (1999)

Oil stability

index (OSI)

Sage, thyme Miura et al. (2002)

TBAR‐assayin lipid‐richmedia

Laurel, sage, rosemary,

oregano, coriander

ylang‐ylang, lemongrass,

basil, rosemary, cinnamon,

lemon, frankincense,

majoram

Baratta et al. (1998a,b)

Thymus pectinatus Vardar‐Unlu et al. (2003)

Deoxyribose

degradation

assay

Artemisia afra, Artemisia

abyssinica, Juniperus procera

Burits et al. (2001)

T. pectinatus Vardar‐Unlu et al. (2003)

DPPH A. afra, A. abyssinica, J. procera Burits et al. (2001)

34 kinds of citrus oils Choi et al. (2000)

Tea tree (Melaleuca alternafolia) oil Kim et al. (2004)

Sage, thyme Miura et al. (2002)

Amazonian basil, common

basil, thyme

Sacchetti et al. (2004)

Melissa oYcinalis de Sousa et al. (2004);

Mimica‐Dukic et al. (2004)

3 Mentha species Mimica‐Dukic et al. (2003)

T. pectinatus Vardar‐Unlu et al. (2003)

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protection of LDL from oxidation may contribute in preventing heart attack

and stroke. It could be shown that essential oils are able to prevent copper‐induced LDL oxidation (Graßmann et al., 2001, 2003; Takahashi et al.,

2003; Teissedre and Waterhouse, 2000). Teissedre and Waterhouse (2000)

explained this protecting eVect with the phenolics content of the investigated

essential oil. Graßmann et al. (2001), however, identified g‐terpinene as the

most active substance in this context. This high antioxidative capacity of

g‐terpinene is in accordance with the findings of Choi et al. (2000) who could

show that the radical‐scavenging activities of citrus oils depends—among

FIGURE 5. Phenolic monoterpenes and diterpenes.

FIGURE 6. Structures of terpinolen and g‐terpinene.


other less important factors—on their g‐terpinene and terpinolene content.

Structures of these monoterpenes (Fig. 6) reveal that they contain no

hydroxyl groups (i.e., antioxidative capacity must be explained by other

structural factors). In a study on tea tree oil (TTO), its AC also was

516 Graßmann

attributed to the nonphenolic compounds a‐terpinene, terpinolen, and g‐terpinene but not to the main component of TTO (i.e., terpinen‐4‐ol, whichcontains a hydroxyl group [Kim et al., 2004]).

Graßmann et al. (2001) proved that g‐terpinene can be enriched in LDL

by preincubating human blood plasma with lemon oil or g‐terpinene and

that the subsequently isolated LDL shows a high resistance against copper‐induced oxidation. Takahashi et al. (2003) showed in later studies that

g‐terpinene, which was added to LDL solutions is able to prevent the

oxidation of LDL.

By monitoring the consumption of endogenous antioxidants like a‐tocopherol and carotenoids it could be shown that the highly lipophilic

g‐terpinene protects the carotenoids from oxidation but has no influence

on a‐tocopherol consumption. This may be due to the good hydrophobic

interaction of g‐terpinene with the LDL particles and its capacity to donate

hydrogen atoms or electrons, or its ability to chelate metal ions. However

the latter (i.e., copper complexation) does not play an important role, since

it could be shown that g‐terpinene is not an eVective copper‐chelator(Graßmann et al., unpublished results). Foti and Ingold (2003) revealed

the underlying mechanism by which g‐terpinene inhibits lipid peroxidation.

The important fact is that the chain carrying peroxyl radicals are HOO�

radicals, which react rapidly with linoleylperoxyl radicals and thereby ter-

minate the chain reaction. Since this mechanism is diVerent from the

mode of action of vitamin E and since vitamin E becomes a prooxidant at

high concentrations, the discovery of g‐terpinene may provide a new

stabilizing substance for edible lipids and may also provide a possibility to

enrich foodstuVs and beverages to increase highly eVective antioxidants in

nutrition.

c. Interaction with Other Antioxidants

An important mode of action of monoterpenes when working as antiox-

idants is to support other antioxidants like a‐tocopherol (e.g., rosemary

extracts show synergistic eVects together with a‐tocopherol) (Wagner and

Elmadfa, 2003). These results show that lipid‐soluble antioxidants can

work together to protect lipids from oxidation; in case of rosemary extracts

this is due to the regeneration of a‐tocopherol. Milde et al. (2004). proved

that not only the cooperation of lipid‐soluble antioxidants can lead to eVec-tive synergisms but also the combination of lipid‐soluble with more water‐soluble antioxidants gives overadditive protection. Therefore, g‐terpinenetogether with rutin results in a synergistic inhibition of copper‐inducedLDL oxidation.

These results again support the idea that ‘‘Health benefits of fruits and

vegetables are from additive and synergistic combinations of phytochemicals’’

(Liu, 2003).


3. Conclusions

Essential oils and their components (i.e., mainly monoterpenes and diter-

penes, possess AC in diVerent in vitromodel systems). In some cases, this can

be explained by their content of phenolics like carnosol, carnosic acid,

carvacrol, or thymol. However, newer results also proved high AC for

essential oils not containing such phenolic substances in noteworthy

amounts. This is due to hydrocarbons like a‐terpinene or g‐terpinene or

terpinolen. Their AC is based on a reaction mechanism that acts by chain

carrying HOO�radicals, which react rapidly with linoleylperoxyl radicals

and thereby terminate the chain reaction. An interesting feature is synergistic

action between terpenoids and other antioxidants like a‐tocopherol or fla-

vonoids like rutin. Therefore, it is essential to test antioxidants not only

separately but also in combination, because health promoting properties of

fruits and vegetables are in all likelihood due to the mixture of secondary

plant metabolites.

C. TETRATERPENES—CAROTENOIDS

The main group of tetraterpenes are the carotenoids, which are abun-

dantly found as pigments in plants. More than 600 carotenoids have already

been isolated from nature, their basic structure being a symmetrical tetra-

terpene skeleton formed by the conjugation of two C20‐units. All carote-

noids can be derived from the acylic unit by diVerent reaction steps involving

hydrogenation, dehydrogenation, cyclization, or oxidation reactions. Figure

7 shows the major transformation reactions.

Carotenoids can be divided depending on their functional groups. Those,

which contain only carbon and hydrogen atoms are called carotenes, those

with at least one oxygen function are referred to as xanthophylls. Figures

8 and 9 show the major carotenoids.

1. Significance for Plant and Humans

Carotenoids perform three major functions in plants, which are given in

the following sections.

a. Accessory Pigments for Light Harvesting

The carotenoids are important components of the light harvesting anten-

nae. With few exceptions, the chloroplasts of all species contain a collection

of main carotenoids namely b‐carotene, lutein, violaxanthin, and neoxanthin

(Bartley and Scolnik, 1995). They absorb light between 450 and 570 nm

thereby expanding the absorption spectra of photosynthesis and enhan-

cing photosynthetic eVectiveness. It is crucial that the carotenoids be

located close to the chlorophyll molecules to ensure an eVective transfer of

energy. The major carotenoids in this context are the xanthophylls lutein,

violaxanthin, and neoxanthin.

FIGURE 7. Transformation reactions during carotenoid synthesis.

FIGURE 8. Structures of lycopene, a‐carotene, and b‐carotene.

518 Graßmann

FIGURE 9. Structures of luteine, b‐cryptoxanthin, and zeaxanthin.

TerpenoidsasPlantAntioxidants

519

520 Graßmann

b. Prevention of Photooxidative Damage

The excited triplet state of chlorophyll, which is generated in the photo-

synthetic apparatus, may initiate photooxidation processes via singlet

oxygen and damage the photosynthetic system. Carotenoid molecules with

nine (or more) conjugated double bonds are able to absorb energy from

triplet state chlorophyll or from singlet oxygen and by this means prevent

generation of the damaging singlet oxygen (Choudhury and Behera, 2001;

van den Berg et al., 2000). Additionally, there is considerable evidence,

which indicates a photoprotective role of the xanthophyll cycle in remov-

ing excess energy from the photosynthetic antennae. Under conditions of

excess excitation energy zeaxanthin accumulates from violaxanthin via two

de‐epoxidation steps. This accumulation allows a rapid nonphotochemical

quenching of chlorophyll fluorescence (Demming‐Adams and Adams, 1996).

Figure 10 shows the reactions involved in the xanthophyll cycle.

Also during N‐limited conditions, which lead to restricted photosynthetic

capacity, the xanthophyll cycle may prevent damage by dissipating excess

light. This is indicated by increased xanthophyll cycle pigments (e.g., in

spinach leaves) (Verhoeven et al., 1997), which were grown under N‐limited

conditions.

c. Pigmentation to Attract Animals for Pollination and Dispersal of Seeds

Carotenoids are not only located in chloroplasts but also in chromoplasts

where they contribute to most of the orange, yellow, and red colors of fruits

or flowers. However, other compounds (e.g., water‐soluble anthocyanins)

also contribute to the color of fruits and flowers. The green of the chlor-

ophylls masks the yellow or reddish colors but they are revealed in the

autumn leaves of many trees.

For humans, carotenoids play an important role in health. The function

of retinol (vitamin A) in vision has been known for a long time. Though

retinol is formed mainly from b‐carotene by symmetrical cleavage, 50 other

carotenoids can function as provitamin A, the important ones being

a‐carotene, b‐cryptoxanthine, and cis‐b‐carotene (Castenmiller and West,

1998; Wagner and Elmadfa, 2003). Observational epidemiological studies

have been very consistent in proving an inverse relationship of higher dietary

levels of fruits and vegetables and the risk of developing certain kinds of

cancer or CVD (Johnson, 2002; Tapiero et al., 2004). It has been suggested

that carotenoids are the chemopreventive agents in fruits and vegetables.

There are several mechanisms by which carotenoids can prevent diseases.

EVects on Human Immune Function Carotenoids exhibit immunomod-

ulatory actions, which could contribute to their assumed anticarcinogenic

eVects. Hughes (1999) describes the ability of b‐carotene to enhance cell‐mediated immune response. Supplementation results in an enhanced activity

of natural killer cells as well as in antigen presenting monocytes.

FIGURE 10. Scheme of the xanthophyll cycle and its regulation by excess or low light.

TerpenoidsasPlantAntioxidants

521

522 Graßmann

Influence on GJC Another biological function of carotenoids is the

support of gap‐junctional communication (GJC). During carcinogenesis,

GJC is lost and this loss may be important for malignant transformation,

and its restoration may reverse malignant processes (Stahl et al., 2002;

Tapiero et al., 2004; Yamasaki et al., 1995). Carotenoids stimulate GJC in

a diVerential and dose‐dependent manner (Stahl et al., 1997); however, the

underlying mechanisms are not yet understood.

Antioxidative Properties Oxidative damage has been discussed in con-

text with two diseases of the elderly (i.e., cataract and AMD). There is

evidence that lutein and zeaxanthin, the only two carotenoids, which have

been identified in the human crystalline lens, may reduce the risk for devel-

oping these most common eye diseases. One possible mode of action is that

the macular pigments filter blue light, which is particularly damaging to the

photoreceptors and to the retinal pigment epithelium. Another hypothesis is

that the antioxidant properties of lutein and zeaxanthin may reduce the

degree to which oxidative stress promotes these diseases since there is

considerable oxidative stress in the eye due to intense light exposure and a

high rate of oxidative metabolism in the retina (Alves‐Rodrigues and Shao,

2004; Ham, 1983; Johnson, 2002; Mares‐Perlman et al., 2002). Antioxidative

properties of carotenoids may also be important in the prevention of cancer

or CVD. The most important aspects of the antioxidant activity of carote-

noids are discussed in the following section. However, one should keep

in mind that the probable influence of carotenoids on disease prevention is

only at the beginning of being elucidated and it is still unclear whether the

antioxidant properties of carotenoids are connected with the prevention

of disease. In a few experiments, the prooxidative eVects of carotenoids

have been shown especially at high oxygen pressure or high carotenoid

concentration (El‐Agamey et al., 2004b; Krinsky, 1998).

2. Antioxidative Properties of Carotenoids

Carotenoids are most possibly involved in scavenging of singlet oxygen

and peroxyl radicals. Additionally, they are able to deactivate sensitizer

molecules, which are involved in the generation of ROS. One example

is the triplet chlorophyll, which emerges during photosynthesis and may

initiate photooxidative processes.

a. Singlet Oxygen Quenching

Singlet oxygen can be generated by electron energy transfer from the

excited state of a sensitizer to oxygen. Sensitizers may be chlorophylls,

riboflavin, porphyrins, and others, and they may induce singlet oxygen

production in biological systems consequentially leading to the damage of

DNA, lipids, proteins, and other biological molecules. Therefore, it is quite


beneficial for plants to possess carotenoids as they are the most eVectivesinglet oxygen quenchers found in nature.

The main mechanism of photoprotection against singlet oxygen by

carotenoids is physical quenching, which occurs by following mechanism:

1O�2 þ carotenoid !3O2 þ3carotenoid�

3carotenoid� ! carotenoidþ thermal energy

Because of the long, conjugated polyene system of carotenoids, they lose

the excess energy via vibrational and rotational interactions with the solvent

(i.e., they dispense it as thermal energy). The carotenoid emanates un-

changed from this reaction, ready to begin another cycle of singlet oxygen

quenching. It has been estimated that each carotenoid can quench 1000

singlet oxygen molecules before it reacts chemically. This chemical quench-

ing is responsible for the destruction of the molecule (Di Mascio et al., 1992;

Edge et al., 1997; Krinsky, 1998).

b. Scavenging of Peroxyl and Other Radicals

Peroxyl radicals are intermediates of the LPO process, which is charac-

terized by a radical chain reaction. This process can be interrupted by

chain‐breaking antioxidants, which are able to react with the lipidperoxyl

radicals. These chain‐breaking antioxidants are, for example, tocopherols,

phenols, or ascorbic acid. An important feature of them is that the resulting

antioxidant radical is too unreactive to propagate the chain reaction of LPO.

The antioxidant radical may be removed by reaction with another radical,

thereby forming a stable product. Another common fate of the antioxidant

radical is to be recycled by another antioxidant. Carotenoids are able to act

as chain‐breaking antioxidants by three pathways (El‐Agamey et al., 2004a;

Krinsky and Yeum, 2003) (i.e., electron transfer, hydrogen abstraction, or

radical addition [adduct formation]). Which of these pathways preferably

proceeds mainly depends on the structure of the carotenoid and on the

environment and system used to assess the antioxidant activity. It is unlikely,

for example, that electron transfer will take place in a highly lipophilic

environment, since this does not facilitate charge separation. One important

point is that the antioxidant activity of carotenoids may be converted in a

prooxidative activity at high oxygen pressure (Burton and Ingold, 1984).

In the physiological range of oxygen pressure the reaction of peroxyl radicals

with carotenoids will lead to a resonance‐stabilized structure that will

terminate peroxidation processes, whereas increasing oxygen tension will

allow the carotenoid radical to react with oxygen, thereby producing radi-

cals, which are able to propagate peroxidation (Burton, 1989; Burton and

Ingold, 1984 ; Palace et al. , 1999; Palozza an d Kri nsky, 1992 ). However,

most data indicate that the prooxidant eVect only arises at 100% oxygen

and not at ambient conditions (21% oxygen) or at physiological or tissue

524 Graßmann

concentrations (1–2% oxygen). Kennedy and Liebler (1992) showed that

b‐carotene provided similar antioxidant protection under an air atmosphere

(150 torr O2) and under physiological conditions (15 torr O2). Thus, there is

only little evidence for the thesis that b‐carotene may act as a prooxidant in

the body (Krinsky, 1998).

c. Oxidation Products of Carotenoids

Looking at the diversity of carotenoids it is obvious that by diVerentoxidation reactions an even greater variety of oxidation products will be

generated. Those oxidation products are likely to be in vivo metabolites;

therefore, not only the carotenoids but also their oxidation or cleavage

products should be taken into consideration. A variety of oxidation pro-

ducts have been detected (e.g., epoxides or apo‐carotenoids) (Grosch et al.,

1976; Handelmann et al., 1991; Kennedy and Liebler, 1991; Stratton et al.,

1993). These compounds may have biological activities and may interfere

with a variety of signaling pathways. Studies have revealed that such sub-

stances have diverse in vivo eVects, like enhancing GJC, inhibiting Naþ‐Kþ‐ATPase, or impairing mitochondrial respiration (Aust et al., 2003; Siems

et al., 2000, 2002). However, this topic is beyond the scope of this contribu-

tion. The most potent product of carotenoid oxidation is retinoic acid

formed by enzymatic cleavage of b‐carotene.

d. Antioxidant Activity of Carotenoids In Vitro

Reactivity Toward Singlet Oxygen The first detected mechanism by

which carotenoids act as antioxidants was their ability to quench sing-

let oxygen (Foote and Denny, 1968). There are numerous investigations

regarding this feature some of which are briefly discussed further.

Conn et al. (1991) investigated diVerent carotenoids on their rate con-

stants for the quenching of singlet oxygen and found that the ability to

quench singlet oxygen increases with increasing number of conjugated dou-

ble bonds. The two carotenoids in the eye (zeaxanthin and lutein) have very

diVerent quenching rate constants. Zeaxanthin (n ¼ 11) seems to be at least

twice as eVective as lutein (n ¼ 10), which may be due to the additional

conjugated double bond in zeaxanthin.

Conn et al. (1991) also supposed that an epoxide group rather than

carbonyl or hydroxyl substituents increases the reactivity of the carotenoid

with respect to singlet oxygen. Di Mascio et al. (1989, 1992) proved that

lycopene quenching rates are higher than those for b‐carotene, therefore it ispossible that opening the b‐ionone ring has a positive eVect on 1O2 quenching.

However, it seems clear that significant diVerences can arise when the

rate constants are determined by diVerent techniques (e.g., the values for

b‐carotene vary up to fourfold depending on the assays) (Di Mascio

et al., 1992).


These results show that carotenoids are extremely good 1O2 quenchers

in vitro but little work has been carried out to test how eVectively they

protect cells against 1O2‐related damage.

Reactivity Toward DiVerent Radical Species Carotenoid antioxidant

activity can be studied by following the bleaching of carotenoids or the

analysis of carotenoid oxidation products during or after their reaction

with diVerent radical species (Palozza and Krinsky, 1992). The results prove

that carotenoids are able to trap oxygen and/or organic free radical inter-

mediates. Oxygen radicals are not the only ones that can be trapped by

carotenoids; lutein, for example, is able to scavenge sulfur radicals (Chopra

et al., 1993). Everett et al. (1996) showed that b‐carotene quenches glutathi-one, sulfonyl, and nitrogen dioxide radicals. Mortensen and Skibsted (1996)

investigated the interaction of b‐carotene with phenoxyl radicals that results

in the formation of a b‐carotene radical cation and an adduct. Phenoxyl

radicals are important species in biological systems and are formed when

phenolic antioxidants react with peroxyl and alkoxyl radicals; the toco-

pherol radical represents the most important phenoxyl radical in biological

systems.

Bohm et al. (1997) reported about the reaction of a‐tocopheroxyl radi-cals with b‐carotene that shows near diVusion controlled rate constants in

hexane. These aspects will be considered later when the interaction of

carotenoids with other antioxidants is discussed.

Depending on the radical species, the antioxidant action of carotenoids

will follow diVerent mechanisms (i.e., either electron transfer or addition

processes will take place). For an overview, see Krinsky and Yeum (2003).

Mortensen et al. (2001) and Rice‐Evans et al. (1997) divided the radical

species in two groups; those that cause mainly electron transfer�CCl3,

�OOCCl3, RSO2

�, NO2

�, C6H6O

�,

and tocopheroxyl radical and those that were shown to lead to addition

processes

RS�, L

�, LOO

�, and O2��

.

AC of Carotenoids in DiVerent Test Systems Another aspect while dis-

cussing antioxidant activity of carotenoids is to compare the diVerent sub-stances in a variety of test systems. b‐Carotene was shown to be able to

decrease nuclear damage induced by xanthin oxidase (XOD)/hypoxanthine

or polymorphonuclear leukocytes (PML). Additionally, b‐carotene is able

to inhibit LPO induced by various systems (e.g., the XOD system) (Palozza

and Krinsky, 1992). Researchers often use liposomes as a model membrane

system to investigate the ability of carotenoids to inhibit LPO.

There have been a number of studies showing the inhibition of LPO

in liposomes or isolated membranes (summarized in Palozza and Krinsky,

1992). However, the results are often inconsistent, which may be explained

526 Graßmann

by the di Verent preparat ions of carote noid ‐ con taining lipos omes or mem-

bran es. For exampl e, studi es from Kenn edy and Liebler (1992) came out

with the result that b‐ carote ne inhibi ts 2,2 0‐ azobis(2,4 0‐ dimet hylvaleron itrile)

(AM VN) ‐ induced peroxida tion of phos phatidy lcholine liposomes at concen-

tration s of app roximatel y 0.1–0.5 mol% . Ho wever, no inhibi tion co uld be

detect ed in AMVN ‐ induced pe roxidation of rat liver microsomes at con-

centra tions of 1.5 mol%. This is explai ned by Liebler (1993) by di Verentprep arations of the lipos omes. In the former case, b‐ carote ne was mixed with

the phosph olipids before lipos ome formati on, an d in the latte r case,

the micr osomal membr anes were pr eformed and b‐ carote ne was addedsubseq uently. Ano ther lipophi lic syst em to measur e an tioxidative e Y cacyis the oxidat ion of LDL that is though t to be impl icated in atheros clerosis

( Chisolm and Steinb erg, 2000 ). Several carote noids have be en test ed on their

abili ty to inhibit LDL oxidat ion in vitro , indica ting that b‐ carote ne an dlycopen e may play a protective role ( Agar wal and Rao, 1998; Fuhr man

et al. , 200 0; Pac ker, 1993 ). Despite this in vitro evidence for protective e V ects,in vivo supplem entation with b‐ carote ne in most invest igations did not

lead to an enhanced in vitro resistance of LDL towards oxidat ion ( Gazia no

et al. , 1995; Jial al an d Fuller , 1995; Jialal and Grund y, 1 993; Pr incen et al. ,

1992; Reave n et al. , 1993 ).

Wo odall et al. (1997) applie d a modified Fenton reaction or free radi-

cals generat ed from 2,2 0‐ azob is‐ isobut yronit rile (AIBN) or from AMV M to

oxidiz e caroten oids. Lycope ne showe d the highest react ivity in the di Ver-ent test systems; howeve r, the reactivi ty of the other tested carote noids (i. e.,

b‐ carote ne, zeaxant hin, isozea xanthi n, ech inenone, lutein, astax anthin, an dcanthaxa nth in) v aried between the systems. This group su ggested ‘‘that

hyd rogen abstr action should be co nsidered as one of the possibl e mechan-

ism s that occur when carote noids are exposed to pero xyl radica ls and other

oxidis ing agents.’’

Siems et al. (1999) used di Verent pro oxidant s in vitro (NaOC l, AIBN or

phot o‐ irradiat ion, UV light in presence of the phot osensi tizer Rose Benga l)

and found that the breakdow n of lycopene an d b‐ carote ne was much faster

than that of lutein and zea xanthin in all systems. The high antiox idant

activity of lycopene in the ‘‘light‐induced oxidation’’ is not surprising, since

lycopene is known to be a very eVective singlet oxygen quencher and it is

possible that under UV‐light or in presence of Rose Bengal singlet oxygen is

the main oxidizing species. Also, Woodall et al. (1997) proved a high AC for

lycopene in three diVerent test systems, and in the case of AIBN as free

radical generator they also found a higher rate of breakdown for lycopene

and b‐carotene than for lutein and zeaxanthin.

Miki (1991) studied b‐ carote ne, lutein, zeaxan thin, astax anthin, tunax-anthin, and canthaxanthin in comparison with a‐tocopherol. He used a

heme‐protein‐Fe2þ‐complex as free radical generator and quantified TBA

production. He found that astaxanthin is the most eYcient scavenger fol-

lowed by zeaxanthin, canthaxanthin, lutein, tunaxanthin, and b‐carotene.


a‐Tocopherol had an higher IC50 value than all tested carotenoids. However,

in the modified test system of Woodall et al. (1997), zeaxanthin showed a

much higher AC than lutein, and astaxanthin and canthaxanthin were the

least eVective. These diVerencesmay be due to the use of a heme‐iron in case ofMiki or the use of diVerent solvents. It is well known that factors like solubili-

ty or steric hindrance that may be of great importance in one environment but

not in the other can have influence on the AC of carotenoids (El‐Agamey

et al., 2004a,b; Packer, 1993). Jorgensen and Skibsted (1993) investigated the

antioxidant eVect of astaxanthin, b‐carotene, canthaxantin, and zeaxanthin

against the peroxidation of methyl esters in diVerent systems (i.e., metmyo-

globin as a water‐based free radical generator in a heterogeneous lipid/water

system and AIBN as a free radical generator in a homogeneous chloroform

solution). In case of the heterogeneous system, each of the carotenoids pro-

tected the methyl esters against oxidation and the antioxidative eVect showedlittle dependence on the structure of the carotenoid. In case of the homoge-

neous solution, however, the stability of the carotenoids in the oxidizing

system depended on the structure and the order of decreasing stability

was shown to be astaxanthin > canthaxanthin > b‐carotene > zeaxanthin.

Another investigation of Mortensen et al. (2001) came to the conclusion

that the reaction of carotenoids with a‐tocopherol also depends on the

environment (see later section).

Miller et al. (1996) assessed the relative antioxidant activities of a range

of carotenoids and xanthophylls through the extent of their abilities to

scavenge the ABTS radical cation. The order was shown to be lycopene >b‐cryptoxanthin � b‐carotene > lutein � zeaxanthin � a‐carotene > echi-

nenone >> astaxanthin � cantaxanthin. It is likely that the scavenging of

the ABTS radical cation is due to the hydrogen‐donating properties of the

carotenoids. This would be in agreement with Woodall and coworkers who

showed the same order of carotenoids in the reaction with AMVN. There-

fore, the thesis of Woodall and coworkers that hydrogen abstraction at the

allylic C‐atoms of carotenoids may contribute to the AC of carotenoids

seems most likely.

Interaction with Other Carotenoids or Antioxidants An important point

regarding antioxidative capacity is the interaction between substances of

diVerent mode of action or polarity. For an overview see Truscott (2001).

Firstly, several carotenoids may show interactions with each other. Edge

et al. (1998) tested the relative one‐electron reduction potentials of diVerentcarotenoid radical cations by monitoring the quenching of one carotenoid

radical cation by another carotenoid. The order was found to be astaxanthin

> cantaxanthin > lutein > zeaxanthin > b‐carotene > lycopene. This means

that lycopene is the most easily oxidized carotenoid and is able to repair all

other carotenoid radical cations. Mortensen et al. (2001) investigated the

oxidation potentials of diVerent carotenoids in Triton X‐100 micelles and

found that lycopene is the easiest carotenoid to be oxidized to its radical

528 Graßmann

cation and astaxanthin is the most diYcult, which is in agreement with the

results from El‐Agamey et al. (2004a). Stahl et al. (1998) proved that mix-

tures of carotenoids are more eVective in protecting liposomes against

oxidative damage and found lycopene and lutein to be the most potent

carotenoids in this respect.

There are several indications that carotenoids also interact with a‐tocoph-erol. b‐Carotene markedly delayed the AIBN‐induced loss of endogenous

microsomal tocopherols (Palozza and Krinsky, 1991), caused a synergistic

inhibition of LPO in combination with a‐tocopherol (Palozza and Krinsky,

1992; Toyosaki, 2002), enhanced a‐tocopherol antioxidant activity as shown

by pulse radiolysis and laser flash photolysis studies (Bohm et al., 1997), and

protected LDL from oxidation in cooperation with a‐tocopherol (Packer,1993). Bohm et al. (1997) proved that a‐tocopheroxyl radicals react rapidlywith b‐carotene with near diVusion controlled rate constants in hexane.

The reactivity of carotenoids towards a‐tocopherol is influenced by the

environment. Mortensen et al. (2001) found that in polar environment the

a‐tocopherol cation is deprotonated, and the deprotonated cation does not

react with carotenoids, whereas in a nonpolar environment like hexane

the protonated a‐tocopherol radical cation is converted to tocopherol by

carotenoids.

Besides interaction with other carotenoids or a‐tocopherol, carotenoidsmay also interact with more hydrophilic antioxidants. The most common

antioxidant in this context is ascorbic acid. Bohm et al. (1997) proved that

ascorbic acid can repair carotenoid radical cations in methanol. This can

be explained by the more polar nature of carotenoid radical cations. Thus,

they may reorientate in biological membranes so that the charge is near the

polar interface and becomes accessible to ascorbic acid (Edge et al., 1997;

Krinsky and Yeum, 2003). This could also explain the adverse eVects ob-

served in several trials where b‐carotene did not protect smokers (who in

general do have lower blood ascorbic acid levels) from lung cancer but

provided protection for nonsmokers.

Yet, with other hydrophilic antioxidants interactions can also take place.

Trombino et al. (2004) investigated the antioxidant eVect of ferulic acid, aloneand in combination with b‐carotene, in isolated membranes and intact cells

and revealed synergistic interactions. Milde and coworkers similarly could

show that LDL is protected in a synergistical manner by rutin and lycopene

or lutein, respectively (unpublished results). These synergistic eVects can be

explained by a diVerent location of hydrophilic and lipophilic antioxidants

in membranes, cells, or LDL, respectively.

3. Conclusions

Carotenoids perform important biological functions in plants as well as

in humans. In plants, their main functions are photoprotection, light har-

vesting in photosynthesis, and pigmentation. For man, the best known


function is that of vitamin A, which is essential for vision. However, it has

been speculated that carotenoids are at least in part responsible for the

health promoting properties of fruits and vegetables. Besides their influence

on human immune function and GJC, they possess remarkable antioxidative

properties. The abilities of quenching singlet oxygen and reacting with a

variety of radical species may help to reduce oxidative stress in humans

and thereby protect them from various diseases like CVD or cancer. Many

in vitro investigations proved the AC of carotenoids; however, there is still

very limited knowledge about the extent to which carotenoids act as anti-

oxidants in vivo. An important point to keep in mind is the interaction of

carotenoids with other antioxidants, especially in the light of the fact that in

vegetal foodstuV combinations always occur.

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Terpenoids as Plant Antioxidants

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