Designer Fruits and Vegetables with Enriched Phytochemicals for Human Health

Designer fruits and vegetables with enriched phytochemicals for human health

Rong Tsao1, Shahrokh Khanizadeh2, and Adam Dale3

1Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, CanadaN1G 5C9 (e-mail: [email protected]); 2Agriculture and Agri-Food Canada, Horticultural Research and

Development Centre, 430 Blvd. Gouin, St-Jean-sur-Richelieu, Quebec, Canada, J3B 3E6; 3Department of PlantAgriculture, University of Guelph, Simcoe Campus, 1283 Blueline Rd. Box 587, Simcoe, Ontario, Canada

N3Y 4N5. Scientific publication number S226 of the Food Research Program, Agriculture and Agri-Food Canada,Guelph, Ontario. Received 12 July 2005, accepted 10 January 2006.

Tsao, R., Khanizadeh, S. and Dale, A. 2006. Designer fruits and vegetables with enriched phytochemicals for human health.Can. J. Plant Sci. 86: 773–786. High dietary intake of fruits and vegetables rich in phytochemicals, particularly those with antiox-idant activity, has been linked to reduced risks of many chronic diseases including cancer and cardiovascular diseases.Nutraceuticals containing such bioactive phytochemicals have been popular and made available in the market. However, exces-sive supplementation with these extracted and sometimes purified phytochemicals may pose new health concerns. Non-processedfruits and vegetables that are known to be rich in bioactive phytochemicals are therefore advantageous for the intact and balancedphytochemical contents. However, production of phytochemicals in fruits and vegetables is affected by many factors. Genotype isa fundamental factor that affects the biosynthesis of phytochemicals, but farming practices and environmental factors such as geo-graphic location, growing season, soil type and mineral status, plant maturity, postharvest storage and processing, can all signifi-cantly affect the concentration of many phytochemicals. Development of fruits and vegetables with elevated concentrations ofknown antioxidant phytochemicals must consider all these factors. This is a challenging task and requires close multi-disciplinarycollaborations among scientists.

Key words: Phytochemicals, antioxidant, fruits, vegetables, polyphenols flavonoids

Tsao, R., Khanizadeh, S. et Dale, A. 2006. Fruits et légumes sur mesure enrichis de composés phytochimiques pour la santéhumaine. Can. J. Plant Sci. 86: 773–786. La consommation d’une grande quantité de fruits et de légumes riches en composés phy-tochimiques, en particulier ceux ayant une activité anti-oxydante, est associée à une réduction des risques relatifs à maintes affec-tions chroniques, dont le cancer et les maladies cardiovasculaires. Les produits nutraceutiques contenant ces composés sont envogue et affluent sur le marché. Prendre une quantité excessive de suppléments renfermant ces substances purifiées après extrac-tion pourrait cependant engendrer de nouvelles préoccupations médicales. Les fruits et les légumes non transformés qu’on sait êtreriches en substances phytochimiques bioactives s’avèrent intéressants en raison d’une concentration naturelle et équilibrée de cescomposés. Maints facteurs influent cependant sur la synthèse des substances phytochimiques dans les fruits et les légumes. Legénotype en est un fondamental, mais les pratiques agricoles et des paramètres environnementaux comme l’emplacement, la sai-son végétative, le type de sol et le bilan minéral, la maturité de la plante, le stockage et le conditionnement après la récolte ont tousune incidence sensible sur de nombreuses substances phytochimiques. On doit prendre en compte tous ces éléments avant de créerdes fruits et de légumes riches en anti-oxydants phytochimiques. Il s’agit là d’un travail complexe appelant la collaboration de sci-entifiques de nombreuses disciplines.

Mots clés: Phytochimique, anti-oxydant, fruits, légumes, flavonoïdes polyphénolés

The newest statistics published by the American CancerSociety in February 2005 (http://www.cancer.org/docroot/STT/stt_0.asp) show that for the first time, cancer has sur-passed heart disease as the top killer of Americans under 85.In Canada, heart disease remains the top killer, however, theCanadian Cancer Society’s (CCS) statistics in 2004(http://www.cancer.ca) indicated that cancer is the numberone cause of early death in Canada, and the number of newcancer cases is increasing. The CCS projected that by 2010,cancer will be the leading cause of death of Canadians. TheCCS statistics for 2005 showed that the number of new can-

cer cases in Canada is growing twice as fast as the popula-tion. While there are many risk factors for cancer that wecannot change, such as age, sex and genetic inheritance,many others, such as lifestyle-related factors can help us sig-nificantly reduce the risks (Table 1). Canadians are simplynot eating enough fruits and vegetables; 60% of us do nothave the recommended amounts of 5–10 servings per day.

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Abbreviations: �-CLAMS, β-carotene–linoleic acidmodel system; FRAP, ferric reducing/antioxidant power;HPLC, high-performance liquid chromatography; LBR,lyophilized black raspberry; ORAC, oxygen radical absorp-tion capacity; PCL, photochemiluminescence; PPO,polyphenol peroxidase; ROS, reactive oxygen species

Presented at the Plant Canada 2005 Symposium“Phytochemicals in Human Health Research: Bioactivityversus Physiological Relevance”.

774 CANADIAN JOURNAL OF PLANT SCIENCE

Approximately 90% of all cancer cases correlate with envi-ronmental factors, including one’s dietary habits, and one-third of all cancers, are avoidable by changing dietary habitsonly (Milner 1994; World Cancer Research Fund 1997).

Some cancers are preventable to a large extent by includ-ing enough fruits and vegetables in the diet (Steinmetz andPotter1991a, b; Block et al. 1992), but it is not the onlyhealth benefit fruits and vegetables can offer. The currentleading cause of death in Canada, cardiovascular and heartdisease, for example is also found to be influenced by con-suming diets rich in fruits and vegetables (Duthie andBrown 1994). In fact, many chronic degenerative diseases,including cancer and cardiovascular diseases have beenlinked to oxidative stresses caused by excessive free radicalsand reactive oxygen species (ROS) such as the superoxideanion (˙O2

–), hydroxyl radical (˙OH) and the peroxy radical(ROO˙). Free radicals are molecules with unpaired elec-trons, therefore short-lived and highly reactive. They reactwith vital biomolecules such as lipids, proteins and nucleicacids (e.g., DNA) by acquiring electron(s) from those mole-cules, chemically oxidizing them. Free radicals and ROS areformed during normal aerobic respiration, metabolism andinflammation via the mitochondrial electron transport chain,phagocytes, microsomal electron transport chain, oxidaseenzymes and transition metals (e.g., iron and copper)(Duthie and Brown 1994; German and Dillard 1998; Nice1997). Damage caused by these endogenous free radicalscannot be avoided. Free radicals also arise from sources out-side (exogenous) the human body. These exogenous freeradicals form from environmental factors such as pollution,sunlight, strenuous exercise, X-rays, cigarette smoking andalcohol (Nice 1997), many of which can be avoided byadjusting our life style. Excess free radicals and ROS areconsidered mediators of disease and the most importantcause of major health problems such as cancer, cardiovas-cular diseases and other chronic degenerative diseasesincluding arthritis, multiple sclerosis, myocardial ischemia-reperfusion injury, immune system decline, hyperplasticdiseases, cataract formation, brain dysfunction, as well asthe aging process (Ames et al. 1993; Keher 1993; Nice1997; German and Dillard 1998). Free radicals and ROS areneutralized by antioxidant defence mechanisms, which con-sist of primary and secondary defences (Nice 1997). Theprimary defence system either prevents the formation of freeradicals or removes them as soon as they are formed.Endogenous enzymes such as the superoxide dismutase andcatalase, and essential vitamins C and E and other foodminor components, e.g., flavonoids, are considered to be

involved in the primary defence system. The secondarydefence system only takes effect when the primary systemfails. It repairs the already damaged biomolecules such asproteins and lipids. It also consists of a series of enzymesand small molecules (tocopherols, β-carotene, and otherdietary antioxidants) (Nice 1997; Tsao and Akhtar 2005).

The physiological functions of phytochemicals in the pre-vention of chronic diseases are highly complex. The actionsof phytochemicals in influencing the development of breastcancer, for example, are likely to involve mechanisms otherthan antioxidant effects. Until recently, only certain essen-tial antioxidants such as vitamins have been recognized asimportant for the maintenance of optimal health and the pre-vention of chronic diseases. However, in recent yearsincreasingly more evidence has shown that other food com-ponents such as polyphenols and carotenoids, independent-ly or in combination with the essential nutrients, may play amajor role in the reduction of the incidence of many chron-ic diseases. Consumption of food rich in antioxidative phy-tochemicals other than vitamins has been clearly linked tothe prevention and reduction of cancer (Steinmetz andPotter 1991a, b; Block et al. 1992) and cardiovascular dis-eases, and stimulation of the immune system (Duthie andBrown 1994; German and Dillard 1998; Heliovaara et al.1994). Many in vitro studies have shown that non-essentialphytochemicals may be stronger antioxidants than antioxi-dant vitamins, and contribute to the total antioxidant activi-ty of fruits and vegetables (Bors et al. 1990; Hanasaki et al.1994; Wang et al. 1996; Cao et al. 1996; Guo et al. 1997;Tsao et al. 2003a, 2005).

PHYTOCHEMISTRY OF POLYPHENOLS AND CAROTENOIDS

Hundreds if not thousands of phytochemicals in fruits andvegetables have been shown to act as antioxidants. Thesenaturally occurring compounds belong to dozens of differ-ent chemical groups, some of which have been recentlyreviewed (Tsao and Deng 2004; Tsao and Akhtar 2005).Among these, polyphenols and carotenoids are majorantioxidant phytochemicals in fruits and vegetables (Larson1997; Cadenas and Packer 2002).

PolyphenolsPolyphenols are secondary plant metabolites that containseveral hydroxyl groups arranged on aromatic rings.Thousands of these compounds have been identified andhundreds have been found in fruits and vegetables.Polyphenol is a highly inclusive class of compounds, and it

Table 1 Modifiable risk factors related to cancerz

• 60% of Canadians do not eat the recommended amounts of fruit and vegetables. • About half of Canadians (54% of women and 44 per cent of men) are physically inactive. • Almost half of Canadians (56% of men, 39 per cent of women) are at an unhealthy body weight. • Rates of physical inactivity are declining slowly but rates of excess body weight are increasing, especially in children. • 18% of Canadians over 12 years of age are heavy drinkers. • In 2002, 21% of Canadians over 12 years of age smoked and 18% of youth aged 15–19. • Tobacco use is declining, but is still high in some groups. zhttp://www.cancer.ca/ccs/internet/standard/0,3182,3172_367655_390750327_langId-en,00.html

TSAO ET AL. — DESIGNER FRUITS AND VEGETABLES 775

Fig. 1. Major polyphenol groups found in fruits and vegetables.


can be classified into several major sub-groups: phenolicacids (benzoic acids and cinnamic acids), ellagic acids, stil-benes, lignans and flavonoids (Fig. 1). Among them,flavonoids share a common structural backbone: two aro-matic rings connected through three carbon atoms, whichform an oxygen-containing heterocycle (Fig. 1). Flavonoidscan be further divided into flavonols, flavones, flavanones,flavanols and procyanidins, isoflavones and anthocyanidins(Fig. 1). Flavonoids are such a diverse group of polypheno-lic compounds that even experts in the field are often con-fused by the different terms within the group. The chemistryof polyphenols is further complicated by the fact that mostof these compounds are stored in fruits and vegetables inconjugated forms with various sugar units (glycosides), andare often polymerized. Although considerable effort hasbeen made to characterize these compounds, more remainsto be done (Manach et al. 2004).

Phenolic AcidsThe two major phenolic acid groups, benzoic acid deriva-tives and cinnamic acid derivatives, are almost ubiquitous infruits and vegetables. Examples of hydroxybenzoic acidsfound in fruits and vegetables include protcatechuic, vanil-lic, syringic and gallic acids. Caffeic and chlorogenic acidsare hydroxycinnamic acids. Caffeic acid is perhaps the mostabundant phenolic acid, and its o-dihydroxy function maycontribute favourably to the antioxidant activity.

Ellagic AcidsEllagic acid derivatives can be considered a special group ofdimeric gallic acid (which is a benzoic acid derivative).Ellagic acid consists of two gallic acid moieties, formedthrough intermolecular carbon-carbon coupling, and sponta-neous dehydration to give the lactone form of ellagic acid.These compounds, often called ellagitannins, are found inmost berries including strawberries and raspberries. Ellagicacid present in berries is generally conjugated. The ellagi-tannins, also referred to as hydrolyzable tannins, are typi-cally high molecular weight compounds (500–2800 Da or >)that are glucosylated. Blackberries and muscadine grapesare also excellent sources of ellagitannins.

StilbenesTwo aromatic rings are connected with a two-carbon doublebond to form this special group of polyphenols. These com-pounds are often found to be at very low concentrations. Themost widely studied stilbene is resveratrol from grapes.

LignansLignans are biosynthesized from two molecules of conifer-yl alcohol. Although some seeds such as flax are high in lig-nan, fruits and vegetables in general are not the best sourceof lignans. However, some fruits such as strawberries andcranberries are known to contain these antioxidants(Meagher and Beecher 2000; Valsta et al. 2003).

FlavonolsThese are the most common flavonoids in fruits and vegeta-bles, with quercetin and kaempferol being by far the most

abundant (Manach et al. 2004). The richest sources offlavonols are onion, kale, broccoli and many berry fruits.These compounds are almost always present in glycosylatedforms, for example, in apples, quercetin alone was found tobe conjugated with at least five different sugar units(quercetin-3-galactoside, glucoside, xyloside, arabinoside,and rhamnoside) (Tsao et al. 2003b).

FlavonesThe flavone, apigenin, is often found in tropical citrus fruits,but only a few crops, for example parsely, which containthese flavonoids are grown in Canada.

IsoflavonesUnlike most of the flavonoids, this special group of com-pounds has the B-ring connected to the 3-C position insteadof 2-C of the heterocycle (C-ring) (Fig. 1). Isoflavones areoften found in legumes. Although the most common sourcesof dietary isoflavones are soybean and its related products,red clovers have been found to contain higher concentra-tions of more biologically available non-glycosylated forms(aglycones) of isoflavones (Tsao et al. 2004, unpublished).A native North American fruit, Osage orange (Maclurapomifera) was found to contain prenylated isoflavones thatare strong antioxidants (Tsao and Yang 2003).

FlavanonesThe major dietary source of flavanones is citrus fruits.Tomato is also known to contain some of these compoundssuch as naringenin.

FlavanolsThis group of flavonoids is known to exist in bothmonomeric and polymeric forms. The former are oftencalled catechins, and the latter, procyanidins. These com-pounds have an astringent taste and are often found in theouter layers (skin) and seeds of fruits. In apple skin forexample, more than half of the total polyphenolics are cate-chins and procyanidins with catechin, epicatechin, pro-cyanidin B1 and B2 being the major flavanols (Tsao et al.2003b). Flavanols are not normally glycosylated; however,procyanidins often have different numbers of catechin orepicatechin units that are connected at different carbon posi-tions.

AnthocyaninsThese are pigments in the epidermal tissues that give fruitsand vegetables some of their colour, often from pink to pur-ple. Anthocyanins are in ionic form, and their colours arepH dependent. Lower pH (acidic) gives dark red colour, andhigher pH (alkalinic) gives dark blue or purple colour. Thesecompounds are often glycosylated or acylated, and thosewithout sugar or other forms of conjugation (glycones) arecalled anthocyanidins.

CarotenoidsCarotenoids are also pigments found in fruits and vegeta-bles, but with distinct yellow, orange and red colour (Fig. 2).Chemically, carotenoids are terpenes with highly conjugat-


ed double bonds. Some carotenoids such as lutein and zeax-anthin from corn and pumpkin, contain oxygen, and areoften called xanthophylls. Unlike some other carotenoids,xanthophylls are not vitamin A precursors, however, asantioxidants, they may possess higher activities (Miki 1991;Palozza and Krinksy 1992). The golden yellow flesh colourof the Canadian Yukon potato was also found to come fromlutein (Tsao and Yang 2006). Some carotenoids such as β-carotene and lycopene from tomato are hydrocarbons.

Quantitative and Qualitative Analyses ofPhytochemical Antioxidants Identification of antioxidant phytochemicals in fruits andvegetables has been difficult, due to the low concentration,and lack of standards. Polyphenols and carotenoids in fruitsand vegetables must be extracted and separated before theycan be identified and quantified. For fresh fruits and vegeta-bles, 50–80% polar organic solvent (e.g., methanol, acetoni-trile, ethanol and acetone) in water has been the mostcommonly used extraction method. Percentages of theorganic solvent often vary depending on the water contentof the sample and the polarity of target compounds. For cer-tain antioxidant phytochemicals, further partitioning withapolar solvent such as diethyl ether, ethyl acetate or evenhexane (for carotenoids) are often necessary. Samples con-taining high concentrations of complex glycosides or esters

of carotenoids may require hydrolysis during or after initialextraction. Separation procedures of naturally occurringantioxidant phytochemicals have been summarized in arecent review (Tsao and Deng 2004). High-performance liq-uid chromatography (HPLC) coupled with detection tech-niques such as UV-Vis diode-array (DAD) and massspectrometry (LC-MS) has been pivotal in the characteriza-tion of polyphenols and carotenoids. Using these tech-niques, one of the authors (RT) has compiled a UV-vislibrary and a MS library based on a large collection ofpolyphenols and carotenoids found in fruits and vegetablescommonly grown in Canada. This data base has been usefulin the confirmation of known, and identification ofunknown, compounds (Tsao and Yang 2003; Tsao et al.2004; Li et al. 2005).

Evaluation of Antioxidant Activity ofPhytochemicals Since it is very difficult to measure the antioxidant capacityof the human body in situ, the antioxidant activities of phy-tochemicals of fruits and vegetables are often measured invitro with different model systems. In the past several years,the authors of this paper have used and modified a few dif-ferent in vitro systems, namely, the ferric reducing/antioxi-dant power (FRAP) assay, the β-carotene–linoleic acid

Fig. 2. Major carotenoids found in fruits and vegetables.


model system (β-CLAMS), the oxygen radical absorptioncapacity (ORAC) and the photochemiluminescence (PCL)method, and found that these systems gave consistent andreproducible results (Tsao et al. 2003a, c; Rekika et al. 2005;Tsao et al. 2005). The ORAC method measures peroxylscavenging capacity and the PCL against the superoxideradical, whereas the FRAP assay measures the redox poten-tial. Hence, a major advantage of the ORAC and PCL assaysis that the scavenging potential of a biologically plausibleradical is being tested. We also found that for fruit extracts,results from FRAP and β-CLAMS often correlated positive-ly and the activities were also proportional to the total phe-nolic content (Tsao et al. 2003a, 2005). One difficulty withmodel systems is that they are often based on differentmechanisms, which leads to considerable variability inresults. In addition, the active form of antioxidants in vivomay not necessarily be in the same form as that found orig-inally in fruits and vegetables, as there are a host of factorsaffecting the metabolism and bioavailability of these antiox-idants. There is no perfect system available to measure the“true” antioxidant capacity of a single antioxidant or a com-plex medium of antioxidant phytochemicals (Ou et al.2002).

FACTORS AFFECTING PHYTOCHEMICALANTIOXIDANTS IN FRUITS AND VEGETABLES

A recent study of 43 fruits and vegetables suggests that theirnutritional value has declined in the past 50 yr (Davis et al.2004). The researchers attributed the decline to the fact thatfarmers have been planting crops designed to improve traitsother than nutritional value, such as size and yield. Otherstudies have also shown that factors such as maturity, UVlight exposure, and postharvest storage and processingmethod, play an important role in phytochemical composi-tion (Parr and Bolwell 2000). Our recent studies on differentapple and strawberry fruits also indicated that the levels ofphytochemical antioxidants were dependent on genetics andwhether or not the skins were consumed (Tsao et al. 2003a,b; Rekika et al. 2005). Plant secondary metabolites are oftenfound in higher concentrations in the outer layers and skinof fruits, and are used as a defence against invading insectsand microorganisms. Conventional growing of fruits andvegetables keep such insect and microbial pressure low, andit is not known how this affects the production of thesedefence chemicals. Hence both breeding and farming prac-tices appear to influence phytochemical composition offruits and vegetables.

Farming PracticesA common question being asked is whether there are differ-ences in nutritional quality between organic and conven-tionally grown foods. The answer is not always clear.Hakkinen and Torronen (2000) found no consistent effect oforganic farming on the level of phenolic content in straw-berries. However, Levite et al. (2000) compared resveratrollevels in wine grapes, and reported an average of 32% high-er concentration of resveratrol in organic grapes than in theconventional grapes. Lombardi-Boccia et al. (2004) ana-lyzed the impact of organic and conventional production

practices on plums. They found that the organic plums con-tained higher concentrations of several minerals includingzinc and magnesium, and higher concentrations of toco-pherols and β-carotene. Total polyphenols in the organicplums managed with a trifolium cover crop were higher insix of nine phenolics measured. Studies on two berry cropsand field corn also showed that organic foods had higherlevels of ascorbic acid and total phenolics (Asami et al.2003). Higher levels of phenolic antioxidants in organiccrops may be caused by altered levels of certain enzymesand regulatory factors as a result of farming practices, i.e.,organic vs. conventional. Carbonaro et al. (2002) found thatorganic peaches contained about one-third higher concen-trations of polyphenolic compounds than conventionalpeaches, while the activity of enzymes such as polyphenolperoxidase (PPO) in organic pears was more than threefoldhigher than that of conventional pears (Table 2).

Similar results were found for vegetables. Ren et al.(2001) investigated the polyphenol content, antioxidantactivity and anti-mutagenicity of five vegetables and foundnot only that the flavonoid concentrations in juices of organ-ic vegetables were 1.3–10.4 times higher than those of con-ventional vegetables, but that the antioxidant activity andantimutagenic activities were also significantly heightened.It is estimated that organic farming has elevated antioxidantlevels in about 85% of the cases studied to date and on aver-age, levels are about 30% higher compared with food grownconventionally (Benbrook 2005).

Other Environmental FactorsEnvironmental factors, such as geographic location, grow-ing season, and soil type and mineral status are also knownto influence levels of plant secondary metabolites. Lesterand Eischen (1996) found that muskmelons grown on finesandy loam soils produced less β-carotene than those onsilty clay loam soils. Soil mineral nutrient content can havea major effect on phenolic accumulation. Ruiz et al. (1998)found that boron deficiency seems to be associated withsubstantial increases in intracellular phenolics in manyspecies. A limited nitrogen supply is typically associatedwith higher levels of phenolics in the plant, and there is atrend towards increased phenolics whenever nutrientresources are low in comparison with fixed carbon avail-ability (Waterman and Mole 1994). Often the faster thegrowth rate of the crop, the lower the concentration ofpolyphenols and vitamins. This phenomenon has beenreferred to as the “dilution effect” (Davis et al. 2004).Kumar et al. (2004) attributed such an effect to up-regulat-ed expression of specific and select classes of genes intomatoes grown under sustainable, lower-nitrogen systemscompared with those grown on black polyethylene mulch.Many of the genes are known as defence genes that controlthe synthesis of secondary defence metabolites.

Wang and Lin (2003) found that strawberries grown incompost-treated soils had a nearly 50% increase in somespecific flavonoids and a 50–100% increase in total antiox-idant activity compared with strawberries grown in soilstreated with fertilisers.


Growing season was another factor that affected totalphenolic content and antioxidant activity of 11 commercialcultivars and 15 advanced breeding lines of spinach(Howard et al. 2002) and blueberries (Howard et al. 2003).Over-winter spinach, which was planted in late fall and har-vested in the spring, had much higher levels of total pheno-lics and antioxidant capacity than spinach planted in earlyfall and harvested in late fall, indicating that growing condi-tions, as well as biotic and abiotic stresses, influenced phe-nolic metabolism (Howard et al. 2002). Otherenvironmental factors, such as temperature and ultravioletand visible light, have been known to affect phenolic syn-thesis in plants, and discussions on these have been summa-rized by Parr and Bolwell (2000).

Dumas et al. (2003) investigated the effects of environ-mental factors (temperature, light) and agricultural tech-niques (variety, water availability and mineral nutrients) onthe antioxidant content of tomatoes, and found that thebiosynthesis of phytochemicals such as lycopene was com-pletely stopped above 32°C, and significantly reducedbelow 12°C. Other compounds such as phytoene and β-carotene were also found to be affected by temperature. Thesame study also showed that light intensity had a positiveeffect on the production of lycopene, vitamin C, and pheno-lics. The authors also indicated that in addition to the signif-icant varietal difference in carotenoids, vitamins C and E,and phenolics, the availability of water in soil also affectedthe concentrations of these antioxidants, although no signif-icant trend was found. Minerals such as nitrogen and potas-sium were found to significantly increase lycopene and totalcarotenoid levels. These compounds were also drasticallyincreased when tomatoes were treated with synthetic growthregulators such as 2-(4-chlorophenylthio)triethyl-amidehydrochloride (CPTA) (Dumas et al. 2003).

Maturity, Postharvest Storage and ProcessingFor small dark-coloured fruits, anthocyanin content increas-es as the fruit ripens. However, the increase in anthocyaninsdoes not always result in higher antioxidant activity.Increase of one particular group of polyphenols is oftenaccompanied by decreases in others. Kosar et al. (2004)studied the effects of maturation on phenolic composition offour strawberry cultivars, and found the highest amounts of

anthocyanins from ripe fruits whereas ellagic acid wasfound as the main phenolic in the green fruits. They alsofound that although the levels of low molecular weight phe-nolic acids such as benzoic and cinnamic acid changed dur-ing maturation, no differences in content of flavonoids ingreen and ripe fruit were detected. Siriwoharn et al. (2004)found that phenolics did not show a marked change withmaturity, with values decreasing slightly from under-ripe toripe. Antioxidant activities, while increasing with ripening,did not show the marked positive change that total antho-cyanins exhibited. Variation in blackberry composition dueto plots, subsampling, sample preparation, and measurementwas highly significant. For example, sample-to-sample dif-ferences and sample preparation were found to be majorcontributors to variation in total phenolic and anthocyanincontent. Variation in anthocyanin content, in particular, wasfound to be influenced by fruit maturity; ripe and overripeberries were more susceptible to sample-to-sample differ-ence, sample preparation, and analytical measurement thanthose of under-ripe berries. Except for ellagic acid, similarobservations were obtained for other polyphenols includingellagitannins and flavonols (Siriwoharn et al. 2004).

Postharvest storage may affect the composition of certainphytochemical antioxidants; however, depending on thestorage conditions, the total antioxidant capacity does notnecessarily change. Phenolics in apple did not change sig-nificantly during cold storage for up to 9 mo (Burda et al.1990; Golding et al. 2001). Antioxidant activity and totalphenolic and anthocyanin content in blueberries showed aslight increase, depending on cultivar, during a marketablecold storage period (3–5 wk) (Connor et al. 2002). Certainpostharvest storage conditions, such as high oxygen treat-ment (60–100% oxygen) may in fact increase total phenolicand anthocyanin content, increase antioxidant activity, andreduce rot (Zheng et al. 2003). Similar results were foundwith vegetables. Modified-atmosphere packaging (7% O2and 10% CO2) of Swiss chard stored in the cold had noeffect on total flavonoid content after 8 d (Gil et al. 1998).Storage conditions such as low temperature may act as aphysical stress that increases the production of defence sec-ondary metabolites. Infection may be another trigger of thisdefence mechanism. Lattanzio et al. (2001) investigated theeffect of microbial infections during low temperature stor-

Table 2. Polyphenol peroxidase (PPO) activity and total polyphenol content of conventional and organic peach and pear samplesz

PPO activity (unit min–1/100 g f.w.)y

Sample Caffeic acid Chlorogenic acid Catechol Total polyphenols

PeachConventional 2451.9 ± 126.4 2053.2 ± 145.0 NDx 21.3 ± 1.6Organic 2174.5 ± 198.2 2655.3 ± 171.2* ND 29.0 ± 1.2**

PearConventional 674.2 ± 50.5 959.1 ± 100.9 557.1 ± 143.2 58.4 ± 2.0Organic 865.1 ± 43.8* 3020.7 ± 235.4*** 401.4 ± 110.3 64.5 ± 1.5*zValues are the average of at least six determinations ± SD.yConventional vs organic: significantly different: * P < 0.05; ** P < 0.01; *** P < 0.001. xND, not detectable. Reproduced from Carbonaro et al. (2002) with permission.


age of apple, and found concentrations of major polypheno-lics and PPO activity in infected apple tissues were signifi-cantly higher than in healthy tissues.

Processing of fruits and vegetables normally leads todecrease in phytochemical antioxidants. Ferreira et al.(2002) found that total phenolic content in pears decreasedby 64% under sun-drying conditions. Freezing, pasteuriza-tion, boiling and microwave cooking can reduce the totalantioxidant levels (Gil-Izquierdo et al. 2002; Guyot et al.2003; Aziz et al. 1998). One exception is perhaps the pro-cessing of tomatoes. Takeoka et al. (2001) found thatalthough vitamin C level in processed tomato was reducedsignificantly, lycopene level increased more than 2.5-foldafter 30 min thermal processing possibly due to increasedrelease of lycopene. The increase in lycopene enhanced theantioxidant activity of processed tomato.

Plant GeneticsGenetics has perhaps the greatest effect on the production ofantioxidant secondary metabolites. Parr and Bolwell (2000)state that “given the natural variation which exists bothwithin a single species and between closely allied species, itseems probable that much can be achieved with respect tomodifying the phenolic composition of crops by the use oftraditional plant-breeding techniques”. Chemical composi-tion is known to be different among cultivars of the sameplant species, and it is for this reason that chemical profilesof different cultivars have been used for taxonomic purpos-es (Grayer et al. 1996). Insect and disease-resistant plantcultivars tend to have higher levels of phenolic compoundsas defence chemicals (Waterman and Mole 1994). A thor-ough investigation of the polyphenolic content of applesgrown in Ontario showed that not only the total concentra-

tions, but individual concentrations of polyphenols also var-ied significantly among the eight cultivars studied (Tsao etal. 2003b, 2005). Total phenolic content ranged from 782 to2012 µg g–1 in the peels, and 164 to 561 µg g–1 in the flesh.The difference was even greater at individual phytochemi-cals level (Table 3). Similar results were found in NewZealand apples (McGhie et al. 2005). Ellagic acid concen-tration was found to differ by twofold across four strawber-ry cultivars in a study by Williner et al. (2003). In anotherstudy of four strawberry cultivars and their hybrids, individ-ual polyphenolic compounds were found to be at signifi-cantly different levels (Kosar et al. 2004). Genotype wasconcluded to be the major factor affecting the concentrationof the antioxidant polyphenols in these strawberries. Tsao etal. (2003a) found that the total anthocyanin and total pheno-lic contents varied significantly among dozens of cultivatedand wild strawberries studied over a 3-yr period, directlycontributing to higher antioxidant levels. Similar resultshave been found in other fruits. Sanchez et al. (2003) foundthat total phenolics in the peel of six pear cultivars rangedfrom 1235 to 2005 µg g–1, and from 28 to 81 µg g–1 in theflesh. Total anthocyanins and phenolics in different culti-vars of four Vaccinium species were also found to be signif-icantly different, and higher antioxidant activity was foundin cultivars with higher anthocyanins or phenolics (Prior etal. 1998). Wang and Stretch (2001) evaluated 10 cranberry(Vaccinium macrocarpon Aiton) cultivars for oxygen radi-cal absorbance capacity (ORAC), anthocyanins, and totalphenolics, and found significant variation among varietiesin total anthocyanin and phenolic contents, which ultimate-ly also affected the antioxidant activity. Similarly, in a studyof 11 cultivars of fresh plums, Kim et al. (2003) found thattotal phenolics varied from 125.0 to 372.6 mg 100 g–1, and

Table 3. Total phenolic concentrations and antioxidant activities of the apple peel and flesh extracts

TPCz TPIy FRAPx PCLw ATO120v

PeelsEmpire 781.6 1016.5 2736 3800 16.9Mutsu 1016.9 1089.4 6820 4643 19.9McIntosh 1163.4 1636.4 6436 5531 31.0Golden Delicious 1265.2 1248.5 9616 5223 22.4Cortland 1322.8 1658.5 11908 4133 31.8Ida Red 1478.8 1762.6 12083 5958 25.9Northern Spy 1548.3 2072.7 10044 6112 35.5Red Delicious 2011.5 2350.4 17851 4112 59.1

FleshEmpire 163.5 177.4 550 1457 8.2Mutsu 197.5 313 1584 1895 38.8Ida Red 236.5 489.3 2749 3069 50.8MacIntosh 255 488.1 2785 2967 58.3Golden Delicious 259.8 416.6 2036 2192 45.0Red Delicious 357.9 534.4 3215 2192 49.0Cortland 364.1 497.7 3660 1629 64.9Northern Spy 560.8 933.6 6425 5049 70.8zTotal phenolic content measured by Folin-Ciocalteu Method in µg gallic acid equivalent/g fresh weight. yTotal phenolic index measured by HPLC. xFRAP values in µM. wAscorbic acid equivalent in µM. vThe relative inhibition values at 120 min, apple peel samples were diluted by a factor of 4 before being assayed. Reproduced from Tsao et al. (2003b, 2005) with permission.


total flavonoids varied from 64.8 to 257.5 mg 100 g–1. Theyalso found that the antioxidant capacity of the plum cultivarscorrelated well with polyphenol content. In raspberries,major differences (>20-fold) were observed in the levels ofpelargonidin type anthocyanins and some proanthocyanidintype tannins among the 14 cultivars (Beekwilder et al.2005). The content of ellagitannins varied approximatelythreefold. The authors also concluded that genetics was themajor factor determining the variation in these phytochemi-cals. In an excellent study of 11 blackberry cultivars andadvanced selections, Siriwoharn et al. (2004) showed thattotal phenolic and total anthocyanin content varied greatlyamong cultivars. Their results showed the potential for cre-ating new cultivars with higher pigment and phenolic con-tents through classical plant breeding.

Genetics also plays an important role in antioxidantenzymes. Different blackberry cultivars showed significant-ly different antioxidant capacities that related to the oxygenradical scavenging enzyme activities in blackberry (Jiao andWang 2000).

The situation is much the same in vegetables. Research onnine different tomato varieties showed that the total pheno-lic content, total antioxidants and lycopene levels differedmarkedly (Martinez-Valverde et al. 2002). The sameauthors also found that quercetin was the most abundantflavonoid and the highest concentration was nearly six timesof that of the lowest. Lycopene levels, too, were significant-ly different among varieties. Genotype was found to play asignificant role in the antioxidant activity among eight broc-coli cultivars (Kurilich et al. 2002). Each genotype was ana-lyzed for carotenoid, tocopherol, ascorbic acid, andflavonoid content. No correlation between ascorbic acid orflavonoid and the antioxidant capacity of the hydrophilicextracts was found, but the carotenoids did correlate withantioxidant capacity of the lipophilic extracts and accountedfor the majority of the variability in that fraction. Theauthors suggested that this may indicate either the presenceof other antioxidant components that have yet to be identi-fied or that the known antioxidants are producing synergis-tic effects. Genetics was also a major factor that affected thetotal phenolic content and antioxidant activity of 11 com-mercial cultivars and 15 advanced breeding lines of spinach(Howard et al. 2002).

As discussed above, levels of antioxidant phytochemicalsdiffer markedly across varieties of a given fruit or vegetable.

This variation, and the recently heightened interest in thehealth benefits of fruits and vegetables, have certainly ledplant breeders to initiate selection of fruits and vegetableswith higher than normal polyphenolic or carotenoid content(Prior et al. 1998; Shim et al. 1999; Yoshinaga et al. 2000;Reyes et al. 2004; Cevallos-Casals et al. 2006). In a study ofgenotypes of red-fleshed plum (Prunus salicina Erhr. andhybrids) and peach [Prunus persica (Batsch) L.], Cevallos-Casals et al. (2006) selected plums that were rich in pheno-lics and anthocyanins, and because of that, had highantioxidant activity.

Concentrations of β-carotene, lycopene and lutein in car-rots can be elevated through traditional breeding (Simon etal. 1989; Simon 1997), and the high levels of these antioxi-dants have been found to be more bioavailable than thosefrom tomato paste. The increased bioavailability of non-provitamin A carotenoid such as lutein did not interfere withthe absorption of β-carotene in human studies (Horvitz et al.2004; Molldrem et al. 2004). Howard et al. (2002) showedthat advanced breeding lines of spinach, which showedincreased disease resistance, had higher levels of total phe-nolics, individual and total flavonoids, and antioxidantcapacity than commercial cultivars. The results of this studyindicate that plant breeders can select for increased phenoliccontent to increase antioxidant capacity in spinach cultivars.

DESIGNER FRUITS AND VEGETABLESFOR BETTER HUMAN HEALTH

Designing Antioxidant-rich Fruits and Vegetablesthrough BreedingThe newly discovered link between high dietary intake offruits and vegetables rich in phytochemicals and loweredrisks of chronic diseases presents a new opportunity to mit-igate such diseases through the development of food cropswith uniformly high levels of naturally occurring antioxi-dants. Although farming practices and environmental ele-ments contribute to phytochemical production in fruits andvegetables, genetics is perhaps the most fundamental factor.A proven cultivar or advanced selection of a fruit or veg-etable with elevated antioxidant concentration could serveas a template for “designer fruits and vegetables” withenriched antioxidant phytochemicals. Many of the currentfruit and vegetable cultivars have been selected on the basisof quality traits such as disease or insect resistance, to which

Table 4. Content of phenolics and oxygen radical absorbing capacity (ORAC) of spinach cultivars harvested in spring and fall 2000

Spring 2000 Fall 2000

cultivar ORACz Phenolicsy ORAC Phenolics

Bolero 13.4 ± 0.1x 2291.8 ± 76 10.9 ± 1.5 1774.1 ± 89.7*Coho 12.2 ± 0.1 2904.6 ± 17.3 12.2 ± 0.4 1848.9 ± 90.9*Fallgreen 20.8 ± 0.4 3608.9 ± 32.6 12.6 ± 1.0* 1547.4 ± 43.9*Ozarka II 14.4 ± 0.3 4544.4 ± 12.1 14.8 ± 0.1 1940.8 ± 105.8*zMicromoles of Trolox equivalents per gram of fresh weight. yMilligrams of chlorogenic acid equivalents per gram of fresh weight. xStandard error of the mean (N = 3). *Indicates significant difference between growing seasons, Student’s t test (P < 0.05). Data extracted from Table 1 of Howard et al. (2002) with permission.


some polyphenolic compounds have been related. However,the levels of composition of these compounds are far fromoptimal, particularly in relation to health benefits.

Increasing levels of antioxidant phytochemicals in fruitsand vegetables can be achieved by the semi-empirical selec-tion of new varieties with improved polyphenolic orcarotenoid composition, through traditional breeding, orthrough genetic engineering. Given the natural variation thatexists both within a single species and between closelyallied species, modification of levels of desired polypheno-lic or carotenoid content in fruits and vegetables seemsattainable through the use of traditional plant-breeding tech-niques. Conventional breeding is a long-term endeavour andonly a few programs have led to commercial production offruits and vegetables enriched with phytochemicals such asthe β-carotene-rich orange-coloured cauliflowers. Most pro-grams are currently at the surveying stage, i.e., determiningphytochemical profiles of antioxidants among differentgenotypes of a fruit or vegetable. Although we know thatvarietal differences in phytochemicals are mostly quantita-tive, establishing a consistently high-antioxidant cultivartakes time due to environment by genotype interaction.

There have been several breeding programs targetingantioxidant-rich fruits and vegetables. Lombard et al. (2005)examined the total flavonol content and individual quercetinconcentrations in five different onion cultivars, and foundsignificantly higher concentrations in red skinned onion ascompared with the yellow-skinned cultivars. Even amongthe four yellow-skinned cultivars, some differences weregreater than twofold (Lombard et al. 2005). The red-fleshedplum (Prunus salicina Erhr. and hybrids) and peach [Prunuspersica (Batsch) L.] cultivars obtained by Cevallos-Casalset al. (2006) focused on anthocyanin content and resulted inincreased antioxidant activities. A study by Maas et al.(1992) also showed that high ellagic acid levels in strawber-ry can be achieved through breeding. The higher antioxidantactivity and bioavailability of carotenoids from carotenoid-rich carrots (Simon et al. 1989; Simon 1997; Horvitz et al.2004; Molldrem et al. 2004) is another good example show-ing that traditional breeding can be used to improve nutri-tional value. Advanced breeding lines of spinach withhigher levels of total phenolics, individual and totalflavonoids, and antioxidant capacity can be achievedthrough traditional breeding (Howard et al. 2002) (Table 4).An orange cauliflower plant found growing spontaneouslyin a Canadian field nearly 30 yr ago was instrumental in thedevelopment of a β-carotene-rich cauliflower (Li et al.2001).

The biosynthetic pathways of polyphenols andcarotenoids are now well understood, and enzymes that reg-ulate the production of specific polyphenols and carotenoidshave been and are still being discovered. All these haveopened the possibility of directly developing new varietieswith specifically modified phytochemical profiles. In partic-ular, the use of molecular biotechnology to fine-tune thecontrol of polyphenolic and carotenoid metabolism, toupregulate desirable metabolic routes or to downregulateundesirable ones is now a very real possibility (Parr andBolwell 2000).

Whole Foods vs. ExtractsIn the past decade, we have witnessed the development andrapid growth of a new market and industry of nutraceuticals,functional foods and natural health products. Dietary sup-plements containing antioxidants extracted from fruits andvegetables have proven to be effective in alleviating manychronic diseases in many case studies (American DieteticAssociation 1995).

Many products extracted from fruits, vegetables and med-icinal herbs are available commercially; however, theeffects of these extracts in a pill or capsule form on healthare not always clear. In fact, due to the complex chemistryof the phytochemicals in fruits, vegetables, grains or herbalplants, many researchers have found that consuming wholefoods is more beneficial. Studies with ellagic acid showedthat inhibition of colon and oesophageal cancer cells wasgreater when the whole black raspberries were used ratherthan ellagic acid alone (Kresty et al. 2001). A “food-based”chemopreventive approach was taken to evaluate theinhibitory potential of lyophilized black raspberries (LBRs)against N-nitrosomethylbenzylamine (NMBA)-inducedoesophageal tumorigenesis in rats, and found that dietaryadministration of LBRs inhibited events associated withboth the initiation and promotion/progression stages of car-cinogenesis.

Synergistic effects among antioxidants are considered tobe significant. For example, Seeram et al. (2005) foundsuperior anti-proliferative, apoptotic and antioxidant activi-ties in pomegranate juice compared with its purifiedpolyphenols, and attributed this to the multi-factorial effectsand chemical synergy of multiple compounds comparedwith single purified active ingredients. In fact, little infor-mation is available regarding possible synergistic or antago-nistic biochemical interactions among polyphenolscontained in fruits and vegetables. Mertens-Talcott et al.(2003) investigated interactions between quercetin andellagic acid, two polyphenolics that are present predomi-nantly in small fruits, on cell death and proliferation-relatedvariables in the MOLT-4 human leukemia cell line. Theyfound that ellagic acid significantly potentiated the effectsof quercetin (at 5 and 10 µmol L–1 each) in the reduction ofproliferation and viability, and in the induction of apoptosis.Significant alterations in cell cycle kinetics were alsoobserved. In a study by Vinson and Jang (2001), a synergis-tic effect was found that the combination of citrus extractand vitamin C produced a synergistic antioxidant effect inan in vitro lipoprotein oxidation model, and in a double-blind, placebo-controlled study with 26 normal and hyperc-holesterolemic subjects. In the in vivo study, the citrusextract and vitamin C, but not vitamin C or vitamin E alone,significantly lowered triglycerides. The combination of cit-rus extract and vitamin C increased the lag time of lipopro-tein oxidation, compared with vitamin C alone or a placebo,and was a significantly better antioxidant than vitamin E.Citrus fruits are known to contain high concentrations offlavonoids such as hesperidin. Polyphenols not only showeda synergistic effect among themselves and with essential vit-amins, they were also found to enhance the effect of anti-cancer drugs such as sulindac and tamoxifen (Suganuma et


al. 1999). Using human lung cancer cell line PC-9, theseauthors found a tea polyphenol (-)-Epicatechin, which isfound in other fruits such as apple (Tsao et al. 2003b),enhanced apoptosis, growth inhibition of PC-9 cells, andinhibition of tumour necrosis factor-α release fromBALB/c-3T3 cells by other tea polyphenols in a dose-dependent manner. They also found that the effects ofpolyphenols on induction of apoptosis were synergisticallyenhanced by other cancer-preventive agents, such as sulin-dac and tamoxifen. The authors concluded that a mixture ofpolyphenols for cancer prevention in humans is more rea-sonable than a single compound, and even more effectivewhen it is used in combination with other cancer preventives(Suganuma et al. 1999).

High doses of purified phytochemical antioxidants cancause adverse effects (Lowe et al. 2003). The bioavailabili-ty of many polyphenols and carotenoids has not been fullyinvestigated, and there have not been any recommendeddaily doses for any specific compounds. Until further toxi-cological studies show otherwise, caution should be takenwhen consuming antioxidant-rich food supplements. Eventhe use of essential vitamin antioxidants during cancer ther-apy is highly controversial. Some data suggest antioxidantscan ameliorate toxic side-effects of therapy without affect-ing treatment efficacy, whereas other data suggest antioxi-dants interfere with radiotherapy or chemotherapy (Seifriedet al. 2003). Overall, current knowledge makes it prematureto generalize and make specific recommendations aboutantioxidant usage for those at high risk for cancer or under-going treatment (Seifried et al. 2003). Polyphenolic antiox-idants at high doses or under certain conditions can alsocause prooxidant activity (that is, promote oxidation).Sakihama et al. (2002) found that flavonoids and dihydrox-ycinnamic acids can nick DNA via the production of radi-cals in the presence of Cu and O2. Lowe et al. (2003)concluded that there is an optimal dose of carotenoids thatresults in maximum antioxidant effectiveness in humancells. Doses higher than optimal were either less effectivethan sub-optimal doses, or actually resulted in increaseddamage to the cell, possible due to prooxidant activity.

CONCLUSIONDirect and rigorous scientific evidence of the dietary bene-fits associated with the consumption of specific polypheno-lic or carotenoid compounds, or even classes of thesephytochemicals, is rare, even though individual antioxidantsin a particular food may be found to contribute to in vitroactivity (Tsao et al. 2005). Improving the levels of com-pounds such as epicatechin and procyanidin B2 in apple(Tsao et al., 2005) may thus have potential medical andsocial benefits. Recent advances in food science and humannutrition, and developments in plant biochemistry, physiol-ogy and molecular biology have reached the stage where ithas become feasible to attempt to modify the profiles ofphytochemical antioxidants, particularly polyphenols andcarotenoid of fruits and vegetables (Parr and Bolwell 2000).

Development of safe and nutritious fruits and vegetablesthat contain high concentrations of phytochemical antioxi-dants requires unprecedented close collaboration among sci-

entists in all related disciplines. It is important for plantbreeders, food chemists, nutritionists, molecular biologists,and medical professionals to work together in order tounveil the many unknowns in the area of phytochemicalsand human health.

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