The Contribution of Phytochemicals to the Antioxidant Potential of Fruit Juices

CHAPTER

THE CONTRIBUTION OF PHYTOCHEMICALS TO THE ANTIOXIDANT POTENTIAL OF FRUIT JUICES

7

Yvonne V. Yuan and Sachitha A. Baduge Ryerson University, Toronto, ON, Canada

7.1 INTRODUCTION The human body is constantly exposed to free radical species, whether through endogenous biochemical processes including oxidative metabolism or exogenous factors such as infection, radiation, or xenobiotics, and diet-related chronic disease risk factors including inflammation, obesity, types I and II diabetes mellitus, carcinogenesis, and cardiovascular diseases (Ryan and Prescott, 2010). Epidemiological data link habitual inadequate intakes of fruits and vegetables with increased risk of ischemic heart disease and stroke, gastric and esophageal cancers on a global basis, accounting for approximately 1.8% of total global disease burden (Lock et al., 2005). These researchers determined that increasing fruit and vegetable consumption to approximately 600 g/day in adults may potentially reduce the worldwide burden of disease for ischemic heart disease and stroke by as much as 31% and 19%, respectively (Lock et al., 2005). On the other hand, when Hung et al. (2004) investigated the relationship between consumption of fruits and vegetables and incidences of cardiovascular disease, cancer, and death from other causes in the Nurses’ Health and Health Professionals’ Follow-up Studies, an inverse relationship between total intake of fruits and vegetables with cardiovascular disease was observed, but not with incidence of all cancers. The strongest inverse relationship was observed with intake of green leafy vegetables versus cardiovascular disease and major chronic disease risk (Hung et al., 2004). The potential beneficial effects of fruit and vegetable consumption likely involve more than one mechanism associated with increased intakes of insoluble and soluble fiber, constituent vitamins and minerals, as well as the myriad secondary metabolite phytochemicals contained within these plant foods. Moreover, fruits are consumed in various manners including cooked or raw; peeled, cored or whole, or juiced. Fruit juices are commonly consumed for convenience, the ability to combine several fruits to enhance flavor profiles or increase nutrient content or for patients who have swallowing difficulties. For consistency and standards of identity, fruit juices are defined by the US Food and Drug Administration (2003) as “the aqueous liquid expressed or extracted from one or more fruits or vegetables, purees of the edible portions of one or more fruits or vegetables, or any concentrates of such liquid or puree”; whereas nectars are defined as “the common or usual name Fruit Juices. DOI: https://doi.org/10.1016/B978-0-12-802230-6.00007-2 © 2018 Elsevier Inc. All rights reserved.

95

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CHAPTER 7 THE CONTRIBUTION OF PHYTOCHEMICALS

in the United States and in international trade for a diluted juice beverage that contains fruit juice or puree, water, and may contain sweeteners”; whereas juice cocktails, drinks, or beverages are defined as a “juice beverage diluted to less than 100% juice with the word ‘juice’ qualified by a term such as ‘beverage’, ‘drink’ or ‘cocktail’.” Many varieties of fruit juices (e.g., blueberry, pomegranate, ac¸ai, black currant, etc.) have attracted a great deal of attention due to the presence of phytochemicals with the capacity to scavenge free radicals in vitro or in vivo, associated with antiproliferative or apoptotic efficacies (Patras et al., 2013). By definition, an antioxidant is a compound or substance that can delay the onset (initiation) or interrupt the progression (propagation) of an oxidizable substrate. Thus, antioxidant activity may occur via several mechanisms including free radical scavenging, singlet oxygen quenching, inactivation of peroxides and other reactive oxygen species (ROS), chelation of transition metal ions, quenching of secondary reaction products and inhibition of prooxidative enzymes, among others. These molecules are broadly classified by their mechanism of action as primary or secondary antioxidants. Primary antioxidants (e.g., tocopherol isoforms and mono- or polyhydroxy phenolic compounds) inhibit oxidation via hydrogen atom transfer (HAT) or single electron transfer (SET) mechanisms (often simultaneously), or as free radical acceptors with the formation of stable nonradical products (Patras et al., 2013). On the other hand, secondary antioxidants (e.g., L-ascorbic acid, citric or phosphoric acids) inhibit or retard oxidation by suppressing oxidation promotors such as transition metal ion catalysts, singlet oxygen, prooxidative enzymes and other oxidants. It is not uncommon for an antioxidant to act via more than one mechanism; e.g., L-ascorbic acid may act as via SET as a reducing agent, a metal ion chelator or oxygen scavenger. As discussed in recent reviews, it is also important to note that antioxidant efficacy is a function of structure
function characteristics, concentration, temperature, type of oxidation substrate, physical state of the system, presence of prooxidants and synergists (Patras et al., 2013; Yanishlieva and Marinova, 2001). There are more than 5000 individual phytochemicals that have been identified; with dietary phytochemicals known to include polyphenols, chelating compounds, antioxidant vitamins (e.g., L-ascorbic acid, tocopherols and tocotrienols) and provitamins (e.g., α- and β-carotene), other carotenoids (e.g., lycopene, β-cryptoxanthin), and carnosine (Liu and Felice, 2007). The polyphenols comprise phenolic acids, flavonoids (diphenylpropanes, C6
C3
C6), hydroxycoumarins, hydroxylated anthraquinones, anthrones, and xanthones; with the former two categories being prevalent in the diet, including fruit juices. Flavonoids can then be divided into flavones (e.g., luteolin (30 ,40 ,5,7-tetrahydroxyflavone), apigenin (40 ,5,7-trihydroxyflavone), tangeritin) and flavonols (e.g., kaempferol, quercetin and its glycosides quercetrin and rutin), flavanones (e.g., naringenin and its glycoside naringin (flavanone-7O-glycoside); hesperetin and its glycoside hesperidin) and chalcones, flavanols (e.g., catechins (e.g., (1)-catechins, (2)-epicatechin, (2)-epicatechin-3-O-gallate), leucoanthocyanidins), isoflavones and (pro-) anthocyanidins (Rice-Evans et al., 1996). On the other hand, phenolic acids, characterized by hydroxylated aromatic rings, include methoxy- and hydroxyl-benzoic acids (C6
C1; e.g., gallic acid derivatives) and cinnamic acids (C6
C3; e.g., coumaric, caffeic and ferulic acids). Recent statistics indicate that approximately 824 million metric tons of fresh fruit were produced globally in 2013; with 81 million metric tons of apples contributing 12% of the total. Apples were the most important fresh fruit, followed by bananas, wine grapes at 77 million metric tons, and oranges at 71 million metric tons (Global Apple Consumption Grows, 2015). The global fruit juice market has progressed considerably from selling generic apple and orange juices. According to recent statistics on the fruit juice market, growth in the retail market has only been 2%; barely

7.1 INTRODUCTION

97

ahead of the population growth (Falguera and Ibarz, 2014). This translates into globally near-static per capita consumption (Ayton, 2015). The Association of the Industry of Juices and Nectars (AIJN, 2014; a European fruit juice association) also confirms that even though the overall fruit juice market started to pick up momentum in 2013, recording a 1% increase to 38.9 billion liters globally, average fruit juice consumption appears to have reached a plateau. This may be associated with negative messaging about the contribution of juice consumption to sugar intake and obesity within the lay media, economic instability, and the ongoing challenge to encourage consumption of breakfast by adults and youth. The lay media has focused on the purported role of sugar-sweetened beverages in contributing to the increased incidence of noncommunicable, diet-related chronic disease risks including obesity and type II diabetes. In response to the negative media attention, the fruit juice industry has taken a strong stance by reviewing the scientific evidence which indicates that there are no data which link consumption of 100% juice beverages to weight gain (AIJN European Fruit Juice Association, 2014; Pasut et al., 2016; Buscemi et al., 2012). Of course, overconsumption of any foodstuff including sugar can potentially contribute to adverse health effects and it is therefore important to drink or eat in moderation. Current scientific data indicates that 100% juice containing no added sugars does not have a causal relationship to the obesity pandemic and that drinking 100% juice has been associated with an overall nutritious diet (O’Neil and Nicklas, 2008; Pereira and Fulgoni, 2010; Nicklas et al., 2008). In recent years, in a bid to promote and increase fruit juice consumption, fruit juice producers and processors have repositioned themselves to appeal to wider demographic groups. For example, Lucozade was originally developed as an effervescent beverage energy source during illness, but 1983 saw the rebranding of the beverage towards the burgeoning energy drink market, resulting in a tripling of sales between 1984 and 1989 in the United Kingdom (Ayton, 2015). Another potential boost to the US fruit juice industry was the release of the 2015
2020 American Dietary Guidelines (US Departments of Health and Human Services and of Agriculture, 2015). The Healthy Eating Patterns and Dietary Guidelines specifically refer to the role that 100% fruit juices can play in the consumption of the required servings of fruits, since 75% of Americans do not consume the recommended amount of fruits and vegetables; and drinking 100% juice is an easy way to boost essential vitamin intake, albeit the contribution of dietary fiber is not at the same level as with whole fruit (Meyering, 2016; US Departments of Health and Human Services and of Agriculture, 2015). The terms “Superfood” and “Superfruit” had their nascence in approximately 2004 as marketing terms within the food and beverage industry to refer to intensely colored vegetables and fruits, particularly berries, with high in vitro antioxidant capacity and potential epidemiological links to positive effects on reducing diet-related chronic disease risk factors (Falguera and Ibarz, 2014). Examples of such superfruits include goji, ac¸ai, cranberry, pomegranate, sea buckthorn, blueberry, blackberry, and raspberry. Consumers in Japan, China and the United States represent the largest markets for antioxidant fortified food and drinks, with superfruit juice sales reaching $2.6 billion in 2009 (Hudson, 2010). Various exotic blends such as Pom Hula (a mixture of 50% pomegranate, 30% pineapple and 20% apple juices from concentrate, as well as natural flavors), Welch’s Dragon Fruit Mango Cocktail (a mixture of pear, mango, dragon fruit, and guava juices from concentrate containing 20% fruit juice) and Bolthouse Farms’ Ac¸ai 1 10 Superblend juice (a mixture of apple juice from concentrate, ac¸ai puree, black currant, lemon, raspberry juices from concentrate, Merlot grape juice, bilberry, hibiscus, blueberry, pomegranate, yumberry juices from concentrate,

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CHAPTER 7 THE CONTRIBUTION OF PHYTOCHEMICALS

mangosteen puree, and goji juice, both from concentrate) are some recent popular superfruit juices and cocktail blends currently sold in the United States (Bouckley, 2014). With increased layperson/consumer and scientific interest in the antioxidant capacity of fruit juices, much still remains to be determined; e.g., what is the contribution of the various secondary metabolite phytochemicals to the antioxidant potential of fruit juices? It is well known that the composition of fruit juices and thereby, antioxidant activities, is a function of whether it is an extract of the whole fruit, or its parts as can be observed with pomegranate juices (Tzulker et al., 2007), or whether the juice is clarified or left cloudy as with different apple juices (Kahle et al., 2005; Oszmianski et al., 2007). Different harvest years (Tzulker et al., 2007) and varieties of fruit (i.e., dessert vs cider apples; Kahle et al., 2005) are also known to influence not only the polyphenol content and composition, but also the antioxidant activity of fruit juices. Moreover, the contribution of different fruit juice constituents to antioxidant activity have been evaluated, namely the potential correlation of L-ascorbic acid, anthocyanins, total polyphenols as well as individual flavonoids to different antioxidant measurements have been studied in several studies (Tzulker et al., ˇ 2007; Piljac-Zegarac et al., 2009). Thus, this book chapter will discuss the contribution of phytochemicals to the antioxidant activity of not only commercially available fruit juices, but also labprepared juices. In vitro and in vivo studies on various types of fruit juices including those “made from concentrate” (MFC), “not from concentrate” (NFC), nectars will be included, as well as juices subjected to temporal storage studies and accelerated aging. It is essential to quantify the proportion of ingested antioxidants that are either excreted unchanged, or absorbed, metabolized and thereby potentially able to impact the antioxidant status and oxidative stress of humans (Wootton-Beard et al., 2011). Garrett et al. (1999) refer to the term “bioaccessibility” as the quantity of antioxidants that have been released from a food matrix and present at the intestinal brush border for potential transport into the cell or in situ effects in the lumen; and “bioavailability” as the quantity of antioxidants which are absorbed from the intestinal tract into the circulation and thereby are able to exert biological effects at the cell and organ level (Granado-Lorencio et al., 2007).

7.2 ANTIOXIDANT PROFILES OF FRUIT JUICES 7.2.1 APPLE JUICES It is well known that the main sources of polyphenols in a typical Western (e.g., North and South American, European, and Scandinavian countries) diet include apples and berries as fruits, and onions as vegetables (Kahle et al., 2005). Indeed, apple juices, whether commercially available in a carton containing an unknown apple cultivar (339 mg gallic acid equivalents (GAE)/L; Gardner et al., 2000; Table 7.1), commercial clear and cloudy juices (110
459 mg/L, respectively; Kahle et al., 2005), fresh-squeezed, pasteurized and filtered juices from dessert (154
178 mg/L) or cider apple cultivars (261
970 mg/L; Kahle et al., 2005), or fresh-squeezed and pasteurized cloudy juices (472
1044 mg/L) or clarified clear juices (250
699 mg/L; Table 7.1) are rich in total polyphenols. Juices made from cider apple cultivars are richer in total polyphenols, as are cloudy apple juices compared to clear juices (Table 7.1). That the polyphenol content of apple juices is rich in flavonoids (ranging from 22% to 91% of total polyphenols; Table 7.1) as well as phenolic acids, reflects the diversity of constituent secondary metabolites including hydroxycinnamic acids, dihydrochalcones, flavan-3-ols, flavanones, and polymeric cyanidins (Table 7.2; Kahle et al., 2005;

Table 7.1 Total Polyphenols, Total Flavonoids, Vitamin C and Antioxidant Activity of Commercial Not From Concentrate (NFC), Made From Concentrate (MFC), and Fresh-Squeezed Fruit Juices, Nectars, and Pulps

Fruit Juice

% Juice

Harvest Location

Vitamin C (g/kg FW)

Total Polyphenolsa (mg GAE/L)

Total Flavonoids (mg/L)

Antioxidant Activityb (mmol Trolox/L)

369

182

1.903

375 391

172 138

2.223 2.158

464 339

282

2.827

110
173

41
78

152
459

76
200

154 165 165 178

88 108 105 110

970 570 830 641 304 261 671

453 300 237 326 166 128 312

1044 472

945 256

References

Pomes Apple (Pyrus malus; Malus domestica) Elstar 1 Goudreinet

100

Elstar Elstar 1 Jonagold 1 Goudreinet Elstar 1 Santana Commercial

100 100

Commercial clear juice Cloudy juice Dessert apples Granny Smith Golden delicious Red delicious Fuji Cider apples Boskoop Bittenfelder Brettacher Winterrambur Kaiser Wilhelm Kaiser Alexander Bohnapfel Cloudy juices Champion Idared

100 100 100

Unknown (purchased in Netherlands)

Unknown (purchased in Aberdeen, Scotland, UK) Unknown (purchased in Wu¨rzburg, Germany)

100 100

0.0007

Granato et al. (2015)

Gardner et al. (2000) Kahle et al. (2005)

100

100

Wroclaw, Poland

Oszmianski et al. (2007) (Continued)

Table 7.1 Total Polyphenols, Total Flavonoids, Vitamin C and Antioxidant Activity of Commercial Not From Concentrate (NFC), Made From Concentrate (MFC), and Fresh-Squeezed Fruit Juices, Nectars, and Pulps Continued

Fruit Juice

% Juice

Clear juices Champion Idared

100

Harvest Location

Vitamin C (g/kg FW)

Total Polyphenolsa (mg GAE/L)

Total Flavonoids (mg/L)

699 250

605 55

Antioxidant Activityb (mmol Trolox/L)

References

Drupes Sour Cherry (Prunus cerasus) Nectar (with apple)

40

Croatia

0.16

1106

3.6

ˇ Piljac-Zegarac et al. (2009)

4.268
5.052

Granato et al. (2015)

3.25

ˇ Piljac-Zegarac et al. (2009) McKay et al. (2015) Granato et al. (2015) ˇ Piljac-Zegarac et al. (2009)

Berries Cranberry (Vaccinium macrocarpon) Nectar

100

Unknown (purchased in Netherlands)

30

Croatia

Cocktail

54

Ocean Spray, USA

795

139

Blueberry (Vaccinium spp.) Nectar

100

963
1981

207
667

42

Unknown, (purchased in Netherlands) Croatia

0.06

1796

3.8

25

Croatia

0.25

1920

5.68

40

Croatia

0.27

1302

3.8

Black currant (Ribes nigrum) Nectar Strawberry (Fragaria spp.) Nectar (with apple)

788
976

0.11

107
175

1547

4.478
8.931

Citrus Orange (Citrus sinensis) Osbeck varieties Moro (blood) Tarocco (blood) Sanguinello (blood) Valencia late (blonde) Washington navel (blonde) Commercial Jaffa orange Florida orange Commercial (MFC)

100

100 100 100 100 100 100 100 100 100 100

100 Commercial (NFC)

100 100 100

100 Fresh squeezed

Unknown (purchased in Netherlands) Lentini, Siracusa, Sicily

100

Unknown (purchased in Aberdeen, Scotland, UK)

421
520

0.47
0.51 0.57
0.78 0.49
0.54 0.58

674
1147 387
1091 383
603 488

0.42

361

0.22 0.24 0.18

755 591 504

USA (purchased in Texas, USA)

61
146

Granato et al. (2015) Rapisarda et al. (1999)

Gardner et al. (2000) 643 443 550

USA 1 Mexico (purchased in Texas, USA) USA 1 Brazil (purchased in Texas, USA) USA (purchased in Texas, USA) Florida USA 1 Mexico (purchased in Texas, USA) Mexico (purchased in Texas, USA) Selangor, Malaysia

1.730
2.049

Vanamala et al. (2006)

488
617 291
478

Vanamala et al. (2006)

235 504

466 1353

30

Ghafar et al. (2010)

Grapefruit (Citrus paradisi) Commercial Pink grapefruit Commercial (NFC)

100 100 100

Unknown (purchased in Aberdeen, Scotland, UK) Unknown (purchased in Texas, USA)

0.19 0.16

Gardner et al. (2000)

535 537 407
522

Vanamala et al. (2006) (Continued)

Table 7.1 Total Polyphenols, Total Flavonoids, Vitamin C and Antioxidant Activity of Commercial Not From Concentrate (NFC), Made From Concentrate (MFC), and Fresh-Squeezed Fruit Juices, Nectars, and Pulps Continued

Vitamin C (g/kg FW)

Total Polyphenolsa (mg GAE/L)

Total Flavonoids (mg/L)

Fruit Juice

% Juice

Harvest Location

Common lime (Citrus aurantifolia) Wild lime (Citrus hystrix) Musk lime (Citrus microcarpa)

100

Selangor, Malaysia

2117

107

100

Selangor, Malaysia

4907

223

100

Selangor, Malaysia

1050

87

Antioxidant Activityb (mmol Trolox/L)

References Ghafar et al. (2010)

Grapes Boˆrdo grapes (Vitis labrusca) Commercial Organic Fresh squeezed

Burin et al. (2010)

Santa Catarina, Brazil 100 100 100

1112
3433 2634
2643 235
21,375

2.51
11.05 8.24
9.08 7.32
8.23

Exotic Fruit Pineapple (Ananas comosus) Commercial

100

Pomegranate (Punica granatum) Commercial Fresh arils Frozen arils Fresh arils Fresh arils Whole fruit Commercial

100 100 100 100 100 100 100

Unknown (purchased in Aberdeen, Scotland, UK) California, USA

Newe Ya’ar, Israel

Unknown (purchased in Netherlands)

0.0008

Gardner et al. (2000) Gil et al. (2000)

358

2566 2117 1808 605
1209 453
1285 2000
11,000 2256
3113

20 14 10 Tzulker et al. (2007) 206
293

20.64
20.77

Granato et al. (2015)

Passion fruit (Passiflora edulis) P. edulis (purple) P. edulis (Frederick) P. edulis (yellow) P. edulis (pink) P. edulis (yellow)

100/Pulp

100

Ecuador

P. edulis (orange)

Pulp extract

Mapou, Mauritius

0.09

574

P. edulis

Frozen pulp extract Frozen pulp

Sao Paulo, Brazil

0.04

740

P. edulis Sims f. flavicarpa Degener

Sarawak, Malaysia

Sao Paulo, Brazil

0.32 0.11

362 317

0.24 0.23

362 298 435

158

FW, fresh weight. a Total polyphenols expressed as gallic acid equivalents. b Antioxidant activity expressed as DPPH• stable free radical scavenging activity as Trolox equivalents.

Ramaiya et al. (2013)

Talcott et al. (2003) LuximonRamma et al. (2003) Genovese et al. (2008) Zeraik and Yariwake (2010)

Table 7.2 Polyphenol Composition of Apple (Pyrus malus) Juices (mg/L) Variety of Juice

Bohnapfel

Kaiser Alexander

Kaiser Wilhelm

Winterrambur

Brettacher

Bittenfelder

Boskoop

Fuji

Cider Apples

Red Delicious

Granny Smith (Purchased in Wu¨rzburg, Germany)

Golden Delicious

Dessert Apples

Champion

Idared

Cloudy

Clear

Cloudy

Clear

Hydroxycinnamic Acids Chlorogenic Caffeic 4-p-Coumaroyl-quinic acid

54 3.8 9.1

38 4.8 14

33 6.1 22

54 2.5 11

488 ND 29

223 3.2 44

448 4.0 140

230 7.3 78

117 4.2 17

81 3.0 50

305 5.5 49

83

78

130

115

16

15

86

80

26

7.6

2.7

7.2

37

45

63

63

36.3

20

136

17

20

3.5

3.4

9.3

4.1

7.1

8.5

25

35

94

28

15

13

35

119 15

118 13

29 9.4

18 7.3

1.6 2.5 1.7 2.8 3.8

1.7 2.8 2.2 3.5 4.7

0.7 1.1 1.0 1.2 0.8

1.0 1.6 1.3 2.1 0.8

29 150 63 524

28 132 60 198

17 28 9.8 208

9.1 14 3.8 39

Dihydrochalcones Phloretin-20 -Oxyloglucoside Phloridzin

Flavanols (2)-Epicatechin (1) Catechin

Flavanones Quercetin Quercetin Quercetin Quercetin Quercetin Quercetin

3 O-glucoside 3 O-galactoside 3 O-xyloside 3 O-arabinoside 3 O-rhamnoside 3 O-rutinoside

ND ND ND 0.5 1.9 ND

ND ND ND ND 1.6 ND

ND ND ND ND ND ND

ND 2.2 ND ND 1.4 ND

ND ND ND ND ND ND

1.0 6.9 5.0 5.0 4.3 0.8

ND 2.9 4.7 0.9 2.8 ND

4.0 8.1 4.5 6.1 4.0 ND

ND 2.4 3.3 0.4 2.8 ND

1.3 1.6 ND 0.4 1.6 ND

1.9 7.1 4.1 4.9 4.6 ND

Procyanidins B1 B2 C1 Polymers References ND, none detected.

Kahle et al. (2005)

Oszmianski et al. (2007)

7.2 ANTIOXIDANT PROFILES OF FRUIT JUICES

105

Oszmianski et al., 2007). Cider apple cultivars made into fresh-squeezed and filtered juices contained greater concentrations of hydroxycinnamic acids, including chlorogenic and 4-p-coumaroylquinic acids in particular, compared to dessert apple cultivars, as well as greater concentrations of the dihydrochalcones, phloretin-20 -O-xyloglucoside and phloridzin (Table 7.2; Kahle et al., 2005). Cloudy Idared and Champion cultivars of apple juices also contained greater amounts of chlorogenic and 4-p-coumaroylquinic acids than clarified counterparts (Oszmianski et al., 2007). When Granato et al. (2015) evaluated the 1,1-diphenyl-2-picrylhydrazyl (DPPH•) stable free radical scavenging activity of a variety of commercially available fresh-squeezed and pasteurized juices, including four apple juices, they reported a range from 1730 μmol Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalents/L for an orange juice sample to at high of 20,766 μmol Trolox equivalents/L for a pomegranate juice, with apple juices containing a variety of cultivars ranging from 1903 to 2827 μmol Trolox equivalents/L (Table 7.1). These workers also reported very close correlations of their DPPH• scavenging data with not only ferric reducing antioxidant power (FRAP; r 5 0.913) assay values, but also cupric-reducing antioxidant activity (r 5 0.891) data, demonstrating the similar antioxidant mechanisms of action between the stabilization of DPPH• by SET or HAT from an antioxidant molecule and the SET involved in the reduction of ferric to ferrous, or cuprous to cupric ions (Patras et al., 2013; Granato et al. 2015). Moreover, the DPPH• scavenging efficacy of these juices was positively correlated with total polyphenolics as GAE (r 5 0.970); total flavonoids (r 5 0.520); total proanthocyanidins (r 5 0.452) as well as nonflavonoid phenolics (r 5 0.900). Strong antioxidant activities of juices were attributable to p-coumaric and gallic acids, (1)-catechin, cyanidin 3-glucoside and quercetin contents of juices (Granato et al., 2015); thus, also helping to substantiate the antioxidant activities of cloudy and clear Champion and Idared apple juices reported by Oszmianski et al. (2007; Tables 7.1 and 7.2). These latter workers reported an approximately twofold greater DPPH• scavenging activity for fresh-squeezed Champion apple juices compared to Idared counterparts, as well as a 10%
25% greater stable free radical scavenging activity of cloudy versus clear juices. However, the DPPH• scavenging efficacy determined by these workers was obtained after only 10 min reaction time without considering whether a steady-state plateau of quenching this free radical was achieved. Indeed, using electron paramagnetic resonance in combination with DPPH•, these workers determined that between 24% (Idared, clear juice) and 83% (Champion, cloudy juice) of the DPPH• was scavenged after 3 min reaction time. Thus, the reaction kinetics are key to interpreting the DPPH• assay, given that rapid kinetics, such as with L-ascorbic acid, occur in 1 min or less; intermediate kinetics (i.e., α-tocopherol) between 5 and 30 min and slow kinetics such as with phenolic acids occurring after 1 h or more (Patras et al., 2013). Indeed, the strong DPPH• scavenging activity of the Champion cloudy juice was ascribed to the high overall total polyphenol and total flavonoid contents (Table 7.1), as well as the contribution of procyanidins and (2) epicatechin (Table 7.2) in this juice (Oszmianski et al., 2007). Interestingly, the DPPH• scavenging activity of the clear Idared juice was observed to be initially weak, but with scavenging of the stable free radical continuing for 30 min or more. The slower reaction kinetics identified for the clear Idared juice was suggested to be associated with procyanidins and larger condensed polymers of epicatechin or catechin (Oszmianski et al., 2007), but given the low concentration of these molecules in this juice, it is perhaps more likely that the elevated levels of phenolic acids, namely chlorogenic and 4-p-coumaroyl-quinic acids played a role in the slower kinetics, as above.

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CHAPTER 7 THE CONTRIBUTION OF PHYTOCHEMICALS

The antioxidant capacity of apple juices assessed in vitro may or may not alter antioxidant status or oxidative stress in vivo following consumption of the juices, when assessed for bioaccessibility and/or bioavailability. For example, when 11 healthy, ileostomy patients consumed 1 L of cloudy apple juice (made from cider apples) containing 407 mg total polyphenols including 157 mg total procyanidins, Kahle et al. (2007) recovered 41.7% of the polyphenols and corresponding metabolites in the ileostomy contents. Two hours postconsumption, 90% of the oligomeric procyanidins, with a degree of polymerization (DP) of 3.4, were recovered from the ileostomy fluids, compared to an initial DP 5 5.7 in the original juice; thus polymeric procyanidins could conceivably reach the colon. The ileostomy fluid was also found to contain 1- and 3-caffeoylquinic acids as isomers of the parent 4- and 5-caffeoylquinic acids in the apple juice as potential products of enterocyte esterase activity (Kahle et al., 2007). Metabolites of the hydroxycinnamic acids, caffeoyl and p-coumaroylquinic acid esters, namely quinic acid was also identified in the ileostomy fluid, thus leading to the absorption of quantities of this phenolic acid. Methyl esters such as methyl caffeate and methyl coumarate were also identified in fluids suggesting the formation of these metabolites via hepatic metabolism and transport back to the lumen via enterohepatic circulation. Metabolites of the dihydrochalcones, namely the aglycone phloretin and phloretin-20 -O-β-glucuronide, but not the parent phloridzin, were also detected in the ileostomy fluids. Thus, following consumption of the cider apple juice polyphenols, 12.7% reached the ileum unmetabolized and 22.3% were recovered as metabolites with the potential to influence the colonic milieu (Kahle et al., 2007). On the other hand, when 12 healthy, nonsmoking subjects consumed 1 L fresh-squeezed cloudy apple juice (Sampion cultivar; containing 248 mg total polyphenols, 30 mg ascorbic acid, 82 mg oligomeric procyanidins, 4.2 mg quercetin glycosides, 52 mg catechin and epicatechin, 82 mg chlorogenic acid and 17 mg/L phloridzin and phloretin xyloglucoside), clear apple juice (containing 270 mg total polyphenols, 0 mg ascorbic acid, 93 mg oligomeric procyanidins, 18.4 mg quercetin glycosides, 44 mg catechin and epicatechin, 58 mg chlorogenic acid, and 46 mg/L phloridzin and phloretin xyloglucoside), or clear apple juice devoid of polyphenols and ascorbic acid, Godycki-Cwirko et al. (2010) reported that serum DPPH• stable free radical scavenging activity was increased over baseline within 1 h postconsumption of the cloudy juice, whereas serum FRAP was increased within 1 h after both cloudy and clear apple juices. Interestingly, serum uric acid was observed to increase over baseline 1 h after consumption of all three types of apple juice; however, serum total phenolics were not influenced by any of the juices, although there was a trend for increased serum quercetin with the clear apple juice. Serum FRAP and DPPH• antioxidant activities were both positively correlated with serum uric acid levels following consumption of all three apple juices. The increased serum antioxidant activity was thus, not associated with changes in serum polyphenols, but rather with uric acid changes, that were attributed to the fructose load from the juices, at approximately 90 g/L.

7.2.2 BERRY JUICES As outlined above, many berry fruits and juice, nectar or cocktail juice blends have come to be marketed and referred to as “superfruit” foods, due to the high levels of total polyphenols, flavonoids, and associated in vitro antioxidant activities of these products (Table 7.1; Granato et al., ˇ 2015; McKay et al., 2015; Ryan and Prescott, 2010; Piljac-Zegarac et al., 2009). In particular, these berries (e.g., cranberries, blueberries, black currants, strawberries) and juices are noted for their anthocyanin content which is responsible for the intense red, purple, or blue coloration (Table 7.3;

Table 7.3 Polyphenol Composition of Anthocyanin-Rich Juices (mg/L) Variety of Juice Cranberry Cocktail (54% Juice; Ocean Spray)

Pomegranate “Wonderful” Fresh Arils (California)

Pomegranate “Wonderful” Frozen Arils (California)

Pomegranate “Wonderful” Commercial (California)

12.7 10.1 45.1

14.4 11.1 102.5

421.3 838.5 302.0

15.3 17.9

8.7 17.9

37.9 83.2

539.2

525.2

417.3

Pomegranate Fresh Arils (Newe Ya’ar, Israel) 2005

Pomegranate Fresh Arils (Newe Ya’ar, Israel) 2006

Pomegranate Whole Fruit (Newe Ya’ar, Israel) 2006

10
210

20,000
37,000

Hydroxybenzoic Acids 4-Hydroxybenzoic Gallic Protocatechuic Vanillic

0.6 0.2 4.6 3.8

Hydroxycinnamic Acids Caffeic Chlorogenic p-Coumaric Ferulic Sinapic

1.8 16.0 9.8 0.6 2.9

Gallagyl-Type Tannins Punicalagin B Punicalagin D Others Ellagic Acid Derivatives Ellagic acid Ellagic acid glucoside Hydrolyzable tannins

30
320

Flavanols Epicatechin Catechin

8.7 1.1 (Continued)

Table 7.3 Polyphenol Composition of Anthocyanin-Rich Juices (mg/L) Continued Variety of Juice Cranberry Cocktail (54% Juice; Ocean Spray)

Pomegranate “Wonderful” Fresh Arils (California)

Pomegranate “Wonderful” Frozen Arils (California)

Pomegranate “Wonderful” Commercial (California)

128.3

59.5

151.1

42.9

38.8

61.1

53.0

46.4

71.4

76.0

23.6

95.2

5.9

3.9

8.5

172

387

Pomegranate Fresh Arils (Newe Ya’ar, Israel) 2005

Pomegranate Fresh Arils (Newe Ya’ar, Israel) 2006

Pomegranate Whole Fruit (Newe Ya’ar, Israel) 2006

20
320

10
340

30
360

Flavanones Quercetin Quercetrin Myricetin

25.0 14.4 8.7

Anthocyanins Peonidin 3-galactoside Peonidin 3-arabinoside Cyanidin 3-arabinoside Cyanidin 3-galactoside Peonidin 3-glucoside Cyanidin 3-glucoside Delphinidin 3,5-diglucoside Cyanidin 3,5-diglucoside Delphinidin 3-glucoside Pelargonidin 3-glucoside

287.1

Total

657

306

References

McKay et al. (2015)

Gil et al. (2000)

173.0 99.8 69.7 22.1 4.8

Tzulker et al. (2007)

7.2 ANTIOXIDANT PROFILES OF FRUIT JUICES

109

McKay et al., 2015; Granato et al., 2015). The total polyphenol content was noted to be quite high among these juice products; e.g., commercially available fresh-squeezed and pasteurized blueberry juice or nectar ranged from 963 to 1981 mg GAE/L (Table 7.1), packaged black currant and strawberry nectars ranged between 1920 and 1302 mg GAE/L, respectively, and fresh-squeezed and pasteurized cranberry juice, packaged nectar and commercial cocktail ranged from 788 to 1547 mg GAE/L. Moreover, the polyphenol content of berry juices, nectars, and cocktails could be seen to contain substantial amounts of flavonoids, ranging from 14% to 18% of total polyphenols in cranberry products (Table 7.1) and 21%
34% of total polyphenols in blueberry juice, as well as phenolic acids, reflecting the diversity of these secondary metabolites including hydroxy-benzoic and -cinnamic acids, flavan-3-ols, flavanones, anthocyanins, procyanidins, and nonflavonoid phenolics (Table 7.3; McKay et al., 2015; Granato et al., 2015). Commercial fresh-squeezed and pasteurized blueberry juices contained greater levels of total proanthocyanidins (range from 869 to 1891 mg/L) than cranberry juices (range from 865 to 1057 mg/L), as well as greater levels of nonflavonoid phenolics ranging from 756 to 1313 mg/L and 682 to 801 mg/L, respectively (Granato et al., 2015). Thus, it is interesting to note that a commercially formulated cranberry cocktail consisting of 54% juice, contained approximately 40.6 mg/L phenolic acids, including hydroxyl-benzoic and -cinnamic acids (Table 7.3; McKay et al., 2015), 9.8 mg/L flavan-3-ols as epicatechin and catechin, 48.1 mg/L flavanones as quercetin, quercetrin and myricetin with a total of 656.5 mg/L of anthocyanins predominated by peonidin 3-galactoside and -arabinoside. Given that proanthocyanidins, as condensed tannins (or oligomeric and polymeric flavan-3-ols) including polyepicatechins and polyepigallocatechins, would likely be hydrolyzed during analysis, the aglycone epicatechin is likely reflective of this group of polyphenols. The chemical analyses of the juices above were performed on a specially formulated cocktail (McKay et al., 2015), juices that had been recently packaged in aluminum foil-enforced cardboard ˇ containers (Piljac-Zegarac et al., 2009), or commercially available fresh-squeezed juices that had been pasteurized prior to packing in glass containers (Granato et al., 2015). However, it is not known to what extent storage conditions may alter the polyphenolic composition of juices, namely, refrigeration storage. Packaged berry juices (cranberry, black currant, blueberry, and strawberry nectars) were analyzed for total polyphenols immediately after opening (Table 7.1), and then again at 2, 4, 9, 15, 22, and 29 days, during which time the juices were stored in the dark at 4 C (Piljacˇ Zegarac et al., 2009). All juices exhibited reductions in total polyphenols after 15 days, albeit the difference was significant for only the black currant and strawberry nectars. Prior to 15 days of storage, total polyphenol content tended to fluctuate with increases in the first 2 days of storage (significant only for strawberry nectar); however, after 15 days, total polyphenol contents were observed to increase once more, such that after 29 days of storage the total polyphenol contents were not different from initial values. These authors suggested that changes observed in the polyphenol content of the juices may be due to an interaction between the Folin
Ciocalteu reagent ˇ with compounds formed during storage (Piljac-Zegarac et al., 2009). Thus, it is noteworthy that despite the lack of difference in total polyphenol concentrations, these workers reported marked reductions in DPPH• scavenging antioxidant activity in a dose-dependent manner over the 29 days of storage. The initial (baseline) DPPH• scavenging efficacy of the berry nectars after 60 min reaction time ranged from 5.68 mM Trolox equivalents for black currant nectar (containing 25% juice; Table 7.1), 3.8 mM for blueberry (42% juice) and strawberry (40% juice) nectars, to 3.25 mM Trolox equivalents for the cranberry nectar (30% juice). Over the course of 29 days at 4 C, the

110

CHAPTER 7 THE CONTRIBUTION OF PHYTOCHEMICALS

DPPH• scavenging efficacy increased transiently after 2 days for blueberry, strawberry, and cranberry nectars, although the difference was not significant compared to baseline; this increase was attributed to potential polymerization of component polyphenols to yield molecules with greater ˇ resonance stabilization (Piljac-Zegarac et al., 2009). Thereafter, the DPPH• scavenging activity markedly decreased after 9 days, before leveling off until 29 days: cranberry and blueberry nectars exhibited an overall 15% reduction, black currant 40%, and strawberry 53% reductions by day 29 which was thought to be due to steric hindrance of SET and HAT functional groups from the increased polymerization of polyphenols in the nectars. It is conceivable that decreased solubility of polymers and condensed tannins may have played a role in the decreased antioxidant activities ˇ observed by these workers. In contrast to the results above with apple juices, Piljac-Zegarac et al. • (2009) found no correlation between total polyphenol content and DPPH antioxidant activity of berry nectars (r 5 0.215), potentially due to the lack of specificity of the Folin
Ciocalteau reagent reacting with not only phenolics, but also sugars and ascorbic acid, etc. Thus, it is interesting to note that strawberry and black currant nectars contained greater concentrations of L-ascorbic acid ranging from 0.27 to 0.25 mg/L compared to cranberry and blueberry nectars at 0.11
0.06 mg/L (Table 7.1). As a labile reducing agent and thereby rapidly acting antioxidant, it is no surprise that ascorbic acid levels dropped to zero within 7 days of storage for blueberry nectar, 9 days for cranˇ berry, 16 days for black currant, and dropped by 58% for strawberry nectar (Piljac-Zegarac et al., 2009). Not surprisingly, the DPPH• scavenging activities of 100% cranberry and blueberry juices were greater than observed for the nectars discussed above, ranging from 1.3
1.6 fold to 1.2
2.4 fold greater, respectively (Granato et al., 2015). These differences may also be partly attributable to the reaction kinetics of component polyphenols since the DPPH• scavenging results above were obtained after 60 min incubation compared to achieving a steady-state plateau in the methodology used by Granato et al. (2015). The DPPH• scavenging efficacy of the fresh-squeezed and pasteurized cranberry juices was ascribed to phenolic acids including hydroxyl-benzoic and -cinnamic acids, and in particular p-coumaric and gallic acids, flavan-3-ols including catechin and the anthocyanin constituents, namely cyanidin 3-glucoside as well as flavanones such as quercetin (Table 7.3; McKay et al., 2015; Granato et al., 2015). The DPPH• scavenging activity of the freshsqueezed and pasteurized blueberry juices was ascribed to the high content of flavonoids and moderate levels of total polyphenols (Table 7.1) and proanthocyanidins as detailed above. Thus, it can be seen that cranberry juices and cocktails in particular are rich in antioxidant molecules with slow kinetics in the DPPH• scavenging assay, namely phenolic acids and anthocyanins. In modeling the effect of digestion on the antioxidant activity of a variety of store-bought juices, Ryan and Prescott (2010) subjected juices to gastric (pH 2.0, pepsin, 37 C, 1 h) followed by small intestinal (pH 7.4, pancreatin, 37 C, 2.5 h) conditions. The FRAP antioxidant activity of Ocean Spray cranberry juice and Long Life Ocean Spray cranberry MFC juice increased by 1.27and 1.11-fold after in vitro digestion, respectively. Interestingly, there were no differences in the FRAP antioxidant activities of apple or orange juices following digestion. These authors suggested that the anthocyanins and polyphenols in general in cranberry juices may increase due to transformations under acidic or alkaline conditions during the in vitro digestion processes. However, the in vitro methodology employed by Ryan and Prescott (2010) was a closed system without any other food matrices present, particularly proteins, which would be expected to influence the activity of the proteolytic enzymes used to mimic the gastric and small intestinal milieu. While in vitro work

7.2 ANTIOXIDANT PROFILES OF FRUIT JUICES

111

may give an indication of in situ bioaccessibility of antioxidant molecules in the gastrointestinal tract pre-absorption, metabolism and excretion, only in vivo work will demonstrate the bioavailability, metabolism and potential bioactivity of these molecules. McKay et al. (2015) performed a single-dose, 24-h, absorption and excretion trial with 10 healthy adults administered 237 mL double-strength 54% juice cranberry cocktail (Table 7.3) and reported a bimodal distribution for peak plasma concentrations for all of the flavanols and flavonols and seven phenolic acids (vanillic, salicylic, caffeic, 4-hydroxybenzoic, 4-hydroxyphenylacetic, ferulic, p-coumaric acids) at 0.5
2.6 h and at 6.1
8.8 h. The time until maximum plasma concentration for the six anthocyanins (Table 7.3) was between 0.9 and 4.7 h. There was considerable interindividual variation in that the plasma of only three subjects exhibited all six anthocyanins. Catechin, anthocyanidin glucuronides and proanthocyanidin-A2 were not detected in the plasma. Similarly, a bimodal distribution in urine was observed for isorhamnetin and three phenolic acids: 4-hydroxybenzoic, 4-hydroxyphenylacetic and 4-hydroxy-3-methoxyphenylacetic acids at 4.8
6.6 and 10
24 h. Total plasma phenolics (i.e., phenolic acids, flavonols and flavanols) was 34.2 μg/mL at 8
10 h, whereas that for urine was 270 μg/mg creatinine occurring 2
4 h earlier (McKay et al., 2015). Both anthocyanins and anthocyanin glucuronides were detected in subject urine, as was proanthocyanidin-A2, with the latter reaching a peak concentration at 11 h. Similar to the bimodal peak plasma concentrations of flavanols, flavonols, and seven phenolic acids above, plasma FRAP antioxidant capacity peaked at 30 min and again at 6 h. Interestingly, there were no correlations between plasma FRAP capacity and the concentration of plasma of phenolics, however the plasma antioxidant activity assessed as total antioxidant performance in the hydrophilic and hydrophobic phases of serum was positively correlated with gentisic acid (r 5 0.285) and 3,4-hydroxyphenylacetic acid (r 5 0.233) concentrations. These authors also demonstrated an 18% increase in the resistance of low density lipoprotein (LDL) to copper-induced oxidation and conjugated diene formation at 8 h versus baseline. Thus, cranberry juice phenolic acids and flavonoids are bioavailable and bioactive to increase plasma antioxidant activity in healthy adults.

7.2.3 CITRUS JUICES Citrus juices, in particular orange and to a lesser extent, grapefruit juice, have been extremely popular as part of the breakfast meal in North America, partly aided by the “Healthy, US-style Eating Pattern” indicating that 1 cup of 100% juice can be part of the recommended 2-cup equivalents of fruit indicated in the American Dietary Guidelines (US Departments of Health and Human Services and of Agriculture, 2015). Similarly, Canada’s Food Guide recommends 7
10 servings of fruits and vegetables for adults daily, with 125 mL or 0.5 cup equivalent to 1 serving of 100% juice and the caveat to consume more vegetables and fruit, than juice (Health Canada, 2011). Orange juice consumption has also been facilitated by the wide variety of products on the market at various price points, including frozen MFC juices, fresh ready-to-drink products MFC or NFC and pasteurized in paperboard cartons, as well bottled fresh-squeezed juices in grocery stores. Moreover, typically two servings of commercially available 100% juices will contain more than the recommended dietary reference vitamin C intake (e.g., 46
132 mg) which ranges between 75 and 90 mg/day for healthy adults. Thus, fresh-squeezed orange juices from blood (e.g., Moro, Tarocco, Sanguinello) and blonde (e.g., Valencia late, Washington navel) cultivars harvested in Sicily containing approximately 118
195 mg and 105
145 mg vitamin C in 250 mL, respectively, more than satisfy the

112

CHAPTER 7 THE CONTRIBUTION OF PHYTOCHEMICALS

dietary reference intake recommendation for vitamin C (Table 7.1; Rapisarda et al., 1999). On the other hand, when Gardner et al. (2000) investigated cartons of pure juices purchased from a local supermarket, unidentified, Florida, and Jaffa orange juices contained between 45 and 60 mg vitamin C in 250 mL. Similarly, cartons of pure grapefruit and pink grapefruit juices contained between 48 and 40 mg vitamin C in 250 mL (Table 7.1). It is highly probable that pasteurization and other processing methods used for the commercially available carton juices decreased the endogenous vitamin C content due to the lability of this water-soluble, reducing agent, and antioxidant vitamin. The role of citrus juices as functional foods stems not only from the fiber and vitamin content, but also the total polyphenol content (Table 7.1; Granato et al., 2015; Rapisarda et al., 1999; Gardner et al., 2000; Ghafar et al., 2010), and in particular, the flavanones (e.g., naringenin and its glycoside naringin, hesperetin and its glycoside hesperidin; Table 7.4; Kelebek et al., 2008, 2009; Rapisarda et al., 1999; Ghafar et al., 2010; Vanamala et al., 2006) and blood orange cultivar anthocyanins (Table 7.4; Kelebek et al., 2008, 2009). The total polyphenol content of 100% orange juices ranged between 361 and 488 mg ferulic acid equivalents/L in fresh-squeezed blonde Valencia and Washington navel juices (Table 7.1) and between 383 and 1147 mg/L in freshsqueezed blood orange cultivars harvested in Sicily (Rapisarda et al., 1999). On the other hand, the total polyphenol content of fresh-squeezed orange juice (unknown cultivar) from fruit harvested in Selangor, Malaysia, was determined to be 1353 mg GAE/L (Table 7.1; Ghafar et al., 2010). Other orange juices (from a variety of cultivars) sold in cartons contained between 504 and 755 mg GAE/L (Table 7.1; Gardner et al., 2000); fresh-squeezed, pasteurized, and sold in glass containers contained 421
520 mg GAE/L (Granato et al., 2015); whereas grapefruit juices sold in cartons contained approximately 535 mg GAE/L for unspecified and pink cultivars (Gardner et al., 2000); and fresh-squeezed lime juices from fruit harvested in Selangor, Malaysia contained 1050, 2117, or 4907 mg GAE/L for musk lime, common lime, and wild lime species, respectively (Table 7.1; Ghafar et al., 2010). Total flavonoid content of orange juices varied from a low of 2.2% of total polyphenols when determined colorimetrically in a fresh-squeezed juice (Table 7.1; Ghafar et al., 2010), to between 14.5% and 28.1% of total polyphenols in fresh-squeezed, pasteurized, and bottled juices (Granato et al., 2015); that of fresh-squeezed lime juices ranged from a low of 4.5% of total polyphenols for wild lime to a high of 8.3% of total polyphenols for musk lime (Ghafar et al., 2010). The total polyphenol composition of citrus juices comprised hydroxy-benzoic and -cinnamic acids as phenolic acids, with ferulic and sinapic acids predominating (Table 7.4; Kelebek et al., 2008, 2009); phenolic acids ranged from 33
60 mg/L in blonde orange juices to 38
140 mg/L in blood orange juices from fruit harvested in Sicily (Rapisarda et al., 1999) and Turkey (Kelebek et al., 2008, 2009). Flavanone content was reflective of the different species of citrus fruit with hesperidin predominating in orange juices ranging from a low of 56 mg/L in fresh-squeezed orange juice of unknown cultivar harvested in Malaysia (Table 7.4; Ghafar et al., 2010), to 113
172 mg/L in fresh-squeezed blood and blonde orange juices harvested in Turkey (Kelebek et al., 2008, 2009), and 329
548 mg/L in MFC orange juice and 180
391 mg/L in NFC orange juice purchased in the United States (Vanamala et al., 2006). On the other hand, naringin was the predominant flavanone in grapefruit juice ranging from 235 to 372 mg/L in an NFC juice purchased in the United States (Table 7.4; Vanamala et al., 2006). Juice from the common lime was similar to that from oranges with hesperidin as the predominant flavanone (Ghafar et al., 2010). Anthocyanins in blood orange juices comprised delphinidin, cyanidin, and peonidin glucosides (Table 7.4) totaling 291 and

Table 7.4 Phenolic Acid, Flavanone and Anthocyanidin Contents of Citrus Juices (mg/L)

33.4

44
80

29.5
54.1

91
117 235
372

329.548

180
391

Common Lime

56.9

Orange (Selangor, Malaysia)

32.59 1.7 112.98 0.22 3.77

46.4
92.1

Grapefruit (Not From Concentrate)

29.8 2.6 143.2 0.51 4.89

38.0
91.2

Orange (Not From Concentrate)

39.91 2.23 171.17 0.95 6.07 32.37

60.1
140.2

Orange (From Concentrate) (Purchased in Texas, USA)

5.23 12.87 6.27 19.84 13.55 57.76

Washington Navel (Blonde Oranges)

6.79 15.08 8.29 26.89 17.3 74.35

Valencia Late (Blonde Oranges)

5.66 8.49 3.52 24.06 18.65 60.38

Sanguinello (Blood Oranges) (Lentini, Sicily)

5.82 0.92 6.74

Tarocco (Blood Oranges)

Sanguinello (Blood Oranges) (Adana, Turkey)

7.54 1.71 9.25

Moro (Blood Oranges)

Moro (Blood Oranges) (Adana, Turkey)

Kozan (Blonde Oranges) (Kozan, Turkey)

Variety of Juice

55.8

166.7

Hydroxybenzoic Acids Gallic Protocatechuic Total

3.33 0.96 4.28

Hydroxycinnamic Acids Caffeic Chlorogenic p-Coumaric Ferulic Sinapic Total

Flavanones Narirutin Naringin Hesperidin Neohesperidin Didymin Apigenin Poncerin Quercetin Total

252.7

181.0

151.26

4.65
6.8 11.7
25.7

260.1
444.5

150.2
180.4

185.7
300.2

244.1

202.3

395
643

11.4
31.4

291
504

11.1
13.7 5.1
7.9 407
522

(Continued)

Table 7.4 Phenolic Acid, Flavanone and Anthocyanidin Contents of Citrus Juices (mg/L) Continued

4.64

11.7

1.57

291.3

43.07

Common Lime

16.1

Orange (Selangor, Malaysia)

16.95

Grapefruit (Not From Concentrate)

132.6

Orange (Not From Concentrate)

1.48

Orange (From Concentrate) (Purchased in Texas, USA)

7.6

Washington Navel (Blonde Oranges)

15.83

Valencia Late (Blonde Oranges)

110.8

Sanguinello (Blood Oranges) (Lentini, Sicily)

2.60

Tarocco (Blood Oranges)

Sanguinello (Blood Oranges) (Adana, Turkey)

12.5

Moro (Blood Oranges)

Moro (Blood Oranges) (Adana, Turkey)

Kozan (Blonde Oranges) (Kozan, Turkey)

Variety of Juice

Anthocyanins Delphinidin 3-diglucoside Cyanidin 3-glucoside Delphinidin 3-(6v-malonyl glucoside) Cyanidin 3-(6v-malonyl glucoside) Cyanidin 3-O-(6v-dioxalyl glucoside) Peonidin 3-(6v-malonyl glucoside) Total References

Kelebek et al. (2009)

Kelebek et al. (2008)

Rapisarda et al. (1999)

Vanamala et al. (2006)

Ghafar et al. (2010)

7.2 ANTIOXIDANT PROFILES OF FRUIT JUICES

115

43 mg/L for Moro and Sanguinello blood oranges harvested in Turkey (Kelebek et al., 2008). Due to the increased popularity of NFC citrus juices and the prominent place of frozen MFC orange juices in North American households one may very well ask, how does processing affect the polyphenol levels and profiles in these juices and does price point have any relevance? Thus, it is noteworthy that the total flavonoid content of MFC orange juices (approximately 53 mg/100 mL) was indeed greater than that of NFC counterparts (approximately 37 mg/100 ml; Table 7.1; Vanamala et al., 2006). There was no relationship between price and total flavonoid content of MFC orange juices or NFC grapefruit juices; but a negative correlation (r 5 20.5) was observed for NFC orange juices. These workers discussed that the juice concentration processes could increase the total flavanone content in the juice pulp due to loss of solubility of these compounds during processing. Differences in flavonoid content of MFC versus NFC juices could also be attributed to variations in growing conditions, postharvest storage, processing techniques, and even the proportions of water used in reconstituting the juices prior to packaging. For example, the naringin content of grapefruit juices was previously observed to vary within a grove of trees and even within a season and certainly from year to year (Vanamala et al., 2006) as fruits from photosynthetic organisms exposed to variable UV-exposure, soil composition, etc. When Granato et al. (2015) investigated the DPPH• scavenging activity of commercial freshsqueezed, pasteurized orange juices in glass containers, the efficacies of orange juices were similar to that of apple juices, as above, at 1.7
2.0 mmol Trolox/L (Table 7.1), and were correlated with the total polyphenolic, total flavonoid and nonflavonoid phenolic content of juices. Thus, these workers determined that the antioxidant activity of orange juices could be attributed to the flavonoid and phenolic composition of the juices, particularly the gallic acid content and cyanidin 3-glucoside present in blood orange juices (Table 7.4; Kelebek et al., 2008). Kelebek et al. (2008) reported that not only was the total polyphenol concentration of Moro blood orange juice greater than that of Sanguinello blood orange juice (Table 7.4), but also the DPPH• scavenging EC50 efficacies at 0.18 versus 0.29 mg/mL, respectively. Similarly, the greater DPPH• scavenging EC50 efficacy of Kozan blonde orange juice at 0.31 mg/mL compared to orange wine (0.46 mg/mL) by these same workers was associated with a greater total polyphenol content (Table 7.4; Kelebek et al., 2009). Gardner et al. (2000) reported that commercially available 100% citrus juices in cartons (e.g., an unknown orange cultivar, Florida orange and Jaffa orange juices, grapefruit and pink grapefruit) exhibited capacity in HAT and SET to reduce Fremy’s salt radical (potassium nitrosodisulfonate) and ferric to ferrous ions, with orange juices being much more efficacious than a vegetable juice. Interestingly, the vitamin C and total polyphenol contents of juices (Table 7.1) were both correlated with the antioxidant activities from the two assays (Gardner et al., 2000). These workers determined that one molecule of L-ascorbate could reduce 2.48 Fremy’s radicals, and therefore, that vitamin C accounted for 65%
100% of the antioxidant activity of the various orange and grapefruit juices, but that ,5% of the antioxidant capacity of apple and pineapple juices could be attributed to vitamin C content. The ability of constituent phenolics to act in HAT or SET with resonance stabilization was also deemed to be key to the antioxidant activity of the citrus juices. Thus, it is noteworthy that when Rapisarda et al. (1999) evaluated the free radical scavenging activities of fresh-squeezed and filtered blonde and blood orange juices from Sicilian fruit, that blood orange varieties exhibited greater DPPH• scavenging EC50 efficacies: Moro, 25
46 μL; Tarocco, 24
49 μL, Sanguinello 40
80 μL compared to blonde orange juice cultivars: Valencia late, 50 μL, and Washington navel, 68 μL. These workers chose a variety of antioxidant assays due

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to the fact that the phenolics in orange juices may be only partially soluble, or even colloidal or suspended, making the choice of hydrophilic or hydrophobic solvents used critical. Thus, the blood orange juice cultivars containing anthocyanins (Table 7.4) were better antioxidants than those from blonde cultivars; moreover, the DPPH• scavenging activities were correlated with total polyphenol content (r 5 0.866) as well as with hydroxycinnamic acid (r 5 0.698), flavanone (r 5 0.425), and anthocyanins (r 5 0.666), with ascorbic acid playing a smaller role (r 5 0.333). Results with a linoleic acid emulsion forced oxidation assay evaluating conjugated diene and hydroperoxide production yielded similar correlation coefficients (Rapisarda et al., 1999). When Malaysian freshsqueezed lime and orange juices were evaluated for DPPH• scavenging EC50 efficacies, not only did wild lime juice have the greatest activity at 35 mg/100 mL, but also the greatest total polyphenols and flavonoid concentrations, with moderate hesperidin (Table 7.1), common lime (EC50 5 78 mg/100 mL), orange (unknown cultivar; EC50 5 96 mg/100 mL) and musk lime (EC50 5 128 mg/100 mL) were lower in total polyphenols and flavonoids (Table 7.1; Ghafar et al., 2010). In contrast to the increase in cranberry juice antioxidant capacity following an in vitro gastric and small intestinal digestion protocol, as above, when Ryan and Prescott (2010) applied the same methodology to a variety of commercially available fresh orange juices of unknown cultivar, there were no differences in the FRAP antioxidant activities after digestion, suggesting that the antioxidant constituents in the orange juices were stable to pH changes and proteolytic enzyme activity. On the other hand, the FRAP antioxidant efficacies of fresh grapefruit juices were decreased following the digestion protocol, reflecting the potential instability and degradation of antioxidant molecules under the conditions studied. These workers discussed the sensitivity of polyphenols to more alkaline conditions such as that employed to simulate the small intestine luminal milieu. It is also possible that polyphenol solubility may have been impacted during the in vitro digestion, and thereby, influenced subsequent DPPH• and FRAP antioxidant assays as discussed above. On the other hand, to truly assess the bioaccessibility of citrus juice polyphenols and thereby, the subsequent bioavailability and bioactivity in vivo, feeding studies such as that performed by Riso et al. (2005) are important. In a crossover design, these workers fed 600 mL fresh-squeezed and pasteurized blood orange juice to 16 healthy female volunteers (20
27 years of age) for 21 or 28 days. The juice contained 75.2 mg/100 mL vitamin C, carotenoids: 67 μg β-cryptoxanthin, 20 μg lutein, 18 μg zeaxanthin, 17 μg lycopene, 10 μg β-carotene and 8 μg α-carotene in 100 mL, 3.5 mg cyanidin 3-glucoside and 1.2 mg cyanidin 3-glucoside-6v-malonyl in 100 mL as key anthocyanins (Riso et al., 2005). After 21 days of treatment, significant increases were observed in plasma vitamin C (up by 28 μmol/L), β-cryptoxanthin (up by 0.5 μmol/L), zeaxanthin (up by 0.04 μmol/L), β-carotene (up by 0.05 μmol/L), and cyanidin 3-glucoside (increased by .10-fold to approximately 8 nmol/L); neither the aglycone or cyanidin 3-glucoside-6v-malonyl were detected in plasma. However, plasma indices of antioxidant protection against oxidative stress were not affected by the blood orange juice consumption: antioxidant activity as the capacity to reduce Cu21 to Cu1, malondialdehyde as a termination product of lipid oxidation and 11-dehydro thromboxane B2 as a marker of systemic platelet activation and inflammation were not significantly different after consumption of the juice for 3 weeks. On the other hand, lymphocytes from subjects treated with blood orange juice for 28 days did exhibit an increase in resistance to ex vivo challenge of with H2O2; moreover, there was an inverse correlation between plasma vitamin C and lymphocyte DNA damage from the H2O2 challenge (r 5 20.49; Riso et al., 2005). Thus, blood orange juice vitamin C, carotenoids,

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and anthocyanins were absorbed into the circulation and exerted a minor effect on indices of oxidative stress in lymphocytes under an ex vivo challenge. Daily intake of anthocyanin-containing juices can increase circulating levels of these molecules as demonstrated with cyanidin 3-glucoside by Riso et al. (2005). It is conceivable that chronic intake of polyphenol rich juices may have an influence on other biomarkers of antioxidant capacity and oxidative stress, such as LDL oxidation, as above, with cranberry juice, or in subjects with elevated diet-related chronic disease risk factors. On the other hand, Riso et al. (2005) did not evaluate the bioavailability and excretion of citrus juice flavanones, as studied by Erlund et al. (2001). Volunteers were recruited to consume a single dose at 8 mL/kg body wt of MFC orange (Pera cultivar; containing 41 mg/L naringenin and 218 mg/L hesperetin) or grapefruit (white March (99%) and white Duncan (1%) cultivars; containing 349 mg/L naringenin) juice amounting to between 400
760 mL and 400
720 mL, respectively. Plasma concentrations of naringenin increased upon consumption of both juices, reaching maximum concentrations of 0.1
1.2 and 0.7
14.8 μmol/L for orange and grapefruit juices, with great interindividual variability. Plasma concentrations of hesperetin after consumption of orange juice ranged between 0.5 and 5.5 μmol/L. Urinary excretion of flavanones largely occurred within 4
8 h, with 83% and 63% of naringenin excreted from orange and grapefruit juices; whereas 65% of hesperetin was excreted in the same time frame from orange juice (Erlund et al., 2001). Flavonoids such as hesperidin, naringin are very likely deglycosylated prior to absorption as aglycones and therefore, differences in intestinal microflora and thereby β-glucosidases and α-rhamnosidases likely contributed to the interindividual variability in bioavailability observed by these workers. Overall, plasma flavanone concentrations have a relatively short half-life with renal clearance of flavanones dose-dependent, thus, Erlund et al. (2001) suggest that plasma flavanone concentrations may not reflect chronic consumption patterns.

7.2.4 GRAPE JUICES Similar to the polyphenol- and anthocyanin-rich berry juices discussed above, grape juices, in particular the products from red grapes, are well known for not only being rich in total polyphenols, but also strong antioxidant activities in vitro (Table 7.1; Burin et al., 2010; Ryan and Prescott, 2010; Leifert and Abeywardena, 2008). The total polyphenol content and composition of grape juices are a function of the grape cultivar, maturity of fruit, growing conditions, weather, contact time with skin and seeds, extraction methods, and processing. For example, grape juice contains a wide variety of polyphenols including phenolic acids such as gallic acid, anthocyanins, as well as mono-, oligo-, and polymeric flavan-3-ols as proanthocyanidins including (1)-catechin, (2)-epicatechin, and associated gallates, as well as resveratrol (trans-3,5,40 -trihydroxystilbene; Leifert and Abeywardena, 2008). Resveratrol is well known to be associated with grape skins and thereby, red wines; whereas proanthocyanidins are found in grape seeds and skins, with the skins exhibiting a greater DP than seeds. The total polyphenol content of seven commercial, two organic, and three homemade grape juices produced in Brazil are summarized in Table 7.1 (Burin et al., 2010). These workers also determined the monomeric anthocyanin concentration of the juices spectrophotometrically to be 26
431, 169
222 and 208
460 mg/L as malvidin-3,5-diglucoside equivalents, respectively. Anthocyanin concentration is known to be influenced by elevated temperatures during extraction and pasteurization, packaging and thereby exposure to light; thus, grape juices in transparent bottles

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have exhibited reductions in color intensity associated with effects on anthocyanins. The DPPH• scavenging activities of grape juices was highly variable, particularly amongst commercial samples, with considerably less variability between the two organic and three homemade grape juices (Table 7.1; Burin et al., 2010). Interestingly, the DPPH• scavenging activities of juices were highly correlated with the total polyphenol content (r 5 0.96) as well as total anthocyanin concentrations (r 5 0.83), albeit slightly less so. Similar to the increase in antioxidant capacity following in vitro gastric and small intestinal digestion discussed above with cranberry juices, Ryan and Prescott (2010) also reported increases in the FRAP antioxidant activities of a commercial red grape juice (1.4-fold increase) as well as an MFC grape juice (1.6-fold). These workers suggested that the antioxidant anthocyanin and other phenolic constituents such as gallic acid may have undergone structural changes as a result of the in vitro digestion conditions thereby increasing the antioxidant activities of these anthocyanin-rich juices.

7.2.5 EXOTIC JUICES 7.2.5.1 Pomegranate juices Pomegranate (Punica granatum L.) juices are amongst the highly pigmented, anthocyanin-rich juices and cocktail blends that have been marketed, and thereby come to be known, as “superfruit” products; thus, giving rise to a plethora of specialty juice products such as those under the Pom Wonderful umbrella, including Pom Hula, as above. Pomegranate juices have been touted for the high levels of total polyphenols, flavonoids, anthocyanins, and in vitro antioxidant activities of these products (Tables 7.1 and 7.3; Gil et al., 2000; Tzulker et al., 2007; Granato et al., 2015; Ryan ˇ and Prescott, 2010; Piljac-Zegarac et al., 2009). The total polyphenol content of 100% pomegranate juices was noted to be quite high, but also highly variable amongst commercially available juices from Californian fruit or purchased in the Netherlands (2256
3113 mg GAE or p-coumaric acid equivalents/L; Table 7.1; Gil et al., 2000; Granato et al., 2015), as well as those produced from fresh (2117 mg p-coumaric acid equivalents/L; Gil et al., 2000; or 453
1285 mg quercetin equivalents/L; Tzulker et al., 2007) or frozen arils (1808 mg p-coumaric acid equivalents/L; Table 7.1; Gil et al., 2000) and in particular, juices produced from the whole fruit comprising the inner pith of the peel, white membranes, and arils (2000
11,000 mg quercetin equivalents/L; Tzulker et al., 2007). The natural variability in total polyphenol contents of 23 pomegranate juices from accessions differing in the color of peel and arils was illustrated by Tzulker et al. (2007) with fruit harvested in 2005 (605
1209 mg quercetin equivalents/L; Table 7.1) and 29 juices from fruit harvested in 2006 (453
1285 mg quercetin equivalents/L). The proportion of total polyphenols as total flavonoids in commercial fresh-squeezed and pasteurized pomegranate juices was reported to range between 9.1% and 9.4%, total proanthocyanidins between 28.4% and 32.4%, and nonflavonoid phenolics between 90.6% and 90.9% (Granato et al., 2015). Pomegranate juice polyphenols have been reported to include derivatives of ellagic acid (27
121 mg/L in commercial and aril juices, or 30
320 mg/L in juice from the whole fruit; Table 7.3; Gil et al., 2000; Tzulker et al., 2007), hydrolyzable tannins (417
539 mg/L in commercial and aril juices), gallagyl-type tannins including punicalagin (2,3-hexahydroxy-diphenoyl-4,6gallagylglucose) B, D, and others (10
210 mg/L in aril juices, 1562 mg/L in commercial juice or

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20
37 g/L in juice from the whole fruit), anthocyanins including cyanidin, delphinidin and pelargonidin glucosides (10
340 mg/L in aril juices, 387 mg/L in commercial juice or 30
360 mg/L in juice from the whole fruit; Table 7.3; Gil et al., 2000; Tzulker et al., 2007). Thus, clearly the polyphenol contents and profiles of pomegranate juices varied between harvest years as well as country and region of harvest: California, United States compared to Newe Ya’ar, Israel, as well as whether just the arils were used to collect juice, or the entire fruit extracted since the peels are known to contain water soluble hydrolyzable tannins such as the punicalagin isomers (Tzulker et al., 2007). This latter point is particularly relevant since the commercial juice extraction process uses hydrostatic pressure to extract juice from the whole fruit including not only the arils, but also the peel, including the husk, rind or pericarp, white membranes and pith of the fruit. Thus, it is noteworthy that commercial juices were distinguished by containing greater amounts of punicalagins and ellagic acid derivatives compared to juices prepared from arils alone (Table 7.3; Gil et al., 2000). These workers also reported that not only were the total polyphenol, but also anthocyanin pigment concentrations greater in juice prepared from fresh arils compared to that from frozen arils. The DPPH• scavenging activities of commercial 100% pomegranate juices from California and Netherlands were quite similar ranging between 20 and 21 mmol Trolox/L; whereas those from fresh and frozen arils ranged from 14 to 10 mmol Trolox/L, respectively (Table 7.1; Gil et al., 2000; Granato et al., 2015). It is noteworthy that the DPPH• scavenging efficacies of aril juices were similar to those of 100% grape juices from Brazil (Burin et al., 2010) and blueberry juices (Granato et al., 2015); but those of juices produced from the whole fruit were approximately 10fold greater than 100% apple and orange juices and 2- to 5-fold greater than grape, blueberry, and cranberry juices (Table 7.1). Gil et al. (2000) also determined that the DPPH• scavenging activities of aril and commercial juices were approximately two- and threefold those of red wine and green tea, respectively. As above, Granato et al. (2015) determined that the DPPH• scavenging activities of a variety of juices, including 2 commercial pomegranate juices, were positively correlated with juice total polyphenol, total flavonoid, proanthocyanidin and nonflavonoid phenolic concentrations. Thus, the pronounced antioxidant efficacies of pomegranate juices was associated with high total polyphenols, and cyanidin 3-glucoside contents (Tables 7.1 and 7.3; Granato et al., 2015). Indeed, Gil et al. (2000) reported that the reduced antioxidant efficacy of juice from frozen arils (Table 7.1) could be attributed to degradation or transformation of anthocyanins as evidenced by the reduced anthocyanin concentration, in particular the cyanidin 3-glucoside levels in these juices (Table 7.3). Moreover, these workers were able to attribute approximately 88% of the antioxidant activity of pomegranate juices as follows: 2.4% to ellagic acid and glycosides, 6.8% to anthocyanins, 30.2% to hydrolyzable tannins and 48.3% to hydrolyzable gallagyl tannins comprising punicalagin isomers and punicalin. The high antioxidant activity of purified punicalagin was attributed to the 16 phenolic hydroxyl groups per molecule (Gil et al., 2000). When Tzulker et al. (2007) evaluated the FRAP antioxidant activities of 2 harvest years, once again the antioxidant activity was positively correlated with the polyphenol contents of aril juices (2005 harvest, r 5 0.95 and 2006 harvest, r 5 0.86) as well as the anthocyanin contents (2005 harvest, r 5 0.7, 2006 harvest, r 5 0.68). Thus, it follows that juices prepared from fruit with arils of a darker color demonstrated greater antioxidant activities. On the other hand, when these workers studied the antioxidant activity of juices prepared from the whole pomegranate fruit, the antioxidant activities were not correlated with anthocyanin concentrations; indeed some of the highest antioxidant activities were from fruit with transparent or pink-colored arils. Thus, it is noteworthy that the

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levels of hydrolyzable tannins including punicalagin isomers were highly correlated with FRAP antioxidant activities (Tzulker et al., 2007). When studying the stability of the total polyphenol content of a pomegranate nectar containing 12% fruit (including a mixture of pomegranate, chokeberry and wild blueberry), while under refrigˇ erated storage (4 C, 29 days), Piljac-Zegarac et al. (2009) reported a relatively low value of 1317 mg GAE/L on day 0 which decreased to low of 846 mg GAE/L on day 15, and then increased to 1596 mg GAE/L on day 29. Thus, these workers surmized that during storage, compounds may have been formed that were reactive with the Folin
Ciocalteu reagent, such that 29 days of refrigerated storage did not reduce the concentration of phenolic compounds. On the other hand, the initial DPPH• scavenging activity of the pomegranate nectar was 3.47 mM Trolox equivalents, which increased after 2 days of storage to approximately 4.2 mM, attributed to the potential polymerizaˇ tion of constituent polyphenols with enhanced resonance stabilization (Piljac-Zegarac et al., 2009); and a final antioxidant activity of 3.67 mM Trolox equivalents after 29 days of refrigerated storage, indicating relative stability during storage. It is noteworthy that these DPPH• scavenging activities of a 12% juice pomegranate nectar are far less than those reported for 100% pomegranate juices from arils, and in particular the whole pomegranate fruit, as above (Table 7.1). When Ryan and Prescott (2010) modeled the in vitro gastric and small intestinal digestion of fresh pomegranate juice and a MFC pomegranate juice counterpart, increases in the FRAP antioxidant activities were observed similar to that of cranberry and grape juices as discussed above. The FRAP antioxidant activities increased 2.34- and 1.64-fold from the initial 8557 and 10,232 μmol/L for the fresh and MFC specimens, respectively. These workers suggested that the antioxidant anthocyanins and other phenolic constituents acid may have undergone structural changes as a result of the in vitro digestion conditions thereby increasing the antioxidant activities of these anthocyaninrich juices. Also, pasteurization during processing of juices may have destroyed some antioxidant constituents, but also increased or made others more available. On the other hand, the work of Seeram et al. (2006) indicated that pomegranate polyphenolics undergo significant metabolism by intestinal bacteria including glucuronidation and subsequent enterohepatic circulation which would not be factored into an in vitro digestion protocol. When these workers administered 180 mL of pomegranate (Pom Wonderful) juice concentrate containing 318 mg punicalagins and 12 mg free ellagic acid to subjects, ellagic acid increased in plasma to a maximum of 0.06 μmol/L within approximately 1 h with a clearance half-life of 0.7 h, thus, ellagic acid was cleared from the plasma within 5 h. There was considerable interindividual variability when plasma of seven subjects was evaluated for ellagic acid metabolites including urolithin (hydroxyl-6H-benzopyran-6-one) A (n 5 2), urolithin B (n 5 1), urolithin A glucuronide (n 5 4), hydroxy-urolithin A (n 5 3) and methyl urolithin A (n 5 1); these ellagic acid metabolites began to appear in the plasma between 0.5 and 6 h. This time course reflects the synthesis and conjugation of urolithin A and B from the gallagyltype tannins by intestinal microflora and subsequent enterohepatic circulation of these metabolites (Seeram et al., 2006). Clearance of gallagyl-type tannins, ellagic acid and metabolites in urine was demonstrated by the appearance of dimethylellagic acid glucuronide (DMEAG), ellagic acid, urolithin A glucuronide, urolithin B glucuronide, with the glucuronides appearing in the second 12-h collection of urine after consumption of the juice concentrate. Thus, subjects absorbed pomegranate juice gallagyl-type tannins including the punicalagin isomers as urolithin metabolites; the presence of DMEAG in urine may be considered as a biomarker of juice intake. Interindividual variability in DMEAG and ellagic acid metabolites may be associated with genetic polymorphisms in key

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metabolic enzymes such as catechol-O-methyl transferase and UDP-glucuronosyltransferases (Seeram et al., 2006). In work to demonstrate the antioxidative bioactivity of bioavailable pomegranate juice polyphenols, Rosenblat et al. (2006) administered 50 mL pomegranate juice (containing 1.5 mmol total polyphenols, comprised of 1561 mg/L punicalagin isomers, 417 mg/L hydrolyzable tannins, 384 mg/L anthocyanins (delphinidin 3,5-diglucoside, cyanidin 3,5-diglucoside, delphinidin 3glucoside, cyanidin 3-glucoside and pelargonidin 3-glucoside) and 121 mg/L ellagic acid derivatives) to 10 control and 10 noninsulin dependent diabetes mellitus (NIDDM) subjects for 3 months. Pomegranate juice consumption by NIDDM subjects did not affect HbA1C levels, but did reduce serum C-peptide levels by 23%; similarly serum lipid peroxides and thiobarbituric acid reactive substances were reduced by 56% and 28%, respectively, in NIDDM subjects compared to before juice treatment, albeit remaining higher than in controls. On the other hand, serum sulfhydryl groups were increased by 12% in NIDDM subjects with treatment over baseline, albeit remaining lower than control subjects. When these workers studied the effects of pomegranate juice treatment on NIDDM subject monocyte-derived macrophages, total cellular peroxides and uptake of oxidized low density lipoproteins (ox-LDL) were reduced by 71% and 39%, respectively, below control levels, while cellular glutathione levels were increased 141% similar to control levels (Rosenblat et al., 2006). Thus, pomegranate juice consumption by NIDDM subjects resulted in significant reductions in serum markers of lipid peroxidation products as indicators of in vivo oxidative stress, as well as increased serum antioxidant capacity from sulfhydryl groups. Juice treatment also reduced monocyte-derived macrophage atherogenic indices as ox-LDL uptake is a precursor to the development of atherosclerotic lesions.

7.2.5.2 Passion fruit juices Passion fruit (Passiflora edulis) has two main commercial cultivars, P. edulis f. flavicarpa, yellow passion fruit and P. edulis Sims, purple passion fruit. The yellow fruit is a mutation or hybrid derived from the purple cultivar. Recent statistics indicate that global production of this exotic fruit was approximately 1.27 million metric tonnes in 2010, up from 1.05 million metric tonnes in 2005, which can no doubt be attributed to the distinct aromas and flavors of the fruit pulp and juice, making it a popular ingredient in tropical fruit juice cocktails and blends (Ramaiya et al., 2013). In general, the purple passion fruit is consumed fresh, whereas the yellow cultivar is used in juice production due to a greater acidity, higher juice yield, and brilliant yellow color. Thus, yellow passion fruit juices and pulps are noted for not only provitamin A carotenoids and xanthophylls, but also total polyphenolics comprising total flavonoids, including flavone glycosides, flavonols, anthocyanins proanthocyanins, as well as phenolic acids and ascorbic acid (Tables 7.1 and 7.5; Ramaiya et al., 2013; Talcott et al., 2003; Luximon-Ramma et al., 2003; Genovese et al., 2008; Zeraik and Yariwake, 2010). The total polyphenol content of frozen, unpasteurized yellow passion fruit juice made from fruit harvested in Ecuador was 435 mg GAE/L (Table 7.1; Talcott et al., 2003), whereas that of the pulp from four cultivars harvested in Malaysia ranged between 298 and 362 mg GAE/L fresh weight (Ramaiya et al., 2013), that of an orange-colored cultivar harvested in Mauritius was 574 mg GAE/L fresh weight (Luximon-Ramma et al., 2003), and those of frozen pulps harvested in Brazil were 158
740 mg GAE/L fresh weight (Zeraik and Yariwake, 2010; Genovese et al., 2008). Interestingly, when yellow passion fruit juice, as above, was subjected to pasteurization at 85 C,

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Table 7.5 Polyphenol Composition of Passion Fruit (Passiflora edulis) Juice and Pulps (mg/L) Variety of Juice

Passion Fruit—Yellow (Ecuador)

Passion Fruit Pulp (Sao Paulo, Brazil)

Passion Fruit Pulp (Mapou, Mauritius)

Passion Fruit Sims f. flavicarpa Degener (Sao Paulo, Brazil)

Hydroxybenzoic Acids p-Hydroxybenzoic Syringic acid derivatives

0.3 4.6

Hydroxycinnamic Acids Chlorogenic Caffeic o-Coumaric p-Coumaric p-Coumaric acid derivatives Ferulic Ferulic acid derivatives Sinapic Flavonoid glycoside

ND 0.3 0.4 0.6 0.5 0.8 0.9 0.6 4.6

Flavones Isoorientin

16.2

Flavanones Quercetin derivatives

23.3

Ellagic Acid Derivatives Ellagic acid Ellagic glucoside Carotenoids Anthocyanidins Proanthocyanidins References

ND ND 92.5 ND 12 Talcott et al. (2003)

Genovese et al. (2008)

Luximon-Ramma et al. (2003)

Zeraik and Yariwake (2010)

ND, none detected.

30 min, the total polyphenol content increased to 464 mg GAE/L, attributed to effects of thermal degradation and polymerization of polyphenolic constituents (Talcott et al., 2003). The proportion of total polyphenols as total flavonoids in the pulp of Mauritian orange-colored passion fruit was reported as approximately 21.1%, or 12.1 mg quercetin equivalents/100 g fresh weight (Luximon-Ramma et al., 2003); whereas that of Brazilian frozen pulps was approximately 3.1%, or 2.33 mg quercetin derivatives/100 g fresh weight (Genovese et al., 2008). Zeraik and

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Yariwake (2010) quantified total flavonoids in Brazilian yellow passion fruit pulp, and reported 15.8 mg rutin equivalents/100 g fresh weight, of which 19%, or 1.62 mg/100 g fresh weight was identified as the flavone glycoside isoorientin. Flavonoids in passion fruit pulp and juices have been noted to comprise such flavone glycosides as isoorientin, orientin, isovitexin, luteolin-6-Cchinovoside, luteolin-6-C-fucoside, as well as schaftoside, isoschaftoside, anthocyanidins, procyanidins, and the flavonol quercetin and its derivatives (Table 7.5; Talcott et al., 2003; Genovese et al., 2008; Zeraik and Yariwake, 2010). Talcott et al. (2003) reported the presence of a wide variety of phenolic acids in Ecuadorean yellow passion fruit juice comprising hydroxybenzoic acids: p-hydroxybenzoic acid and syringic acid derivatives totaling 4.9 mg/L (Table 7.5); and hydroxycinnamic acids: caffeic, coumaric acid isomers and derivatives, ferulic acid and derivatives, and sinapic acid ranging from 0.3
0.9 mg/L to a total of 4.1 mg/L. Luximon-Ramma et al. (2003) determined that the pulp of Mauritian orange-colored passion fruit contained 12 mg proanthocyanidins/L, as cyanidin chloride equivalents (Table 7.5), but did not provide a detailed component analysis. Given that the recommended dietary reference vitamin C intake for healthy adults ranges between 75 and 90 mg/day, consuming juice from several Malaysian passion fruit cultivars would provide between 27.5 and 80.0 mg ascorbic acid/250 mL (Table 7.1; Ramaiya et al., 2013), whereas pulp from Mauritian orange-colored passion fruit would provide approximately 22.5 mg/250 mL (Luximon-Ramma et al., 2003), and that from Brazilian frozen pulp would provide approximately 10 mg/250 mL (Genovese et al., 2008). As above, given the lability of ascorbic acid as a reducing agent and antioxidant, processing of fruit and frozen storage of pulps likely degraded this vitamin and antioxidant. When Talcott et al. (2003) evaluated the total carotenoid content of Ecuadorean yellow passion fruit juice at 378, 401, 427, and 470 nm, the total across all four wavelengths was approximately 92.5 mg/L (Table 7.5), but this is only an estimate as each wavelength corresponds to one or more carotenoids; e.g., ζ-carotene has absorbance maxima at 378, 400, and 425 nm. While up to 13 carotenoids have been identified in the rind, flesh, and juice of yellow passion fruits, ζ-carotene has been identified as the major form and was thought to be likely responsible for the predominant absorption maxima and spectral bands in the juice as quantified by Talcott et al. (2003). As lipid-soluble, potential antioxidant molecules, carotenoids, and in particular, β-carotene as a provitamin A source, are noted for quenching singlet oxygen ROS. These authors reported that the oxygen radical absorbance capacity (ORAC) antioxidant activity of unpasteurized, yellow passion fruit juice was 17.2 μM Trolox equivalents/mL, compared to 18.2 μM Trolox/mL for the pasteurized juice (85 C, 30 min). When the yellow passion fruit juice was fractionated into hydrophilic and lipophilic phases, the ORAC activities were 14.5 and 10.9 μM Trolox equivalents/mL for the unpasteurized juice, respectively and 13.5 and 13.4 μM Trolox/mL for the pasteurized juice (Talcott et al., 2003). The ORAC antioxidant activity assay is another example of HAT to stabilize an azo dye stable free radical or the peroxy radicals derived from the azo dye (Patras et al., 2013). Ramaiya et al. (2013) reported the DPPH• scavenging activities of Malaysian passion fruit pulp extracts using EC50 (effective concentration for 50% radical quenching) values: P. edulis (Purple), 548 μmol Trolox equivalents/L; P. edulis (Frederick), 927; P. edulis (Yellow), 524, and P. edulis (Pink), 778 μM Trolox/L. On the other hand, Genovese et al. (2008) reported that the DPPH• scavenging activity of Brazilian frozen passion fruit pulp was 0.80 μmol Trolox equivalents/g fresh weight; whereas the capacity of the same sample to prevent β-carotene bleaching in a linoleic acid emulsion was 0.40 μmol Trolox equivalents/mL. The FRAP antioxidant activity of Mauritian orange-colored passion fruit pulp was reported as 3.0 μmol Fe (II)/g fresh weight (LuximonRamma et al., 2003).

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Talcott et al. (2003) determined that the ORAC antioxidant activity of yellow passion fruit juice was negatively correlated (r 5 20.62) with the carotenoid content, which is not surprising given that the ORAC assay is conducted under aqueous conditions, and therefore, carotenoids could not be expected to exert protective antioxidant effects. On the other hand, when the yellow passion fruit juice was separated into hydrophilic and lipophilic fractions, as above, the additive antioxidant activity (25.4 μM Trolox equivalents/mL) was 1.47-fold that of the parent juice, perhaps indicating interactions amongst the antioxidant compounds. Interestingly, Genovese et al. (2008) demonstrated that the DPPH• scavenging activities of assorted Brazilian frozen fruit pulps, including passion fruit pulp, was positively correlated with the total polyphenol contents (r 5 0.74), but there were no correlations observed with the β-carotene bleaching assay (r 5 0.41). Similarly, Luximon-Ramma et al. (2003) demonstrated that the FRAP antioxidant activities of 17 exotic fruit pulps harvested in Mauritius, including an orange-colored cultivar of passion fruit, were positively correlated with total polyphenols (r 5 0.95), proanthocyanidins (r 5 0.92), and total flavonoids (r 5 0.69), but poorly correlated with the ascorbic acid content (r 5 0.04). Thus, the total polyphenol and proanthocyanidin contents of fruit pulps were determined to play a greater role in antioxidant activity compared to flavonoids and ascorbic acid. The negative correlation between the DPPH• EC50 scavenging activities of a variety of Malaysian passion fruit pulps with total polyphenols (r 5 0.82) simply reflects the fact that a lower EC50 indicates greater efficacy (Ramaiya et al., 2013). Thus, the antioxidant activities of passion fruit juices and pulps could be seen to be related to the polyphenol contents and profiles similar to the correlative relationships observed with apple, orange, cranberry, grape, and pomegranate juices, nectars, and cocktails, as discussed above. When evaluating the stability of frozen, unpasteurized Ecuadorean passion fruit juice antioxidant constituents during pasteurization (85 C, 30 min), refrigeration for 24 h, followed by accelerated storage conditions (37 C, 28 days), Talcott et al. (2003) reported that while the initial juice preparation did not contain any appreciable L-ascorbic acid (,5 mg/L), fortification of juice with 450 mg/L L-ascorbic acid followed by pasteurization resulted in a 25% loss and a complete loss of L-ascorbic acid after 14 days under accelerated storage conditions. On the other hand, total polyphenolics were quite stable to pasteurization and accelerated storage with only a 6.7% increase over the initial 435 mg GAE/L (Table 7.1) upon pasteurization, followed by decreases after 14 and 28 days of accelerated storage to levels similar to the unpasteurized control, 422 mg GAE/L. Total carotenoids at each wavelength monitored (378, 401, 427 and 470 nm) were not affected by pasteurization, but did exhibit decreases ranging from 14.8% at 378 nm to a maximum of 41.6% at 427 nm, after 28 days of accelerated storage, with the greatest losses occurring after 14 days with nonsignificant changes by day 28 (Talcott et al., 2003). The ORAC antioxidant activity of the yellow passion fruit juice exhibited a 14% increase, as above, upon pasteurization associated with the relative stability of hydrophilic polyphenolics and other aqueous compounds, since the ORAC antioxidant activity of the hydrophilic fraction did not significantly change with pasteurization or accelerated storage conditions over 28 days. On the other hand, the lipophilic fraction of the yellow passion fruit juice experienced a 22.9% increase upon pasteurization which remained unchanged during accelerated storage over 28 days. Thus, these workers attributed the increased overall ORAC antioxidant activity of the yellow passion fruit juice upon pasteurization to lipophilic compounds, namely the carotenoid and xanthophylls constituents of the juice.

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7.3 CONCLUSION The fruit juice industry has greatly diversified product lines comprising different cultivars of apples and oranges, exotic fruits, and superfruit berries in juices, nectars, and cocktails in order to reach a wider consumer demographic. Cloudy and clear juices from dessert and cider apples, juices and nectars from anthocyanin-rich berries (e.g., cranberry, blueberry, black currant, and strawberry), juices from many citrus fruit species and cultivars, grapes, as well as juices from exotic fruit such as pomegranates and passion fruit have demonstrated significant SET and HAT antioxidant activities in vitro. These antioxidant activities could then be attributed to specific groups of polyphenols in the juices including total polyphenols, total flavonoids, anthocyanins and proanthocyanidins. Moreover, much of the antioxidant activity was retained during pasteurization, refrigeration, and accelerated storage studies, as well as in vitro digestion. Of key importance to the potential for these juices to positively impact the antioxidant status or oxidative stress in vivo, the absorption, metabolism, and excretion of parent polyphenols and metabolites was demonstrated for various berry, citrus, grape, and pomegranate juices. Moreover, positive impacts on serum, LDL, NIDDM and atherogenic risk factors could be demonstrated from consumption of juices from apple cultivars, cranberry, citrus cultivars, and pomegranates. On the other hand, parts of the fruit extracted; natural variation associated with different crop years, climate differences, growing conditions, country of origin; as well as processing methods (i.e., pasteurization, hydrostatic pressure) all have an effect on the polyphenol and other small molecule profile and content of juices, and thereby antioxidant activities.

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FURTHER READING S´anchez-Moreno, C., Cano, M.P., de Ancos, B., Plaza, L., Olmedilla, B., Granado, F., et al., 2003. Effect of orange juice intake on vitamin C concentrations and biomarkers of antioxidant status in humans. Am. J. Clin. Nutr. 78 (3), 454
460.