Potential Health Benefits of Citrus - American Chemical Society

Chapter 12

Antioxidant Effects of Citrus Flavonoid Consumption John R. Burgess and Juan E . Andrade

Downloaded by UNIV OF AUCKLAND on May 5, 2015 | http://pubs.acs.org Publication Date: June 9, 2006 | doi: 10.1021/bk-2006-0936.ch012

Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907-2059

Grapefruit juice is a rich source of nutrients and citrus flavonoids which are proposed to provide important health benefits. Many flavonoids are good antioxidants in vitro and this property is one that is hypothesized to mediate their biological effects in vivo. In two studies we tested the effects of citrus flavanones and grapefruit juice on lipid oxidation and the antioxidant defense system in vivo. The first tested the dose-response effect of naringenin consumption in rats adequate or deficient in vitamin E and selenium on growth and multiple measures of antioxidant defense, lipid oxidation and oxidative stress. The second was a human intervention trial of three months in duration testing the effects of regular consumption of grapefruit juice on antioxidant nutrient status and oxidative stress in young adult women. No evidence of antioxidant or pro-oxidant activity was observed, however, in rats inhibition of adipose accumulation was observed.

© 2006 American Chemical Society

In Potential Health Benefits of Citrus; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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162 Our work has focused on understanding adaptations to oxidative stress involving the endogenous antioxidant defense system and the potential role of non-nutrient phytochemicals in modulating this system. The results of two studies testing the effect of citrus phytochemicals on this system will be presented. This chapter will include: i) a brief summary of the multifaceted antioxidant defense system with a focus on the enzyme and antioxidants of interest to our lab, ii) a discussion of the evidence that flavonoids are good antioxidants in vitro, iii) a reminder of the extensive metabolism of phytochemicals in small intestine and liver, iv) a summary of the results of our rat feeding study, v) followed by a discussion of the results of the intervention study that we conducted with grapefruit juice in humans. The chapter will conclude with thoughts on lessons learned for future research. The multifaceted endogenous antioxidant defense system in mammals includes primary antioxidants that help break the chain reaction initiated with the formation of reactive oxygen species (ROS) (1). These include both dietary antioxidants as well as endogenously synthesized compounds. Secondary antioxidants include enzymes which detoxify ROS and those that help support the function of the primary antioxidants. The defense system also includes enzyme systems that repair the damage caused by excessive ROS such as lipases like phospholipase (PL) A which catalyzes the removal of oxidized lipids from the membrane, as well as regenerating systems that form replacement molecules. Finally, a robust antioxidant defense system which is found in healthy organisms exhibits the ability to adapt, up-regulate key components of its system to protect itself in the face of greater oxidative stress. Figure 1 illustrates the multifaceted nature of the antioxidant defense system in the plasma membrane. The interaction of a ROS with polyunsaturated fatty acids (PUFA) in the membrance can lead to the initiation of lipid peroxidation forming a lipid peroxyl radical esterified in the membrane (LOO). If not removed this chemical species can propagate the chain reaction, but alternatively it can be reduced by an electron supplied by tocopherol or ubiquinol. Regeneration of these fat-soluble antioxidants can take place via the action of quinone reductases (QR) or ascorbate (ASC). The resulting lipid hydroperoxide can be released from the phosholipid membrane via PLA , and after diffusing to the soluble portion of the cell be reduced by peroxidases utilizing reducing equivalents supplied by glutathione (GSH). Coupled interactions with GSHreductase and glucose-6-P dehydrogenase (G6PDH) help maintain a constant supply of electrons from NADPH to fully support the system (2,3). We use a dietary deficiency of vitamin E and selenium in rats as a model to study the components of this system which will upregulate to defend the cells of the animals from oxidative stress. In this model we have seen significant enhancements in QR, G6PDH, PLA as well as ASC and GSH synthesis (5-5). 2

2

2



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Figure 1. The multifaceted endogenous antioxidant defense system. Abbreviations: radical form (•), endogenous free radical (En-X*), exogenous free radical (Ex-X ), ascorbate (ASC), vitamin E (vit E), coenzyme Q (CoQ), phospholipases A (PLAJ, fatty acid hydroperoxide (LOOH), cytochrome b reductase (Cyt b reductase) quinone reductase (QR), glutathione peroxidase (GSH-Px), fatty acid alcohol (LOH), glutathione (GSH), glutathione disulfide (GSSG), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6PDH), 6-phospho-gluconate (6-P-gluconate), glucose-6-phosphate (Glucose-6P), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP). 0

2

5


5


164 A key question for understanding the potential health benefits of nonnutrients substances in foods is what effects do they have on the multifaceted endogenous antioxidant defense system? We know that fruit and vegetable intake is associated with lower incidence of chronic disease, that the essential nutrients in these fruit and vegetables can't account for all of the benefits observed, that flavonoids are proposed to contribute to these beneficial effects (6), and that these natural compounds in plants are good antioxidants in the test tube. The proposed antioxidant mechanisms forflavonoidsare: peroxyl radical scavenging, chelation of transition metals, and enhancements of the endogenous antioxidant defense system (7). The first two mechanisms have been shown to occur in vitro and are hypothesized to occur in vivo. The third mechanism is proposed to occur in vivo (8). As outlined by Williams et al. (9), and using quercetin, the most abundant flavonoid in foods, as a model, the structural characteristics that confer the strongest antioxidant properties for flavonoids in vitro are: viscinal diol on the B-ring, 2,3-unsaturation on the C ring, as well as the 4-oxo group (Figure 2). To illustrate this point Table 1 shows the results of a total peroxyl radical antioxidant potential (TRAP) assay represented in lag time in which equimolar concentrations of a variety of flavonoids were evaluated in vitro. The assay utilizes the azo-dye initiator AAPH and the chemical dye dichloroflourescein diacetate as the visualization agent (JO). Note Trolox, a water-soluble form of vitamin E used as a standard in these types of assays- illustrates a lag time of about 500 seconds. Epigallocatechin gallate (EGCG) which contains an additional hydroxyl group on the B-ring as well as a gallic acid moiety is a stronger antioxidant than quercetin, but the citrusflavanoneswhich have fewer functional hydroxyl groups on the B ring and lack the 2,3 unsaturation on the C ring are less strong. Although many flavonoids are good antioxidants in vitro whether this property mediates their health benefits in vivo is debated because of their known extensive metabolism. Overall absorption of flavonoids is estimated to vary considerably based on the amounts excreted in the urine (//). Contributing to this variation is the well-documented metabolism of the flavonoid structure which can occur via first-pass metabolism, by the colonic bacteria, and via the liver (12,13). These modifications include: removal of the sugar (most naturally occurring forms are glycosylated); conjugation of hydroxyl groups with glucuronic acid, sulfate, or glycine; O-methylation; and ring opening via colonic bacteria. Thus, for mostflavonoidsvery little of the parent compound is found intact in blood or tissues. So the question that we wanted to address was whether flavonoids act through enhancing the endogenous antioxidant defense system. We used the established model of vitamin E and Se deficiency that shows strong adaptive properties. We also had previously shown that a synthetic antioxidant (tBHQ at


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Table 1. Total Peroxyl Radical Antioxidant Potential (TRAP) In vitro Determination of Dietary Flavonoids and t-BHQ


Treatment

N

Lag Time (s) c

SE

Trolox

24

498

EGGC

4

973°

39

Quercetin

4

679*

19

(-)Epicatechin

4

403

d

35 12

t-BHQ

4

380

d

Naringenin

4

321

e

Hesperetin

4

268 '

45

23 6

NOTE: Results are expressed as mean lag times (s, seconds) and standard errors (SE). Means with different (a, b, c, d, e) superscript letters are significantly different after One-way ANOVA and post-hoc testing with Tukey (P

Potential Health Benefits of Citrus - American Chemical Society

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