**2.1. Lycopene**

which are nonoxygenated molecules such as lycopene and β-carotene; (2) xanthophylls, which are oxygen-containing molecules such as astaxanthin and fucoxanthin (**Figure 1**) [3]. The daily consumption of carotenoid-rich foods would be beneficial for human health because of their high antioxidant, anticancer, and antiatherosclerotic activities [4–6]. Because carotenoids contain numerous conjugated double bonds, many kinds of geometrical isomers are theoretically possible (**Figure 1C**, **E** and **F**). In general, carotenoids in plants occur predominantly in the (all-*E*)-configuration, whereas the *Z*-isomers are present in the human body and processed foods in considerable quantity, for example, over 50% of total lycopene is present as the *Z*-isomers in serum and tissues [7–9]. Data from several studies have shown that the *Z*-isomerization of carotenoids induced changes in important properties, such as the bioavailability, antioxidant activity, and anticancer activity [10–13]. However, these outcomes vary depending on the type of carotenoid: there were cases where the beneficial effects of carotenoids increased or reduced by the Z-isomerization [10–15]. For example, *Z*-isomers of lycopene and astaxanthin have higher bioavailability than the all-*E*-isomers [12, 16], whereas *Z*-isomers of β-carotene have lower bioavailability than the all-*E*-isomers [14]. Furthermore, the results may depend on the evaluation method used. For instance, when the antioxidant activity of β-carotene was evaluated based on oxidation of the low-density lipoprotein (LDL), the all-*E*-isomer showed higher antioxidant activity than the 9*Z*-isomer [17], whereas the 9*Z*-isomer showed higher antioxidant activity when evaluated based on antiperoxidative activity [18]. Moreover, the beneficial effects of carotenoids differ between the *Z*-isomers. For example, when the antioxidant activity of fucoxanthin was evaluated in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity assay, the order of activity was 13*Z*-isomer ≈ 13′*Z*-isomer > all-*E*-isomer > 9′*Z*-isomer [19]. The above findings indicate that a good understanding of the effects of *E*/*Z*-isomerization on functional changes is important for increasing the beneficial effects of carotenoid ingestion and for the industrial processing of carotenoids. The objective of this chapter is to highlight the impact of *E*/*Z*-isomerization of carotenoids on their bioavailabilities, antioxidant activities,

140 Progress in Carotenoid Research

**Figure 1.** Chemical structures of (A) (all-*E*)-lycopene, (B) (all-*E*)-β-carotene, (C) (all-*E*)-astaxanthin, (D) (all-*E*)-

fucoxanthin, (E) (9*Z*)-astaxanthin, and (F) (13*Z*)-astaxanthin.

Lycopene is an acyclic carotene (C40H56) that is principally responsible for the bright-red color found abundantly in vegetables and fruits such as tomatoes, guava, and watermelons [3, 9]. Lycopene shows an especially strong antioxidant activity among carotenoids [6] and can significantly reduce the risks for arteriosclerosis, atherogenesis, and many types of cancer (such as prostate and esophageal cancer) [4, 5]. Therefore, in recent years, the use of lycopene in health foods and supplements, and as a natural functional pigment has attracted attention. It is well documented that the bioavailability and antioxidant activity of lycopene are changed by *Z*-isomerization. Most previous findings have demonstrated that the *Z*-isomerization of lycopene results in "positive" health effects.

Data from both *in vitro* and *in vivo* tests have suggested that *Z*-isomers of lycopene are more bioavailable than the all-*E*-isomer. Testing conducted using a diffusion model [20], bile acid micelles [21, 22], human intestinal Caco-2 cells [23], and lymph-cannulated ferrets [21, 22] has provided strong evidence supporting the higher bioavailability of the *Z*-isomers. Moreover, in humans, the ingestion of foods rich in lycopene *Z*-isomers resulted in a measurable increase in blood lycopene concentrations compared to a sample abundant in the (all-*E*)-isomer [12, 24–27]. For example, Cooperstone et al. [12] investigated the effects of ingesting red tomato juice, which mainly contained (all-*E*)-lycopene (90% all-*E*-isomer) and *tangerine* tomato juice, which mainly contained *Z*-isomers of lycopene (94% *Z*-isomers), on plasma lycopene concentrations. Lycopene from the *tangerine* tomato juice showed approximately 8.5-fold greater bioavailability than lycopene from the red tomato juice. Unlu et al. [25] reported that when comparing two tomato sauces—one rich in all-*E*-lycopene (95% all-*E*-isomer) and the other rich in (*Z*)-lycopene (45% *Z*-isomers)—that the *Z*-isomer-rich tomato sauce was approximately 1.5 times more bioavailable than the all-*E*-isomer-rich sauce. In general, the uptake of carotenoids into intestinal mucosal cells is aided by the formation of bile acid micelles [21, 22, 24, 27]. Thus, it is believed that because lycopene *Z*-isomers are more soluble in bile acid micelles than the all-*E*-isomer, they are preferentially incorporated into enterocytes and efficiently form chylomicrons [21, 22]. Indeed, very recently, several reports showed that the solubility of lycopene in oils, organic solvents, and supercritical CO2 (SC-CO<sup>2</sup> ) was significantly improved by *Z*-isomerization [28–32]. However, Richelle et al. [33] showed by human oral-dosing tests that the (9*Z*)- and (13*Z*)-isomers were less efficiently absorbed than the 5*Z*- and all-*E* isomers or were converted into 5*Z*- and all-*E* isomers.

against various diseases such as cancer and atherogenesis [4, 17]. Furthermore, β-carotene is very important as a retinol precursor, with a high conversion rate [3, 4]. It is also well documented that the bioavailability and antioxidant activity of β-carotene as well as its antiatherogenic activity are changed by *Z*-isomerization. Most previous studies have shown that the *Z*-isomerization results in "negative" effect for bioavailability. In contrast, β-carotene *Z*-isomerization has shown both "positive" and "negative" effects on antioxidant activity, depending on the evaluation method, and "positive" effects have been shown in terms of anti-

Effects of *Z*-Isomerization on the Bioavailability and Functionality of Carotenoids: A Review

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Data from several *in vitro* and *in vivo* tests have indicated that *Z*-isomers of β-carotene are less bioavailable than the all-*E*-isomer. For example, *in vivo* tests using Caco-2 cells, HSC-T6 cells, and liver microsomes [14], as well as *in vivo* tests using ferrets [41] and gerbils [42] have shown this phenomenon. In humans, the intake of *Dunaliella salina* and *Dunaliella bardawil* rich in (9*Z*)-β-carotene showed lower β-carotene bioavailability than foods rich in the all-*E*-isomer [43–48]. The effects of the *Z*-isomer content on the bioavailability were opposite between lycopene and β-carotene. Generally, after carotenoids are extracted from the food matrix and incorporated into mixed micelles, bioaccessible carotenoids can be internalized by enterocytes [21, 22, 24, 27]. The main absorption site of carotenoids is in the duodenum, and several proteins that are temporarily present at the apical membrane mediate selectivity in terms of carotenoid uptake [27, 49–51]. *In vitro* experiments with Caco-2 cells showed that carotenoid transport decreased in the following order: β-carotene ≈ α-carotene (50% inhibition) > β-cryptoxanthin ≈ lycopene (20% inhibition) > lutein: zeaxanthin (1:1) (7% inhibition) [49]. Because carotenoid *Z*-isomers have higher solubility than the all-*E*-isomers [28–32], they can incorporate into bile acid micelles more efficiency [21, 22]. Therefore, it is considered that *Z*-isomers of β-carotene have lower transport efficiency in Caco-2 cell than the all-*E*-isomers [13]. However, a few studies have suggested that *Z*-isomers of β-carotene have higher bioavailability than the all-*E*-isomers, as evaluated using human intestinal Caco-2 cells [52] and ferrets [53]. The use of different delivery systems with the cell model system and animal

Several studies have been conducted to compare the antioxidant activities of (all-*E*)-β-carotene and the *Z*-isomers, and the degree of antioxidant activity detected varied according to the assay method. Namely, the 9*Z*-isomer showed higher antioxidant activity than the all-*E*-isomer when evaluated in terms of the sensitivity to external oxidants [54], the antiperoxidative activity [18], and oral dose testing in rats [55]. However, the opposite results (or no significant differences) were observed when the antioxidant activities were evaluated by measuring the oxidation of LDL [17] or in TEAC assay [10, 56], and PSC assay [56, 57]. Rodrigues et al. [57] reported that β-carotene *Z*-isomers were less efficient as peroxyl radical scavengers than the corresponding all-*E*-isomers: the *Z*-isomers presented the values about 20% lower than that found for the all-*E*-isomer, and they addressed that the negative effect may be due to the decreasing of the orbital overlap. Based on the above findings, it is difficult to conclude

whether antioxidant activity is enhanced by *Z*-isomerization of the all-*E*-isomer.

Moreover, as additional "positive" effects of β-carotene *Z*-isomers, it has been reported that the 9*Z*-isomer has higher antiatherogenic activity [58] and antiatherosclerotic activity

atherogenic activity.

species might have caused discordant results [15].

Several previous reports have shown that lycopene *Z*-isomers have higher antioxidant activity than the all-*E* isomer and that the relative activities of the isomers varied depending on the assay method [10, 11]. Böhm et al. [10] compared the antioxidant activity of (all-*E*)-lycopene with four unknown *Z*-isomers by measuring their abilities to reduce radical cations of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (TEAC assay), and each *Z*-isomer showed higher antioxidant activity than the all-*E*-isomer. Müller et al. [11] evaluated the antioxidant activities of (all-*E*)-, (5*Z*)-, (9*Z*)-, (13*Z*)-, and (7*Z*,9*Z*,7′*Z*,9′*Z*)-lycopene using four different *in vitro* assay, namely the TEAC assay, the ferric-reducing antioxidant power (FRAP) assay, the peroxyl radical-scavenging capacity (PSC) assay, and the heme-induced peroxidation of linoleic acid in mildly acidic emulsions mimicking postprandial lipid oxidation in the gastric compartment (MbFeIII-LP) assay. No significant changes were observed among the isomers in the TEAC and FRAP assay. However, the lycopene *Z*-isomers showed higher antioxidant activities than the all-*E* isomer in the PSC assay (9*Z*-isomer > 5*Z*-isomer ≈ 7*Z*,9*Z*,7′*Z*,9′*Z*-isomer > 13*Z*-isomer > all-*E*-isomer) and in the MbFeIII-LP assay (5*Z*-isomer > all-*E*-isomer ≈ 9*Z*-isomer ≈ 13*Z*-isomer ≈ 7*Z*,9*Z*,7′*Z*,9′*Z*-isomer). In TEAC assay, Böhm et al. [10] found that *Z*-isomers of lycopene had higher antioxidant activity, but Müller et al. [11] concluded that no significant differences occurred among the isomers. These discrepant findings may be explained by the fact that different concentrations of the isomers were used in each study [10, 11].

Based on the above findings, *Z*-isomerization effectively promotes the beneficial effects of lycopene. Among the *Z*-isomers, (5*Z*)-lycopene would have the highest bioavailability [33] and antioxidant activity [11]. Furthermore, the 5*Z*-isomer has the highest stability of the *Z*-isomers [34–36]. Therefore, regarding lycopene, it is very important to increase the 5*Z*-isomer level and its ingestion. As the *Z*-isomerization method to increase (5*Z*)-lycopene efficiently, heating in some alkyl halides [37] and some kinds of oils such as sesame oil [9, 38], light irradiation with photosensitizers [39], and catalytic treatment [20, 40] were effective. Moreover, to our best knowledge, because the effect of ingesting *Z*-isomer-rich lycopene on inhibiting the development of diseases such as atherogenesis and cancer has not been clarified, further research in that field is expected in the future.
