Flavonoid C-glycosides in Citrus Juices from Southern Italy

Chapter 9

Flavonoid C-glycosides in Citrus Juices from Southern Italy: Distribution and Influence on the Antioxidant Activity Davide Barreca, Ersilia Bellocco, Ugo Leuzzi, and Giuseppe Gattuso* Dipartimento di Scienze Chimiche, Università di Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy *E-mail: [email protected].

Flavonoid C-glycosides are key constituents of the flavonoid fraction of Citrus juices. By drawing on the results presented over the past few years by our research group, a survey on the presence of these derivatives in the juices from eleven different Citrus species cultivated in Southern Italy shows that, so far, 12 individual derivatives have been identified and quantified, in variable amounts ranging from traces to 30–60 mg/L in the richer juices (C. sinensis, C. bergamia and C. medica). In addition, and in-depth study on C. reticulata × C. paradisi juice led to a critical analysis of the role these flavone derivatives play in the antioxidant activity of fresh Citrus juices.

Introduction Der Mensch ist, was er ißt, or man is what he eats (L. A. Feuerbach (1)). Over the past few years, growing consumer awareness about the beneficial effects of a healthy diet has determined a very fast market expansion for dietary supplements and functional foods. Research on nutraceuticals has been the driving force behind this boom, providing evidence on the health-improving action of nutritious food, as well as relentlessly pursuing hitherto unexploited dietary sources. Many supplements and functional foods have been prepared taking advantage, as the key ingredient, of citrus or citrus-derived products, being Citrus species among the best-known natural sources of health-beneficial compounds (2–6). In particular, aside from ascorbic acids, citrus fruits are extremely rich in flavonoids, a broad group of polyphenolic compounds whose radical scavenging © 2014 American Chemical Society

(7), chronic diseases-preventing (8, 9) abilities and biological activity (10–12) have been largely demonstrated. Furthermore, many studies highlighted their antiviral (13), antimicrobial (14, 15), and anti-inflammatory (16), antiulcer (17) and antiallergenic (18) properties. Citrus juices are generally characterized by the presence of several flavonoid components, belonging to the flavanone, flavone, flavonol, flavanol and dihydrochalcone subclasses. However, the general trend observed showed that flavanone derivatives are usually the most abundant individual components, whereas flavones are the ones that are seen with the largest variability. Flavonoids in Citrus juices are seldom found as their aglycones. Being the juice an aqueous environment, they often occur as glycosylated derivatives, whereas the less polar aglycones, along with the equally lipophilic polymethoxyflavones, are located in the essential oil vesicles in the flavedo layer of the peel. So far, the vast majority of the glycosylated flavonoids identified in the juices bear, as saccharide substituents, only a very limited set of hexoses, namely D-glucose and L-rhamnose, either as monosaccharides or as the disaccharides rutinose and neohesperidose (α-(1→6)-L-rhamnopyranosyl-β-D-glucopyranose and α-(1→2)-L-rhamnopyranosyl-β-D-glucopyranose, respectively), both composed of glucose and rhamnose, but differing in the position of the interglycosidic linkage. Furthermore, the saccharide substituents may be found linked to the alglycone skeleton either as O-glycosides or as the less conventional C-glycosides. The latter derivatives possess significantly different chemical properties than their O-linked counterparts, especially (vide infra) in terms of their resistance to acidic hydrolysis. Interestingly, with very few notable exceptions, in Citrus C-glycosyl substituents are found only on flavone aglycones. Even though C-glycosides are the main flavonoid components in other plant species (e.g., millet), they have enjoyed less attention than the better-known Oglycosides. Different classes of flavonoids (and their derivatives) have often a different functions, and C-glycosyl flavonoids have been shown to play, in vivo, the role of insect feeding attractants, antimicrobial agents and UV-protective pigments (19). In vitro, on the other hand, they can prevent tissue oxidation, and cancer development, but they have been also suspected of preventing thyroidal iodine uptake (19). Within this frame, there is surely still room for further investigations, both on the natural plant sources of C-glycosyl flavonoids, and the activity of these interesting compounds.

Results and Discussion C-Glycosyl Flavonoids in Citrus Juices We have been involved, in the past decade, in a long-term project aimed at characterizing the flavonoid and furocoumarin fraction of the juice of fruits from Citrus species grown in Southern Italy (Sicily and Calabria regions (20),). To this end, we have optimized a reverse-phase HPLC-DAD-ESI-MS-MS analytical protocol that allows us to identify and quantify flavonoid components 190

in a single chromatographic course (21). Fresh juice samples are prepared by hand-squeezing fruits that were preliminary peeled to avoid contamination by the lipophilic components present in the flavedo and albedo sections. The juice is then centrifuged, and the supernatant is diluted with DMF (1:1) prior to injection, thus avoiding any preliminary extraction procedure. Analytes separation, carried out on a C-18 column with a water/acetonitrile gradient, takes advantage of simultaneous DAD detection at 278, 310 and 325 nm, that allows for a first discrimination between flavones, flavanones and furocoumarins. In fact, at 278 nm both flavones and flavanones have a strong – and comparable in intensuty – absorption band (the so-called ‘band II’), while at 325 flavones have a much stronger ‘band I’ absorption than the one observed for flavanones. In such a manner, comparison of the two chromatograms provides immediate indication on the nature of the aglycone of the various flavonoid derivatives. In addition, detection at 310 nm helps in identifying the possible presence of furocoumarins components. The nature of the glycosidic linkage can be assessed by parallel acidic digestion of the juice (22). Treatment of the juice sample with 6M HCl in methanol/water mixture at 90°C for two hours results in the complete hydrolysis of the O-linked saccharide substituent, whereas C-linked saccharides do not react under these experimental conditions. Inspection of the hydrolyzed juice by HPLC, and comparison of the resulting chromatogram with the one obtained for the fresh sample, allows for the unambiguous assignment of a C-glycosidic bond to those compounds whose peak has not disappeared. Definitive structural elucidation is carried out by means of ESI-MS-MS. MS2 techniques have proved to be a powerful and convenient technique in flavonoid identification. Careful analysis of the fragmentation pattern of the analytes provides evidence on the nature of the aglycone, the nature and number of saccharides bound to the aglycone, the position of the substituents on the aglycone core. Moreover, it provides unambiguous evidence on whether the sugar units are present as mono- or disaccharides and, in the latter case, the position of interglycosidic bond. This subject has been discussed in detail in previous publications by our group (3), and has been extensively reviewed (23). Quantification of the flavonoid component is obtained by means of selected reaction monitoring (SRM) (24). In the course of our studies, we carried out in-depth investigation on eleven different Citrus species (Table I). Flavonoid C-glycosides have been found in all the species investigated (collectively shown in Figure 1) and with the exception of phloretin 3′,5′-di-C-glucoside 12 – which is a dihydrochalcone – they were assigned a flavone skeleton. On reverse-phase columns, they generally elute with shorter retention times than their corresponding O-glycosides. Furthermore, as it may be expected di-C-glycosyl flavones elutes earlier than the corresponding mono-C-glycosyl flavones. As for the latter, 8-C-glycosyl flavones have usually shorter retention times than the corresponding 6-C-glycosyl derivatives. Of all the species investigated, C. bergamia (27, 28), C. sinensis (21, 25), C. limon (22) and C. medica (22) were found to be the richest in C-glucosyl flavones, with bergamot presenting also the highest variety of individual compounds (Table II). In addition, at first glance two compounds stand out 191

as being the main C-glucosides in most of the species investigated, namely, apigenin-6,8-di-C-glucoside (vicenin-2, 2) and diosmetin-6,8-di-C-glucoside (lucenin-2 4′-O-methyl ether, 3). C. bergamia was found to be very rich in both vicenin-2 2 and lucenin-2 4′-O-methyl ether 3, containing in the three cultivars examined, variable amounts of 38.0–58.4 mg/L for the former, and 22.1–66.8 mg/L for the latter, with the "Femminello" variety being the one with the highest content. The other components are present in lower amount, ranging from the remarkable 5.1–10.1 mg/L determined for scoparin 6 to the small amounts observed for stellarin-2 (chrysoeriol 6,8-di-C-glucoside, 4, 0.5–1.3 mg/L).

Table I. Citrus species Investigated Binomial name

Cultivar(s)

Common name(s)

Ref.

C. sinensis

Moro Tarocco

blood orange

(21, 25)

tangelo, mapo

(26)

bergamot

(27, 28)

C. limetta

Mediterranean sweet lemon, limetta

(29)

C. japonica

kumquat

(30)

C. aurantium

sour orange, bitter orange

(24)

C. myrtifolia

myrtle-leaved orange, chinotto

(31)

C. reticulata × C. paradisi C. bergamia

Femminello Fantastico Castagnaro

C. limon

Femminello Interdonato Monachello

lemon

(22)

C. medica

Diamante

citron

(22)

C. reticulata

tangerine

(22)

C. deliciosa

clementine

(22)

A fairly different picture emerges for C. sinensis (Moro and Tarocco varieties) (21, 25). In this case, along with vicenin-2 2 (37.0–53.0 mg/L), stellarin-2 4 was found to be present in high amount (22.13±0.88 mg/L), with lucenin-2 1 (10.48±0.56 mg/L) and scoparin 6 (7.14±0.88 mg/L) following closely.

192

Figure 1. Flavone-C-glycosides found in the juice of Citrus spp. investigated. Trivial names are indicated in parentheses. In the case of C. medica and C. limon (citron and lemon (22)), lucenin-2 4′O-methyl ether 3 by far surpasses vicenin-2 2, with 36–60 mg/L vs. 9–16 mg/L in lemon, and 61–68 mg/L vs. 6–8 mg/L in citron. On the contrary, in the juice of C. reticulata and C. clementina it is vicenin-2 2 that is more abundant than lucenin-2 4′-O-methyl ether 3, with 23–27 mg/L vs. 6–8 mg/L for the former, and 4–6 mg/L vs. 1–3 mg/L for the latter. 193

Table II. C-Glycosyl flavonoids (1–12, Figure 1) found in the juice of investigated Citrus spp. (mg/L)

194

C. sinensis

C. reticulata × C. paradisi

C. limetta

C. japonica

C. aurantium

C. myrtifolia

C. bergamia

C. limon

C. medica

C. reticulata

C. clementina

1

10.48±0.56

0.10±0.012

n.o.

n.o.

0.12±0.05

n.o.

2.1–3.5(b)

–

–

–

–

2

37.0–53.0(a)

3.89±0.111

0.37±0.02

tr.

1.54±0.21

0.58±0.04

38.0–58.4(b)

9–16(c)

6–8

23–27

4–6

3

2.0–11.7(a)

n.o.

0.75±0.05

tr.

0.45±0.03

0.20±0.02

22.1–66.8(b) 36–60(c)

61–68

6–8

1–3

4

22.13±2.27

n.o.

n.o.

n.o.

n.o.

n.o.

0.5–1.3(b)

–

–

–

–

5

n.o.

n.o.

n.o.

n.o.

n.o.

n.o.

2.1–3.2(b)

–

–

–

–

6

7.14±0.88

n.o.

0.10±0.03

n.o.

n.o.

n.o.

5.1–10.1(b)

–

–

–

–

7

n.o.

n.o.

0.10±0.04

n.o.

n.o.

n.o.

1.3–4.8(b)

–

–

–

–

8

n.o.

n.o.

n.o.

tr.

n.o.

n.o.

n.o.

–

–

–

–

9

n.o.

n.o.

n.o.

0.26±0.01

n.o.

n.o.

n.o.

–

–

–

–

10

n.o.

n.o.

n.o.

0.60±0.03

n.o.

n.o.

n.o.

–

–

–

–

11

n.o.

n.o.

n.o.

0.70±0.05

n.o.

n.o.

n.o.

–

–

–

–

12

n.o.

n.o.

n.o.

19.94±0.40

n.o.

n.o.

n.o.

–

–

–

–

Range determined for Tarocco and Moro cultivars. (b) Range determined for Femminello, Fantastico and Castagnaro cultivars. (c) Range determined for Femminello comune, Inerdonato and Monachello cultivars. n.o.: not observed; fields marked as "–" refer to compounds not investigated.

(a)

Data in our hands lend themselves to critical analysis. It has been often stressed that the flavonoid chromatographic profile of a given Citrus juice can be used both as a fingerprint to identify the species from with it originates and/or potential adulteration, and as a tool to reveal (or confirm) taxonomical relations between different species. It is interesting to observe that, even restricting the analysis to the C-glucosyl flavones, few clear connection become evident. Lemon and citron, which are known to be related (32), share a similar profile, with the diosmetin derivative lucenin-2 4′-O-methyl ether 3 as the main compound. C. limetta (29), which is also related to these two species, has a similar profile, albeit the amount of C-glucosyl flavones present in the juice is so much lower that it is not safe to speculate – basing only on these data – on its taxonomic connection. In a similar fashion, orange, tangerine (22) and clementine (22) all display the apigenin derivative vicenin-2 2 as their main flavone C-glucoside component. Bergamot, which is a hybrid of citron and sour orange, presents – even if in larger amount than in the parent species – both the C-glucosides that characterize the two species, lucenin-2 4′-O-methyl ether 3 and vicenin-2 2, respectively. Myrtleleaved orange, which is a mutation of sour orange, possesses a C-glucosyl flavone profile that is almost superimposable to that of its parent species, C. aurantium. C. japonica (kumquat (30, 33)) possesses a totally different set of C-glycosyl derivatives. The most prominent compound is not a flavone, but rather a dihydrochalcone, namely phloretin 3′,5′-di-C-glucoside. This is a rather unique compound in the Citrus genus, setting kumquat apart from the rest of the commonly grown species. In addition, a wide variety of mono- and di-C-glucosyl flavones was detected in kumquat juice, albeit in low amount (typically, < 1 mg/L). Among these, derivatives that seldom are seen in the Citrus genus were identified, such as the acacetin 6- or 8-C-neohesperidosides. In light of its peculiarity, it is not surprising that for about a century kumquat varieties had been allotted a separate genus, Fortunella (34).

Antioxidant Activity Studies The antioxidant activity of Citrus juices descends from the concerted action of a wide variety of compounds able to quench ‘free radicals’ with different mechanism and efficiency. Best known among these are vitamin C and the broad family of the flavonoids. As mentioned above, C-glucosyl flavones have not been studied intensively for their antioxidant activity. In order to shed light on the contribution this subclass of compounds provides to the radical scavenging and reducing activity of the juice, we decided to turn to preparative HPLC to separate the various flavonoid subclasses. Tangelo (C. reticulata × C. paradisi) juice (26) was selected as a good candidate, owing to the good variety displayed by its juice. In fact, it was shown to contain C-glucosyl flavones (lucenin-2 1 and vicenin-2 2), an O-rutinosyl flavonol (rutin), a number of di- and tri-O-glycosyl flavanones (neoriocitrin, narirutin, neohesperidin, didymin and narirutin 4′-O-glucoside) and polymethoxyflavones (sinensetin, nobiletin and tangeretin), thus allowing to collect a significant set of representative compounds. 195

Six broad fractions were collected (Figure 2): the first one containing all the flavonoids (henceforth referred to as FP, flavonoid pool fraction), and five additional ones containing each all the members of a single flavonoid subclass, that is di-C-glucosyl flavones (fraction I), O-triglycosyl flavanones (II), O-diglycosyl flavonols (III), O-diglycosyl flavanones (IV) and polymethoxyflavones (V) (26). The fractions were collected, evaporated to dryness, and then redissolved in the stipulated amount of solvent needed to restore the original concentration the analytes had in fresh juice.

Figure 2. Fractions collected from tangelo juice by preparative RP-HPLC.

The six fractions were subjected to DPPH• (2,2-diphenyl-1-picrylhydrazyl), ABTS•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and HO• radical scavenging assays, as well as to ferric reducing antioxidant power (FRAP) assessment, and compared to the results obtained for the crude juice (Figure 3). It is evident, from the data presented, that the different subclasses contribute to different extents to the activity of the juice. In the case of DPPH• quenching, the flavonoid pool accounts for ca. 50% percent of the activity of crude juice (CJ, 5.85 μM Trolox [6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid] Equivalents). Within the flavonoid pool, the C-glucosyl flavones (fraction I), the O-diglycosyl flavonols (III) and the O-diglycosyl flavanones (IV) were the subclasses responsible for the activity, whereas the remaining two (i.e., II and V) displayed little or no activity at all (26). A similar trend was observed for the ABTS•+ radical cation quenching, although in this case most of the activity of the crude juice (14.80 mM TE) could be ascribed to the flavonoid pool (ca. 80%). Again, it was fractions I, III and IV that contributed the most to the activity measured for the FB fraction (26). 196

Tangelo juice turned out also an excellent hydroxyl radical scavenger (17.20 mM TE). In this latter case, the FP fraction was found to be responsible for two thirds of the activity (ca. 65%), with again the same trend observed for the different subclasses (i.e., I, III, IV >> II, V). As for the reducing activity, the juice was found to be very efficient in the FRAP assay, but the flavonoid pool appeared not to play a key role in this process (FP = ca. 25% of CJ). Still, the C-glucosyl flavones were found to be the most active among the five subclass fractions (ca. 50% of CJ) (26).

Figure 3. Radical scavenging and reducing activity of the fraction from tangelo juice (data from ref. (26)). CJ: crude juice; FP: flavonoid pool; I: di-C-glucosyl flavones; II: O-triglycosyl flavanones; III: O-diglycosyl flavonols; IV: O-diglycosyl flavanones; V: polymethoxyflavones.

These results allowed us to draw some conclusions. Firstly, it was made evident the polymethoxyflavones and O-triglycosyl flavanones possess a fairly limited efficiency in the in vitro radical scavenging processes employed in this study. This should not come as a surprise, given that DPPH•, ABTS•+ and HO• radical quenching rely on H• radical transfer (HAT mechanism (35),), and both these subclasses do not possess highly reactive free phenolic OH groups. A second interesting point came from the analysis of the activity of the other three classes as a function of their abundance in tangelo juice (I+III: ~4 mg/L and IV ~30 mg/L, respectively (26)). The evidence that even though O-diglycosyl flavanones are significantly more abundant than the flavones and the flavonols but display an overall similar activity, demonstrates that the derivatives that bear a double bond in the 2,3-position of the central pyrone C ring of the aglycone are the ones that exert the highest antioxidant activity. In fact, the presence of such double bound 197

determines a very high degree of conjugation within the flavonoid skeleton, with the consequence that the ArO• radicals generated upon H• transfer enjoy a much higher delocalization and therefore stabilization.

Conclusions The consumption of fresh and processed Citrus products has been shown time and again to be a staple of healthy diets, owing to the abundance in compounds that positively influence well-being and disease prevention. Among the many derivatives that play such a role (i.e. ascorbic acid, flavonoids, anthocyanins, carotenoids, etc.), flavonoid C-glycosides are being studied for their remarkable antioxidant activity. Data discussed in the present chapter, collected over the years on Citrus species grown in Southern Italy, demonstrate that indeed they are among the most efficient radical scavengers within the flavonoid family, deserving more attention and further investigation in light of their potential exploitation in food and food supplement industry.

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