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Article

HPLC-UV-MS Profiles of Phenolic Compounds and Antioxidant Activity of Fruits from Three Citrus Species Consumed in Northern Chile

by
Anghel Brito
1,
Javier E. Ramirez
1,
Carlos Areche
2,
Beatriz Sepúlveda
3 and
Mario J. Simirgiotis
1,*
1
Laboratorio de Productos Naturales, Departamento de Química, Facultad de Ciencias Básicas, Universidad de Antofagasta, Av. Coloso S-N, Antofagasta 1240000, Chile
2
Departamento de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago 7800024, Chile
3
Departamento de Ciencias Químicas, Universidad Andrés Bello, Campus Viña del Mar, Quillota 980, Viña del Mar 2520000, Chile
*
Author to whom correspondence should be addressed.
Molecules 2014, 19(11), 17400-17421; https://doi.org/10.3390/molecules191117400
Submission received: 9 August 2014 / Revised: 12 September 2014 / Accepted: 22 September 2014 / Published: 29 October 2014
(This article belongs to the Special Issue Natural Antioxidants and Ageing)
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Versions Notes

Abstract

:
Peels and edible pulp from three species of citrus including Citrus aurantifolia (varieties pica and sutil) and Citrus x lemon var. Genova widely cultivated and consumed in Northern Chile (I and II region) were analyzed for phenolic compounds and antioxidant activity for the first time. A high performance electrospray ionization mass spectrometry (HPLC-UV-ESI-MS) method was developed for the rapid identification of phenolics in extracts from peels and juices of all species. Several flavonoids including one kaempferol-O-hexoside (peak 16) and one hesperidin derivative (peak 22) three quercetin derivatives (peaks 4, 19 and 36), five isorhamnetin derivatives (peaks 5, 23, 24, 26 and 29) four luteolin derivatives (peaks 14, 25, 27 and 40), seven apigenin derivatives (peaks 2, 3, 12, 20, 34, 35 and 39), seven diosmetin derivatives (peaks 7–9, 17, 21, 31 and 37), three chrysoeriol derivatives (peaks 10, 18 and 30), and four eryodictiol derivatives (peaks 6, 13, 15 and 38) were identified in negative and positive mode using full scan mass measurements and MSn fragmentations. Ascorbic acid content was higher in the pulps of the varieties Genova and Sutil (60.13 ± 1.28 and 56.53 ± 1.06 mg ascorbic acid per g dry weight, respectively) while total phenolic content was higher in Pica peels followed by Sutil peels (34.59 ± 0.81 and 25.58 ± 1.02 mg/g GAE dry weight, respectively). The antioxidant capacity was also higher for Pica peels (10.34 ± 1.23 µg/mL in the DPPH assay and 120.63 ± 2.45 µM trolox equivalents/g dry weight in the FRAP assay). The antioxidant features together with the high polyphenolic contents can support at least in part, the usage of the peel extracts as nutraceutical supplements, especially to be used as anti-ageing products.

Graphical Abstract

1. Introduction

The accumulation of oxidative damage due generally to intracellular ROS concentration has been proposed to be causal to ageing as defined by the Free radical Theory of Ageing, which has been subject to recent debate [1,2]. Fruits and vegetables are considered highly protective for human health against oxidative-stress related diseases and ageing [3]. There is evidence that a fruit extract with high vitamin C and polyphenol content was effective in decreasing intracellular ROS concentration and in protecting lipid, DNA and mitochondrial functionality from the damage induced by free radicals [4]. However, recent studies have demonstrated that the biological activities of polyphenols is not only related to their role as antioxidants but also to other pathways involved in cellular metabolism and cellular survival [5]. In this context and in the framework of the ageing process, citrus crops are one of the most important in the world, as producers of high vitamin C and polyphenolic content, and the fruits are among the most valuable functional diets, highly consumed globally in the form of fresh as well as processed juices and shown to lower oxidative stress-related diseases and ageing [6]. Furthermore, an extract of pummelo (Citrus maxima) was able to attenuate increased ROS levels in aged cells [7], while epidemiological studies reveal a strong correlation between high levels of citrus fruit consumption and low incidence of age-related diseases such as cardiovascular diseases and cancer [8]. Besides, citrus fruits showed cytoprotective action which is enhanced by the presence of antioxidants including vitamin C and flavonoids [9]. Citrus fruits are grown all over the world in more than 140 countries and production has increased enormously from an average of 48 million tons a year in the decade of 1970 to more than 100 million in 2005 [10]. Fruitpeels and seeds from lemon are major agro-industrial wastes in canned fruit manufacture and fruit juice. Peels are a source of molasses cold-pressed oils, flavonoids, pectin and also limonoids [11]. The flavonoids present in citrus pulps and peels are two main classes: the polymethoxylated flavones and the glycosylated flavanones [11,12,13,14]. These secondary metabolites exist in the form of flavanone glycosides mainly as diosmin, naringin, hesperidin and neohesperidin derivatives and possess anticonvulsion, antimutagenic, antiinflammatory, antioxidant, blood lipid lowering and anti-carcinogenic activities among others [8,12,15,16]. In Northern Chile (I and II region), the three cultivated and more widely consumed Citrus species are limón de Pica (Pica Lemon, Citrus aurantifolia (Christm) Swingle var Pica), a tart and tangy lime most famous for its use in Pisco Sour cocktails and produced mostly exclusively in the Town of Pica, which is located in an oasis in the Atacama desert, limón Sutil (Citrus aurantifolia (Christm) Swingle) var sutil and limón Genova (Citrus x limon (L.) Burm) var. Genova (Figure 1). In the last few years, several mixtures of phenolics have been analyzed with advanced LC-MS/MS equipment [17,18,19,20,21] including various publications dealing with the on-line HPLC-MS detection of flavonoids, phenolic acids and alkaloids in citrus fruits [12,13,14,15,22,23]. The advantage of HPLC-MS is its sensitivity and selectivity, and the ability to use tandem mass spectrometry for the observation of aglycones and diagnostic fragments [17,18]. As a continuation of our studies on antioxidant activity and phenolic composition of Chilean fruits [24,25], in the present work we have analyzed for the first time peels and edible pulps from three citrus varieties (pica, sutil and genova), highly consumed in northern Chile, for phenolic compounds using HPLC-UV-MS and compared their vitamin C content and antioxidant capacities. We demonstrate in this work that extracts from those citrus fruits have high polyphenolic content and antioxidant capacities and therefore could be useful as nutraceuticals or antioxidant supplements against oxidative stress and ageing.
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Figure 1. Pictures of Pica lemon (right), Sutil lemon (middle) and Genova lemon (left) from northern Chile.
Figure 1. Pictures of Pica lemon (right), Sutil lemon (middle) and Genova lemon (left) from northern Chile.
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2. Results and Discussion

2.1. Total Phenolic, Ascorbic Acid, Flavonoid Contents and Antioxidant Capacity of Citrus Extracts

In the present study, we assessed the polyphenolic profile of peels and pulps from citrus consumed in Chile, and evaluated their antioxidant capacity as well as the total phenolic, total flavonoid and vitamin C content by spectrophotometric methods. The fruits were collected from a local market in Antofagasta and the peels, pulp and seed-free juices were collected. The peels and pulps were extracted with methanol and the resulting extracts were processed by solid phase extraction. Antioxidant capacity of the extracts was measured and correlated with the total phenolic, flavonoid and ascorbic acid contents. Total flavonoid and phenolic contents were previously determined for several citrus [26] including non edible peels [10,27]. The methods for assessment of antioxidant effects were critically reviewed and the most important advantages and shortcomings of each method were highlighted because the capacity of antioxidants has been assessed by various methods, but they often give inconsistent and conflicting results since comparisons between laboratories are difficult [28,29]. Furthermore, several studies show that total phenolic compounds, ascorbic acid and antioxidant capacity strongly differed between genotypes of each individual fruit [30]. In this study antioxidant activity was determined by three in vitro commonly used assays, namely DPPH free radical scavenging assay, ferric reducing assay and superoxide anion scavenging activity (Figure 2), while ascorbic acid content and total phenolic content of fresh fruit juices were determined by volumetric and Folin-Ciocalteu reagent method respectively. We have employed these three different antioxidant assays plus the Folin-Ciocalteu method which is an electron transfer based assay and gives reducing capacity, for the quantification of the antioxidant capacities of the fruit extracts, since no single assay will accurately reflect all of the radical sources or all antioxidants in a mixed or complex plant extract. The total phenolic, vitamin C and flavonoid contents as well as the extraction yield and antioxidant capacity measured by the bleaching of DPPH radical and ferric reducing antioxidant power are given in Figure 2 and Table S1, (supplementary materials). Ascorbic acid was higher in C. limon var Genova while total phenolic content was higher in Pica peels (60.13 ± 1.28 mg/g ascorbic acid and 34.59 ± 0.81 mg/g GAE respectively, Figure 2). In the DPPH assay, the peels and pulp of Pica lemon exhibited stronger scavenging potential when compared to the other citrus varieties (Figure 2 and Table S1). TPC, TFC values and DPPH activity showed acceptable correlation (R2 = 0.624 and 0.587, respectively, Figures S1 and S2) as well as good correlation was observed between antioxidant assays (Figures S3–S5). Ferric reducing potential was also higher in pica peels and pulp, followed by Sutil peel and pulp. The total flavonoid content of Sutil lemon peel was close to that reported for a sample of Citrus limon (L.) Burm., from Taiwan (11.9 ± 0.66 mg/g) [27] while the total phenolic content of Genova pulp was similar to that reported for an alcoholic extract of peels from Citrus limon (L.) Burm., from Algeria (12.11 ± 0.62 mg GAE/g dry weight) [10]. The vitamin C content of the pica peel and pulp were higher than that reported for Tahiti lime (6.84 ± 0.3 and 41.4 ± 0.9 mg AA/100 g fresh weight, respectively) and sweet lime (22.6 ± 0.8 and 60.2 ± 2.2 mg AA/100 g fresh weight, respectively) [31]. However, in this assay both peel and pulp of Chilean varieties Genova and Sutil showed higher AA content than pica lemon (Table 1). Genova pulp showed values three times higher to that reported for citrus lemon from China (233.44 ± 2.52 mg AA per liter) [32]. Several articles have shown that harvest times and environmental parameters (light conditions, temperature, irrigation, fertilization or cultivation systems) can affect the antioxidant capacity in fruits [30,33], thus further studies using several different parameters and on the cultivation of Citrus in Northern Chile should be carried out to show the variation on the production and accumulation of bioactive compounds and antioxidant activities of these fruits.

2.2. Identification of Phenolic Constituents in Citrus Fruits by HPLC-UV and ToF-ESI-MS/MS

Phenolics occurring in pulp and peel extracts from three Citrus species consumed in northern Chile were separated by HPLC and UV/vis spectra were obtained using a diode-array detectorand electrospray mass spectrometry (ESI-MS) in full scan mode and tandem MSn fragmentations. HPLC fingerprints were generated (Figure 3 and Figures S6–S8) and phenolic compounds subsequently analyzed by ESI-MSn (Table 1).
Molecules 19 17400 g002 550
Figure 2. (a) Total Phenolic Content (TPC); (b) Total Flavonoid Content (TFC); (c) Ascorbic acid (AA) content; (d) Scavenging of the 1,1-diphenyl-2-picrylhydrazyl Radical (DPPH); (e) Ferric Reducing Antioxidant Power (FRAP) and (f) Superoxide Anion scavenging activity (SA), of peels and pulp of three citrus edible species from Chile.
Figure 2. (a) Total Phenolic Content (TPC); (b) Total Flavonoid Content (TFC); (c) Ascorbic acid (AA) content; (d) Scavenging of the 1,1-diphenyl-2-picrylhydrazyl Radical (DPPH); (e) Ferric Reducing Antioxidant Power (FRAP) and (f) Superoxide Anion scavenging activity (SA), of peels and pulp of three citrus edible species from Chile.
Molecules 19 17400 g002
Abreviations: ppe: pica peel, ppu: pica pulp, supe: sutil peel, supu: sutil pulp, gepe: genova peel; gepu: genova pulp, GA: gallic acid. Total phenolic content (a) expressed as mg gallic acid equivalents (GAE)/g dry weight; Total flavonoid content (b) expressed as mg quercetin/g dry weight; Ascorbic acid content (AA) (c) expressed as mg vitamin C/g dry weight; Antiradical DPPH activities (d) are expressed as IC50 in µg/mL for extracts and compounds; Ferric reducing power (FRAP) (e) expressed as µM trolox equivalents/g dry weight; Superoxide anion scavenging activity (SA) (f) expressed in percentage scavenging of SA at 100 µg/mL.
Table
Table 1. Identification of phenolic compounds in Citrus fruits and aerial parts by LC-UV, LC-MS and MS/MS data.
Table 1. Identification of phenolic compounds in Citrus fruits and aerial parts by LC-UV, LC-MS and MS/MS data.
PeakRt (min)HPLC DAD λ Max (nm)[M−H]−/[M+H]+ (m/z)MS2 Ions (m/z)MSn Ions (m/z)[2M−H]− or [2M+H]+ (m/z)Tentative IdentificationSpecies/Plant Part
13.5-191145129, 111 Citric acidPpe, gpe, gpu, spe, spu
24.2268, 334739431 (M-neohesperidose)311 (431–120) Apigenin 7-O-neohesperidoside-6-C-glucosideSpu
317.5268, 334577/579431269, 225 Apigenin 7-O-neohesperidoside (Rhoifolin)Supu
49.0255, 354771/773609 (rutin)463, 301, 179, 151 Quercetin 7-O-glucoside-3-O-rutinosideSupu
510.1250, 268, 342639315 (M-di-glucose)301, 179, 151 Isorhamnetin-3-O-di-glucosideSupu
610.9285, 325757595595, 449, 287 Eriodictyol-4'-O-neohesperidoside-7-O-glucosideSpu, spe, ppe
712.0250, 268, 342623/625503(M-120)383, 3121247Diosmetin 6,8-di-C-glucoside (Lucenin-2,4'-methyl ether)Ppe
812.2250, 268, 342607/609563299 (Diosmetin), 284 Diosmetin 7-O-neohesperidoside (Neodiosmin)Ppe
914.1250, 268, 342607/609563299 (Diosmetin), 284 Diosmetin 7-O-neohesperidosidePpe
1016.3 623/625503(M-120)383, 3121247Chrysoeriol 6,8-di-C-glucoside (Stellarin-2)Gpe
1116.2 357/359194151 3-(2-hydroxy-4-methoxyphenyl)-propanoic acid hexosePpu, gpe, gpu, spe, ppe
1218.5268, 334577/579431269, 225, 201 Apigenin 7-O-rutinoside (Isorhoifolin)Ppe, ppu, gpe, gpu, spe
1331.5285, 325595449 (M-rhamnose)287 (M-neohesperidose) Eriodictyol-7-O-neohesperidosideSpu, spe, gpu, ppu
1419.4254, 267593/595285/287 (Luteolin)241, 1751187/1189Luteolin 7-O-rutinosidePpe, gpe
1519.1285, 325595/597287/289 (eriodictyol)151, 135, 1071189/1191Eriocitrin (Eriodictyol-7-O-rutinose)Ppe, ppu, gpe, gpu, spe
1623.5250, 268, 342607/609563299 (Diosmetin), 284 Diosmetin-7-O-rutinoside (Diosmin)Gpe, gpu, spe, spu
1721.0250, 268, 342461/463341/343(M-120)298 Diosmetin 8-C-glucoside (Orientin 4'-methyl ether)Spe, ppu
1822.0255, 268, 345461/463341/343 (M-120)298 Chrysoeriol 8-C-glucoside (Scoparin)Spe, spu
1922.3254, 354609/611301 (M-rutinose)179, 151 RutinPpu, gpe, spe
2023.0268, 334593/595512473 (M-120), 353 (M-240), 297 Apigenin 6,8-di-C-glucoside (Vicenin-2)Ppe, gpe, spe
2120.9262, 362593/595285/287 (Kaempferol) 1187/1189Kaempferol-3-O-Rutinosespe
2223.9285, 332609/700301/303 (Hesperetin)286, 177, 151 Hesperidin (hesperetin 7-O-rutinoside)Ppe, ppu, gpe, gpu, spe, spu
2324.1250, 268, 342623/625315 (M-rutinose)301, 179, 151 Isorhamnetin-3-O-rutinosidePpe, ppu, gpe, gpu, spe
2424.7250, 342477315 (M-glucose)300, 179, 151 Isorhamnetin-3-O-glucosidePpe
2525.0254, 267609/611489 (M-120)369 (M-240) Luteolin 6,8-di-C-glucoside (Lucenin-2)Ppe, ppu, spe, spu
2626.0250, 268, 342461301 (M-rhamnose)301 (M-rhamnose) Isorhamnetin-3-O-rhamnosidePpu, spe
2727.1254, 267447327 (M-120)299 Luteolin 8-C-Glucoside (orientin)Ppe, spu
28 285, 362577/579271177, 151, 119, 107 Naringin (naringenin 7-O-neohesperidoside)ppe, spe, gpu, ppu, gpe
2929.7250, 268, 342491315, 301301 7-O-Methyl-Isorhamnetin-3-O-glucosideSpu, spe, gpu, ppu
3031.1255, 268, 345607/609563299 (Chrysoeryol), 284 Chrysoeriol 7-O-neohesperidosideSpu, spe, gpu, ppu
3120.0250, 268, 342461/463341 (M-120)/343298 Diosmetin-6-C-glucosideGpu, spe, gpe
3233.8254, 354463301 (M-glucose)268, 179, 151 Quercetin 3-O-glucoside (Isoquercitrin)Spe, ppu
3327.2268, 334473/475429323, 161, 221 Apigenin 7-O-6''Acetyl-glucosidePpu, gpe, gpu, spe
3438.9268, 334431/433311 (M-120)283 Apigenin-8-C-glucoside (vitexin)Spe
3540.1268, 334431/433311 (M-120), 283283 Apigenin-6-C-glucoside (isovitexin)ppu
3638.8254, 354301179, 151179, 151 QuercetinPpu, spe
37 250, 268, 342299179, 151179, 151 Diosmetinppu
3841.8285, 325287179151, 135, 125, 107 EriodictyolGpe, ppu, spe
3942.0268, 334269179225, 201, 151 Apigenin gpe, spe, ppe
4042.0254, 267285/287269 (M-16)243, 241, 217 LuteolinPpe, ppu, gpe, spe, spu, spe
Species and fruit parts: ppe: pica peel; ppu: pica pulp; gpe: genova peel; gpu: genova pulp; spe: sutil peel; spu: sutil pulp.
Molecules 19 17400 g003 550
Figure 3. HPLC-UV chromatograms of Citrus fruits from northern Chile. (a) Pica lemon peel; (b) Pica lemon pulp (c) Genova lemon peel (d) Genova lemon pulp; (e) Sutil lemon peel and (f) Sutil lemon pulp, monitored at 280 nm. Peak numbers refer to those indicated in Table 1.
Figure 3. HPLC-UV chromatograms of Citrus fruits from northern Chile. (a) Pica lemon peel; (b) Pica lemon pulp (c) Genova lemon peel (d) Genova lemon pulp; (e) Sutil lemon peel and (f) Sutil lemon pulp, monitored at 280 nm. Peak numbers refer to those indicated in Table 1.
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Preliminary analysis of UV-vis spectra obtained for the peaks gave a first indication of the family of phenolic compounds [34,35,36,37]. The structures of these compounds were proposed based on UV maxima (Table 1) as well as fragmentation pattern using ESI-MS-MS experiments (key aglycone fragments of 179 and 151 for quercetin and its methyl derivatives, 257, 243 for kaempferol, 241, 175 for luteolin, 225, 201, 149 for apigenin, 151, 135 for eriodictyol and 151, 177 for naringenin) [38]. Some compounds were identified by co-elution with standards while several flavonoids such as diosmin, diosmetin, naringin, vitexin, hesperidin and their MSn fragments (Table 1) were coincident with those compounds already reported to occur in several Citrus species [12,13,14,15,22,23]. In addition, the loss of 162 Daltons was indicative of hexose (glucose or galactose, the most common sugars found in flavonoids) the loss of 146 Daltons was indicative of rhamnose, the loss of 133 Daltons was indicative of pentose (xylose or arabinose, the most common pentoses found in natural products), and the loss of 308 Daltons indicative of compounds having the disaccharide structure rutinose or neohesperidose linked thorough an -O-glycosidic bond [39]. In the MS identification of C-glycosides, the key fragmentations used were [M-60]−, [M-90]−, [M-120]− and [M-240]− [13], while branched O- and C-glucosides were identified as described previously by counting the losses for both types of compounds [40]. The 40 compounds detected and identified or tentatively identified are listed in Table 1, along with UV-visible and MS data. Figure 2 shows structures of several compounds identified while Figure 3, Figure 4, Figure 5 and Figure 6 show structures and full MS and MSn spectra of some representative compounds. The identification of all detected and tentatively characterized compounds present in citrus fruit extracts is explained below.
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Figure 4. Structures of main flavonoids identified in Citrus species from Northern Chile.
Figure 4. Structures of main flavonoids identified in Citrus species from Northern Chile.
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Abreviattions: Glu: glucose; Neo: neohesperidose; Rut: rutinose.

2.2.1. Flavonol O-Glycosides

Peaks 2 (Figure 3) and 3 were identified as apigenin (λ max 268, 334) derivatives. Peak 3 produced a molecular anion at m/z 577 and a apigenin MS2 fragment at m/z 269 (MS3: 225), which pointed out to the presence of apigenin 7-O-neohesperidoside (rhoifolin) [22]. Peak 4 with a pseudomolecular ion at m/z 771 (Figure 3), which in turn produced a rutin ion at m/z 609 (M-hexose) and MS3 ion at m/z 463 (quercetin 3-O-glucose, also showing a MS4 ion at m/z 301 and MS5 ions at m/z 179, 151) was proposed as an O-glycosylated rutin derivative (quercetin 7-O-glucoside-3-O-rutinoside). Peak 5 with a [M−H]− ion at m/z 639 (Figure 4) and MS2 ions at m/z 315 (M-324 Daltons, di-hexose moiety) was tentatively identified as isorhamnetin-3-O-di-glucoside [22]. Peak 8 (Figure 5) (λ max 250, 268, 342) was identified as diosmetin 7-O-neohesperidoside ([M−H]− ion at m/z 607 and MS2 ions at m/z 563: diosmetin-hexose, and m/z 299, diosmetin aglycone) while the isomer of the latter, peak 9 was associated to diosmetin 7-O-rutinoside (diosmin) (MS ions at m/z 463: diosmetin-hexose and 299: diosmetin) both compounds already reported in Citrus species [13,41]. Peak 12 with a [M−H]− ion at m/z 577 (Figure 3) and MS2 ions at m/z 431 (M-rhamnose) and 269 (M-rutinose) was tentatively identified as apigenin 7-O-rutinoside (isorhoifolin) [22].
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Figure 5. MS spectra of peaks 2, 4, 12, and 28. (Refer to Table 1).
Figure 5. MS spectra of peaks 2, 4, 12, and 28. (Refer to Table 1).
Molecules 19 17400 g005
Molecules 19 17400 g006 550
Figure 6. MS spectra of peaks 5, 23, 24, 26, 29 and 32. (Refer to Table 1).
Figure 6. MS spectra of peaks 5, 23, 24, 26, 29 and 32. (Refer to Table 1).
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In a similar manner, isomer compounds 19 (Figure 5) and 22 showing molecular anions at m/z 609 in their full MS spectra were identified as rutin (quercetin-3-O-rutinose, λ max 254, 354 nm), producing a daughter MS ion at m/z 463, by loss of rhamnose moiety and a quercetin daughter ion at m/z 301, with MS3 at m/z 179, 151) by loss of rutinose) [40] and hesperidin (hesperetin 7-O-rutinoside, λ max 285, 332 nm, hesperetin ion at m/z 301, by loss of rutinose) [42,43]. Peak 23 showing a [M−H]− ion at m/z 623 (Figure 4) and a MS2 ion at m/z 315 (isorhamnetin, λ max 250, 268, 342 nm) was identified as isorhamnetin-3-O-rutinoside [22], while peak 24 with a molecular anion at m/z 477 (Figure 4) and a MS2 ion at m/z 315 was identified as isorhamnetin-3-O-glucoside [44,45], and peak 31 (Figure 7) ([M−H]− ion at m/z 461, MS2 ions at m/z 563, 299 (chrysoeriol), 284) was identified as chrysoeriol 7-O-neohesperidoside [13]. Peak 26 (Figure 4) was identified as isorhamnetin 3-O-rhamnoside ([M−H]− ion at m/z 461, MS2 ion at m/z 301, produced by loss of rhamnose moiety) [46]. Peak 29 (Figure 4) was identified as 7-O-methyl-isorhamnetin-3-O-glucoside ([M−H]− ion at m/z 491 MSn ions at m/z 315, 301) while peak 32 (Figure 4) was tentatively identified as quercetin-3-O- hexose ([M−H]− at m/z 463, MS2 at m/z 301, produced by loss of hexose moiety) [47]. Peak 16 with two UV maxima at 262, 362 nm in the DAD spectrum and showing a molecular anion at m/z 593 in the negative ESI spectra (Figure 1) was identified as a kaempferol 3-O-rutinoside [42,48]. Cleavage of this kaempferol glycoside gave the anion aglycone at m/z 285 ([M−H]−) [22]. An isomer of the latter compound 16 identified with peak 14 showing a [M−H]− ion at m/z 593 was identified as luteolin 7-O-hexoside (UV Max 254, 267 nm, luteolin MS ion at m/z 285) [41]. Peaks 21 and 30 were tentatively identified as the isomers diosmetin 7-O-neohesperidose and chrysoeriol 7-O-neohesperidose, respectively [22]. Peak 33 was identified as apigenin 7-O-6''acetyl-glucoside ([M−H]− at m/z 473 and MS2 at m/z 429 (M-acetyl moiety, Figure 7) [41].
Molecules 19 17400 g007 550
Figure 7. MS spectra of peaks 13, 17, 25, 31, and 33. (Refer to Table 1).
Figure 7. MS spectra of peaks 13, 17, 25, 31, and 33. (Refer to Table 1).
Molecules 19 17400 g007
Flavonol C-glycosides. Peaks 7, 17 (Figure 7), and 31 were identified as C-glycosyl derivatives of diosmetin (λ max 250, 268, 342), while peaks 10 and 18 as C-glycosyl derivatives of chrysoeriol [13]. Isomer compounds identified with peaks 17 (Figure 7) and 18 both with a [M−H]− ion at m/z 461 and characteristic MSn ions at 341 (M-120) and 298 were identified as diosmetin 8-C glucoside (orientin 4'-methyl ether) and chrysoeriol 8-C-glucoside, respectively [23]. Identities of the isomers were confirmed by spiking experiments with standard compounds. In addition, related di C-glycosylated compounds identified with peaks 7 (Figure 5) and 10 showed both a pseudomolecular ion at m/z 623 and an adduct [2M−H]− ion at m/z 1247. The fragmentation observed at m/z 503, 383, 312 (M-120, M-240) lead us to the presence of the C-glycosides diosmetin 6,8-di-C-glucoside and chrysoeriol 6,8-di-C-glucoside (stellarin-2) [40]. In a similar manner, peaks 25 (Figure 7) and 27 (Figure 8) ([M−H]− ions at m/z 609 and 447, respectively) were tentatively identified as luteolin 6,8-di-C-glucoside (MS2 ions at m/z 489: M-120, 369: M-240) and luteolin 8-C-Glucoside [13] (MS2 ions at m/z 327: M-120 and 299), respectively. Peak 20 with a molecular anion at m/z 593 and typical C-glycosyl fragments at m/z 503, 473, 413 and 383 was assigned as apigenin-6,8-di-C-β-d-glucopyranoside (vicenin II, Figure 9), [40] identity further confirmed by spiking experiments with standard compound, while peaks 34 and 35 with pseudomolecular ions at m/z 431 and fragments at m/z 311 (M-120), 283 were identified as vitexin (apigenin-8-C-β-d-glucopyranoside) and isovitexin (apigenin-6-C-β-d-glucopyranoside), using also co-injection with standard compounds.
Molecules 19 17400 g008 550
Figure 8. MS spectra of peaks 27, 36, and 37. (Refer to Table 1).
Figure 8. MS spectra of peaks 27, 36, and 37. (Refer to Table 1).
Molecules 19 17400 g008
Molecules 19 17400 g009 550
Figure 9. MS spectra of peaks 7, 8, and 19. (Refer to Table 1).
Figure 9. MS spectra of peaks 7, 8, and 19. (Refer to Table 1).
Molecules 19 17400 g009

2.2.2. Flavanone Glycoconjugates

Several known flavanones and their glycosyl derivatives were identified in Chilean Citrus (Table 1). Peak 6 was identified as eriodictyol-4'-O-neohesperidoside-7-O-glucoside [41] ([M−H]− ion at m/z 757 and MS2 ions at m/z 595 (M-glucose), 449 (M-308, neohesperidose), and 287 (M-glucose and neohesperidose), while a related compound peak 13 (Figure 8) was identified as eriodictyol-7-O-neohesperidoside ([M−H]− ion at m/z 595, MS2 at m/z 449 and 287) [13] and the isomer of the later compound peak 15 was identified as eriocitrin (eriodictyol-7-O-rutinose, MS2 at m/z 287: eriodictiol) [41]. Peak 28 (Figure 3) was identified as naringin (naringenin 7-O-neohesperidoside) ([M−H]− ion at m/z 579, releasing a naringenin MS2 ion at m/z 271, produced by loss of a neohesperidose moiety) [23,49,50].

2.2.3. Flavones C and O Glycosides

Peak 2 produced a molecular anion at m/z 739 and MSn ions at m/z 431 (M-neohesperidose), and 311 (−120) and was identified asapigenin 7-O-neohesperidoside-6-C-glucoside [41].

2.2.4. Flavonoid Aglycones

Peaks 36–39 (Figure 8) showing characteristic UV spectra and [M−H]− ions at m/z 301, 299, 287, 269 and 285 (Table 1) were identified as the flavone aglycones quercetin, diosmetin, eriodictyol, apigenin, and luteolin respectively, [41,51,52,53]. MS-MS analysis of all of those compounds showed characteristic daughter ions (Table 1). The typical fragment ions at m/z 179 and 151 (Figure 3c) produced by rings A and B of the flavone structure were confirmed by RDA rearrangement of 5, 7-dihydroxy-flavones using deuterium labelling experiments [54].

2.2.5. Other Compounds

Peak 1 was identified as citric acid ([M−H]− ion at m/z 191.1)[23] while peak 11 showing a [M−H]− ion at m/z 357 in the MS spectra (MS2 at m/z 194, 151) was identified as 3-(2-hydroxy-4- methoxyphenyl)-propanoic acid hexose, a compound previously reported in Citrus [23].

3. Experimental Section

3.1. Chemicals and Plant Material

Folin–Ciocalteu phenol reagent (2 N), Na2CO3, AlCl3, FeCl3, NaNO2, NaOH, quercetin, sodium acetate, HPLC-grade water, HPLC-grade acetonitrile, reagent grade MeOH and formic acid were obtained from Merck (Darmstadt, Germany). Rutin, diosmin, diosmetin, vicenin II, orientin, vitexin, isovitexin, eriodictyol, luteolin, hesperidin, naringin (all standards with purity higher than 95% by HPLC) were purchased either from ChromaDex (Santa Ana, CA, USA) or Extrasynthèse (Genay, France). Gallic acid, TPTZ (2,4,6 tri (2 pyridyl) 1,3,5 triazine), Trolox and DPPH (1,1-diphenyl-2-picrylhydrazyl radical) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO,USA). Ripe fruits of Citrus aurantifolia (Christm) Swingle var. Pica (local name: Limón de Pica), (Citrus limon (L.) Burm) var Genova (local name: Limón Genova), Citrus aurantifolia (Christm) Swingle var. sutil (local name: Limón Sutil), were purchased at La Vega de Antofagasta fruit Market, Chile in March 2012. Examples were deposited at the Laboratorio de Productos Naturales, Universidad de Antofagasta, Antofagasta, Chile, with the numbers Cavarp-20120315 and CL-20120315 and Cavarsut-20120315, respectively.

3.2. Sample Preparation

Fresh citrus fruits were washed, the peels manually separated and the pulp deseeded. Each peel and pulp-juice (100 g) were separately homogenized in a blender with 100 mL of 0.1% HCl in MeOH and extracted thrice for one hour each time under dark using an ultrasonic bath. The extracts were combined, filtered and the solvent removed in vacuo (40 °C). The remaining aqueous partitions were separately loaded in an Amberlite XAD-7 column (5 cm × 15 cm) and rinsed with 100 mL ultrapure water. The phenolic compounds present in each partition were then eluted with 100 mL of 0.1% HCl in MeOH obtaining 0.62, 0.91 and 0.63 g of Pica, Sutil and Genova pulp extracts, respectively, and 0.76, 0.69 and 0.54 g of Pica, Sutil and Genova peel extracts, respectively. The extracts (aprox. 2 mg) were re-dissolved in 2 mL 0.1% HCl in MeOH, filtered through a 0.45 μm micropore membrane (PTFE, Waters) before use and 10 μL were injected into the HPLC instrument for analysis.

3.3. HPLC Analysis

A Merck-Hitachi (LaChrom, Tokyo, Japan) instrument equipped with an L-7100 pump, an L-7455 UV diode array detector, a D-7000 chromato-integrator and a column compartment was used for analyses. The sample was separated on a Purospher star-C18 column (250 mm × 5 mm, 4.6 mm i.d., Merck, Germany). The mobile phase consisted of 10% formic acid in water (A) and acetonitrile (B). A gradient program was used for HPLC-DAD and ESI-MS as follows: 90% A in the first 4 min, linear gradient to 75% A over 25 min, then linear gradient back to initial conditions for the other 15 min. The mobile phase flow rate was 1 mL/min. The column temperature was set at 25 °C; the detector was monitored at 520 nm for anthocyanins and 320–280 nm for other compounds while UV spectra from 200 to 600 nm were recorded for peak characterization.

3.4. Mass Spectrometric Conditions

An Esquire 4000 Ion Trap mass spectrometer (Bruker Daltoniks, Bremen, Germany) was connected to an Agilent 1100 HPLC instrument via ESI interface for HPLC-ESI-MS analysis. Full scan mass spectra were measured between m/z 150 and 2000 μ in positive ion mode for anthocyanins and negative ion mode for other compounds. High purity nitrogen was used as nebulizer gas at 27.5 psi, 350 °C and at a flow rate of 8 L/min. The mass spectrometric conditions for negative ion mode were: electrospray needle, 4000 V; end plate offset, −500 V; skimmer 1, −56.0 V; skimmer 2, −6.0 V; capillary exit offset, −84.6 V; and the operating conditions for positive ion mode were: electrospray needle, 4000 V; end plate offset, −500 V; skimmer 1, 56.0 V; skimmer 2, 6.0 V; capillary exit offset, 84.6 V; capillary exit, 140.6 V. Collisionally-induced dissociation (CID) spectra were obtained with a fragmentation amplitude of 1.00 V (MS/MS) using ultrahigh pure helium as the collision gas.

3.5. Antioxidant Assessment

3.5.1. Free Radical Scavenging Activity

The free radical scavenging activity of the extracts was determined by the DPPH assay as previously described [55], with some modifications. DPPH radicals absorb at 517 nm, but upon reduction by an antioxidant compound, absorption decreases. Briefly, 50 μL of processed SPE MeOH extract or pure compound prepared at different concentrations was added to 2 mL of fresh 0.1 mM solution of DPPH in methanol and allowed to react at 37 °C in the dark. After thirty minutes the absorbance was measured at 517 nm. The DPPH scavenging ability as percentage was calculated as: DPPH scavenging ability = (Acontrol − Asample/Acontrol) × 100. Afterwards, a curve of % DPPH bleaching activity versus concentration was plotted and IC50 values were calculated. IC50 denotes the concentration of sample required to scavenge 50% of DPPH free radicals. The lower the IC50 value the more powerful the antioxidant capacity. If IC50 ≤ 50 μg/mL the sample has high antioxidant capacity, if 50 μg/mL < IC50 ≤ 100 μg/mL the sample has moderate antioxidant capacity and if IC50 > 200 μg/mL the sample has no relevant antioxidant capacity. Gallic acid (from 1.0 to 125.0 μg/mL, R2 = 0.991) and quercetin (from 1.0 to 125.0 μg/mL, R2 = 0.993) were used as standard antioxidant compounds, and were determined to have IC50 values of 1.1 µg/mL (6.8 µmol/L) and 7.5 µg/mL (24.8 µmol/L), respectively.

3.5.2. Ferric Reducing Antioxidant Power

The determination of ferric reducing antioxidant power or ferric reducing ability (FRAP assay) of the extracts was performed as described by [56] with some modifications. The stock solutions prepared were 300 mM acetate buffer pH 3.6, 10 mM TPTZ (2,4,6-tri (2-pyridyl)-s-triazine) solution in 40 mM HCl, and 20 mM FeCl3·6H2O solution. Plant extracts or standard methanolic Trolox solutions (150 μL) were incubated at 37 °C with 2 mL of the FRAP solution (prepared by mixing 25 mL acetate buffer, 5 mL TPTZ solution, and 10 mL FeCl3·6H2O solution) for 30 min in the dark. Absorbance of the blue ferrous tripyridyltriazine complex formed was then read at 593 nm. Quantification was performed using a standard calibration curve of antioxidant Trolox (from 0.2 to 2.5 μmol/mL, R2: 0.995). Samples were analyzed in triplicate and results are expressed in μmol TE/gram dry mass.

3.5.3. Superoxide Anion Scavenging Activity

The enzyme xanthine oxidase is able to generate superoxide anion radical (O2.−) “in vivo” by oxidation of reduced products from intracellular ATP metabolism. The superoxide anion generated in this reaction sequence reduces the nitro blue tetrazolium dye (NBT), leading to a chromophore with a maximum of absorption at 560 nm. Superoxide anion scavengers reduce the speed of generation of the chromophore. The Superoxide anion scavenging activities of isolated compounds and fractions were measured spectrophotometrically in a microplate reader as reported previously [47]. All compounds, and berry extracts were evaluated at 100 μg/mL. Values are presented as mean ± standard of three determinations.

3.6. Polyphenol, Flavonoid and Vitamin C Contents

The total polyphenolic contents (TPC) of citrus fruits were determined by the Folin-Ciocalteau method [34] with some modifications. An aliquot of each processed SPE extract (200 μL), was added to the Folin–Ciocalteau reagent (2 mL, 1:10 v/v in purified water) and after 5 min of reaction at room temperature (25 °C), 2 mL of a 100 g/L solution of Na2CO3 was added. Sixty minutes later the absorbance was measured at 710 nm. A calibration curve was performed with the standard gallic acid (concentrations ranging from 16 to 500 μg/mL, R2 = 0.999) and the results expressed as mg gallic acid equivalents/g dry mass.
Determination of total flavonoid content (TFC) of the methanolic extracts was performed as reported previously [57] using the AlCl3 colorimetric method. Quantification was expressed by reporting the absorbance in the calibration graph of quercetin, which was used as the flavonoid standard (from 0.1 to 65.0 μg/mL, R2 = 0.994). Results are expressed as mg quercetin equivalents/g dry mass. Vitamin C content was determined by redox titration with a potassium iodate solution, after the samples were homogenized and acidized with sulphuric acid (20%) and in the presence of IK, using starch as an indicator. The end point of the reaction was the appearance of a blue colour [31]. The ascorbic acid content was expressed as mg of ascorbic acid per 100 g fresh weight (FW). All spectrometric measurements were performed using a Unico LQ2800 UV-vis spectrophotometer (Unico instruments, Co., Ltd., Shanghai, China) or a Pharo spectroquant 300 (Merck, Darmstadt, Germany).

3.7. Statistical Analysis

The statistical analysis was carried out using the originPro 9.0 software packages (Originlab Corporation, Northampton, MA, USA). The determination was repeated at least three times for each sample solution. Analysis of variance was performed using ANOVA. Significant differences between means were determined by Student’s t-test (p values <0.05 were regarded as significant).

4. Conclusions

The LC-UV and ESI-MSn system used in this work allowed the detection and the tentative identification of 40 phenolic compounds in three edible pulps and peels of citrus fruits widely consumed in Chile. Several flavonoids including one kaempferol-O-hexoside (peak 16) and one hesperidin derivative (peak 22) three quercetin derivatives (peaks 4, 19 and 36), five isorhamnetin derivatives (peaks 5, 23, 24, 26 and 29) four luteolin derivatives (peaks 14, 25, 27 and 40), seven apigenin derivatives (peaks 2, 3, 12, 20, 34, 35 and 39), seven diosmetin derivatives (peaks 7–9, 17, 21, 31 and 37), three chrysoeriol derivatives (peaks 10,18 and 30), and four eryodictiol derivatives (peaks 6, 1315 and 38) were identified in negative and positive mode using full scan mass measurements and MSn fragmentations. The results obtained pointed out that the methodology developed is appropriate for rapid analysis and identification of C and O-glycosil flavonoids in extracts from native citrus fruits and can be potentially used for other edible South American fruits. The highest total phenolic content was found in Pica peels which also showed the highest total flavonoid content (34.59 ± 0.81 mg/g GAE dry weight and 23.06 ± 1.57 mg quercetin/g dry weight, respectively). The vitamin C pattern for citrus species was similar but with higher content in Genova pulps, followed by the pulp of the variety Sutil (60.13 ± 1.28 and 56.53 ± 1.06 mg ascorbic acid g dry weight, respectively). The antioxidant capacity was also higher for Pica peels (10.34 ± 1.23 µg/mL in the DPPH assay and 120.63 ± 2.45 µM trolox equivalents/g dry weight in the FRAP assay).The antioxidant properties and high content of flavonoids in the peels can make this waste material a good source of nutraceutical and healthy phenolic compounds, especially to be used as anti-ageing products, due to the high content of polyphenols. However, further research is needed to explore other biological activities of Citrus peels to support their potential in the human diet.

Supplementary Materials

Supplementary materials can be accessed at: https://www.mdpi.com/1420-3049/19/11/17400/s1.

Supplementary Files

Supplementary File 1

Acknowledgments

Financial support from FONDECYT (Project 1140178) is gratefully acknowledged.

Author Contributions

Carlos Areche, Beatriz Sepúlveda and Mario J. Simirgiotis designed research; Anghel Brito, Javier E. Ramirez, Mario J. Simirgiotis and Beatriz Sepúlveda performed research and analyzed the data; Mario J. Simirgiotis, and Carlos Areche wrote the paper. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Labuschagne, C.F.; Brenkman, A.B. Current methods in quantifying ROS and oxidative damage in Caenorhabditis elegans and other model organism of aging. Ageing Res. Rev. 2013, 12, 918–930. [Google Scholar] [CrossRef] [PubMed]
  2. Amaro-Ortiz, A.; Yan, B.; D’Orazio, J.A. Ultraviolet radiation, aging and the skin: Prevention of damage by topical cAMP manipulation. Molecules 2014, 19, 6202–6219. [Google Scholar] [CrossRef] [PubMed]
  3. Pennington, J.A.T.; Fisher, R.A. Food component profiles for fruit and vegetable subgroups. J. Food Comp. Anal. 2010, 23, 411–418. [Google Scholar] [CrossRef]
  4. Giampieri, F.; Alvarez-Suarez, J.M.; Mazzoni, L.; Forbes-Hernandez, T.Y.; Gasparrini, M.; Gonzàlez-Paramàs, A.M.; Santos-Buelga, C.; Quiles, J.L.; Bompadre, S.; Mezzetti, B.; et al. Polyphenol-rich strawberry extract protects human dermal fibroblasts against hydrogen peroxide oxidative damage and Improves mitochondrial functionality. Molecules 2014, 19, 7798–7816. [Google Scholar] [CrossRef] [PubMed]
  5. Giampieri, F.; Alvarez-Suarez, J.M.; Battino, M. Strawberry and Human Health: Effects beyond Antioxidant Activity. J. Agric. Food Chem. 2014, 62, 3867–3876. [Google Scholar] [CrossRef]
  6. Mulero, J.; Bernabe, J.; Cerda, B.; García-Viguera, C.; Moreno, D.A.; Albaladejo, M.D.; Avilés, F.; Parra, S.; Abellán, J.; Zafrilla, P. Variations on cardiovascular risk factors in metabolic syndrome after consume of a citrus-based juice. Clin. Nutr. 2012, 31, 372–377. [Google Scholar] [CrossRef] [PubMed]
  7. Buachan, P.; Chularojmontri, L.; Wattanapitayakul, S.K. Fruits on Human Endothelial Cells: Enhancing Cell Migration and Delaying Cellular Aging. Selected Activities of Citrus Maxima Merr. Nutrients 2014, 6, 1618–1634. [Google Scholar] [CrossRef] [PubMed]
  8. Benavente-Garcia, O.; Castillo, J. Update on uses and properties of citrus flavonoids: New findings in anticancer, cardiovascular, and anti-inflammatory activity. J. Agric. Food Chem. 2008, 56, 6185–6205. [Google Scholar] [CrossRef] [PubMed]
  9. Mokbel, M.S.; Hashinaga, F. Evaluation of the antioxidant activity of extracts from buntan (Citrus grandis Osbeck) fruit tissues. Food Chem. 2006, 94, 529–534. [Google Scholar]
  10. Dahmoune, F.; Boulekbache, L.; Moussi, K.; Aoun, O.; Spigno, G.; Madani, K. Valorization of Citrus limon residues for the recovery of antioxidants: Evaluation and optimization of microwave and ultrasound application to solvent extraction. Ind. Crops Prod. 2013, 50, 77–87. [Google Scholar] [CrossRef]
  11. Bocco, A.; Cuvelier, M.-E.; Richard, H.; Berset, C. Antioxidant Activity and Phenolic Composition of Citrus Peel and Seed Extracts. J. Agric. Food Chem. 1998, 46, 2123–2129. [Google Scholar] [CrossRef]
  12. Liu, E.-H.; Zhao, P.; Duan, L.; Zheng, G.-D.; Guo, L.; Yang, H.; Li, P. Simultaneous determination of six bioactive flavonoids in Citri Reticulatae Pericarpium by rapid resolution liquid chromatographycoupled with triple quadrupole electrospray tandem mass spectrometry. Food Chem. 2013, 141, 3977–3983. [Google Scholar] [CrossRef]
  13. Abad-García, B.; Garmón-Lobato, S.; Berrueta, L.A.; Gallo, B.; Vicente, F. On line characterization of 58 phenolic compounds in Citrus fruit juices from Spanish cultivars by high-performance liquid chromatography with photodiode-array detection coupled to electrospray ionization triple quadrupole mass spectrometry. Talanta 2012, 99, 213–224. [Google Scholar] [PubMed]
  14. Di Donna, L.; Taverna, D.; Mazzotti, F.; Benabdelkamel, H.; Attya, M.; Napoli, A.; Sindona, G. Comprehensive assay of flavanones in citrus juices and beverages by UHPLC-ESI-MS/MS and derivatization chemistry. Food Chem. 2013, 141, 2328–2333. [Google Scholar] [CrossRef]
  15. He, D.X.; Shan, Y.; Wu, Y.H.; Liu, G.Z.; Chen, B.; Yao, S.Z. Simultaneous determination of flavanones, hydroxycinnamic acids and alkaloids in citrus fruits by HPLC-DAD-ESI/MS. Food Chem. 2011, 127, 880–885. [Google Scholar] [CrossRef] [PubMed]
  16. Patil, J.R.; Jayaprakasha, G.K.; Murthy, K.N.C.; Chetti, M.B.; Patil, B.S. Characterization of Citrus aurantifolia bioactive compounds and their inhibition of human pancreatic cancer cells through apoptosis. Microchem. J. 2010, 94, 108–117. [Google Scholar] [CrossRef]
  17. Steinmann, D.; Ganzera, M. Recent advances on HPLC/MS in medicinal plant analysis. J. Pharm. Biomed. Anal. 2011, 55, 744–757. [Google Scholar] [CrossRef] [PubMed]
  18. Wright, P. Metabolite identification by mass spectrometry: Forty years of evolution. Xenobiotica 2011, 41, 670–686. [Google Scholar] [CrossRef] [PubMed]
  19. Mattoli, L.; Cangi, F.; Ghiara, C.; Burico, M.; Maidecchi, A.; Bianchi, E.; Ragazzi, E.; Bellotto, L.; Seraglia, R.; Traldi, P. A metabolite fingerprinting for the characterization of commercial botanical dietary supplements. Metabolomics 2011, 7, 437–445. [Google Scholar] [CrossRef]
  20. Aliferis, K.A.; Chrysayi-Tokousbalides, M. Metabolomics in pesticide research and development: Review and future perspectives. Metabolomics 2011, 7, 35–53. [Google Scholar] [CrossRef]
  21. Kang, H.J.; Yang, H.J.; Kim, M.J.; Han, E.S.; Kim, H.J.; Kwon, D.Y. Metabolomic analysis of meju during fermentation by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS). Food Chem. 2011, 127, 1056–1064. [Google Scholar] [CrossRef] [PubMed]
  22. Gattuso, G.; Barreca, D.; Gargiulli, C.; Leuzzi, U.; Caristi, C. Flavonoid Composition of Citrus Juices. Molecules 2007, 1641–1673. [Google Scholar]
  23. García-Salas, P.; Gómez-Caravaca, A.M.; Arráez-Román, D.; Segura-Carretero, A.; Guerra-Hernández, E.; García-Villanova, B.; Fernández-Gutiérrez, A. Influence of technological processes on phenolic compounds, organic acids, furanic derivatives, and antioxidant activity of whole-lemon powder. Food Chem. 2013, 141, 869–878. [Google Scholar] [CrossRef] [PubMed]
  24. Brito, A.; Areche, C.; Sepúlveda, B.; Kennelly, E.J.; Simirgiotis, M.J. Anthocyanin characterization, total phenolic quantification and antioxidant features of some Chilean edible berry extracts. Molecules 2014, 19, 10936–10955. [Google Scholar] [CrossRef] [PubMed]
  25. Ramirez, J.E.; Zambrano, R.; Sepúlveda, B.; Simirgiotis, M.J. Antioxidant Properties and Hyphenated HPLC-PDA-MS Profiling of Chilean Pica Mango Fruits (Mangifera indica L. Cv. piqueño). Molecules 2014, 19, 438–458. [Google Scholar] [CrossRef]
  26. Abeysinghe, D.C.; Li, X.; Sun, C.D.; Zhang, W.S.; Zhou, C.H.; Chen, K. Bioactive compounds and antioxidant capacities in diferent edible tissues of citrus fruit of four species. Food Chem. 2007, 104, 1338–1344. [Google Scholar] [CrossRef]
  27. Huang, Y.-S.; Ho, S.-C. Polymethoxy flavones are responsible for the anti-inflammatory activity of citrus fruit peel. Food Chem. 2010, 119, 868–873. [Google Scholar] [CrossRef]
  28. Niki, E. Antioxidant capacity: Which capacity and how to assess it? J. Berry Res. 2011, 4, 169–176. [Google Scholar]
  29. Prior, R.L.; Wu, X.; Schaich, K. Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements. J. Agric. Food Chem. 2005, 53, 4290–4302. [Google Scholar] [CrossRef] [PubMed]
  30. Kevers, C.; Pincemail, J.; Defraigne, J.O.; Dommes, J. Antioxidant capacity of small dark fruits: Influence of cultivars and harvest time. J. Berry Res. 2014, 4, 97–105. [Google Scholar]
  31. De Moraes Barros, H.R.; Pinto de Castro Ferreira, T.A.; Genovese, M.I. Antioxidant capacity and mineral content of pulp and peel from commercial cultivars of citrus from Brazil. Food Chem. 2012, 134, 1892–1898. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Xua, G.; Liu, D.; Chen, J.; Ye, X.; Ma, Y.; Shi, J. Juice components and antioxidant capacity of citrus varieties cultivated in China. Food Chem. 2008, 106, 545–551. [Google Scholar] [CrossRef]
  33. Akhatou, I.; Fernández-Recamales, Á. Nutritional and Nutraceutical Quality of Strawberries in Relation to Harvest Time and Crop Conditions. J. Agric. Food Chem. 2014, 62, 5749–5760. [Google Scholar] [CrossRef] [PubMed]
  34. Simirgiotis, M.J. Antioxidant Capacity and HPLC-DAD-MS Profiling of Chilean Peumo (Cryptocarya alba) Fruits and Comparison with German Peumo (Crataegus monogyna) from Southern Chile. Molecules 2013, 18, 2061–2080. [Google Scholar] [CrossRef] [PubMed]
  35. Mabry, T.J.; Markham, K.R.; Thomas, M.B. The Systematic Identification of Flavonoids, Part II; Springer-Verlag: Berlin, Germany, 1970. [Google Scholar]
  36. Sun, J.; Liang, F.; Bin, Y.; Li, P.; Duan, C. Screening non-colored phenolics in red wines using liquid chromatography/ultraviolet and mass spectrometry/mass spectrometry libraries. Molecules 2007, 12, 679–693. [Google Scholar] [CrossRef] [PubMed]
  37. Simirgiotis, M.J.; Theoduloz, C.; Caligari, P.D.S.; Schmeda-Hirschmann, G. Comparison of phenolic composition and antioxidant properties of two native Chilean and one domestic strawberry genotypes. Food Chem. 2009, 113, 377–385. [Google Scholar] [CrossRef]
  38. Fabre, N.; Rustan, I.; de Hoffmann, E.; Quetin-Leclercq, J. Determination of flavone, flavonol, and flavanone aglycones by negativeion liquid chromatography electrospray ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12, 707–715. [Google Scholar] [CrossRef] [PubMed]
  39. Abu-Reidah, I.M.; Arráez-Román, D.; Segura-Carretero, A.; Fernández-Gutiérrez, A. Profiling of phenolic and other polar constituents from hydro-methanolic extract of watermelon (Citrullus lanatus) by means of accurate-mass spectrometry (HPLC–ESI–QTOF–MS). Food Res. Int. 2013, 51, 354–362. [Google Scholar] [CrossRef]
  40. Simirgiotis, M.J.; Schmeda-Hirschmann, G.; Borquez, J.; Kennelly, E.J. The Passiflora tripartita (Banana Passion) fruit: A source of bioactive flavonoid C-glycosides Isolated by HSCCC and characterized by HPLC-DAD-ESI/MS/MS. Molecules 2013, 18, 1672–1692. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  41. Lin, L.-Z.; Harnly, J.M. Identification of the phenolic components of chrysanthemum flower (Chrysanthemum morifolium Ramat). Food Chem. 2010, 120, 319–326. [Google Scholar] [CrossRef]
  42. Engels, C.; Gräter, D.; Esquivel, P.; Jiménez, V.M.; Gänzle, M.G.; Schieber, A. Characterization of phenolic compounds in jocote (Spondias purpurea L.) peels by ultra high-performance liquid chromatography/electrospray ionization mass spectrometry. Food Res. Int. 2012, 46, 557–562. [Google Scholar]
  43. Yu, E.A.; Kim, G.-S.; Jeong, S.W.; Park, S.; Lee, S.J.; Kim, J.H.; Lee, W.S.; Bark, K.-M.; Jin, J.S.; Shin, S.C. Flavonoid profile and biological activity of Korean citrus varieties (II): Pyunkyul (Citrus tangerina Hort. ex Tanaka) and overall contribution of its flavonoids to antioxidant effect. J. Funct. Foods 2014, 6, 637–642. [Google Scholar] [CrossRef]
  44. Han, L.; Liu, X.; Yang, N.; Li, J.; Cai, B.; Cheng, S. Simultaneous chromatographic fingerprinting and quantitative analysis of flavonoids in Pollen Typhae by high-performance capillary electrophoresis. Acta Pharm. Sin. B 2012, 2, 602–609. [Google Scholar] [CrossRef]
  45. Carazzone, C.; Mascherpa, D.; Gazzani, G.; Papetti, A. Identification of phenolic constituents in red chicory salads (Cichorium intybus) by high-performance liquid chromatography with diode array detection and electrospray ionisation tandem mass spectrometry. Food Chem. 2013, 138, 1062–1071. [Google Scholar] [CrossRef] [PubMed]
  46. Ferreres, F.; Grosso, C.; Gil-Izquierdo, A.; Valentão, P.; Andrade, P.B. Phenolic compounds from Jacaranda caroba (Vell.) A. DC.: Approaches to neurodegenerative disorders. Food Chem. Toxicol. 2013, 57, 91–98. [Google Scholar] [CrossRef] [PubMed]
  47. Simirgiotis, M.J.; Ramirez, J.E.; Schmeda Hirschmann, G.; Kennelly, E.J. Bioactive coumarins and HPLC-PDA-ESI-ToF-MS metabolic profiling of edible queule fruits (Gomortega keule), an endangered endemic Chilean species. Food Res. Int. 2013, 54, 532–543. [Google Scholar] [CrossRef]
  48. Perestrelo, R.; Lu, Y.; Santos, S.A.O.; Silvestre, A.J.D.; Neto, C.P.; Câmara, J.S.; Rocha, S.M. Phenolic profile of Sercial and Tinta Negra Vitis vinifera L. grape skins by HPLC-DAD-ESI-MSn: Novel phenolic compounds in Vitis vinifera L. grape. Food Chem. 2012, 135, 94–104. [Google Scholar] [CrossRef]
  49. Nagy, T.O.; Solar, S.; Sontag, G.; Koenig, J. Identification of phenolic components in dried spices and influence of irradiation. Food Chem. 2011, 128, 530–534. [Google Scholar] [CrossRef] [PubMed]
  50. Vallverdú-Queralt, A.; Jáuregui, O.; Medina-Remon, A.; Andrés-Lacueva, C.; Lamuela-Raventós, R.M. Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry. Rap. Commun. Mass Spectrom. 2010, 24, 2986–2992. [Google Scholar] [CrossRef]
  51. Simirgiotis, M.J.; Silva, M.; Becerra, J.; Schmeda-Hirschmann, G. Direct characterisation of phenolic antioxidants in infusions from four Mapuche medicinal plants by liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionisation tandem mass spectrometry (HPLC-ESI–MS). Food Chem. 2012, 131, 318–327. [Google Scholar] [CrossRef]
  52. Surco-Laos, F.; Dueñas, M.; González-Manzano, S.; Cabello, J.; Santos-Buelga, C.; González-Paramás, A.M. Influence of catechins and their methylated metabolites on lifespan and resistance to oxidative and thermal stress of Caenorhabditis elegans and epicatechin uptake. Food Res. Int. 2012, 46, 514–521. [Google Scholar] [CrossRef]
  53. Vallverdú-Queralt, A.; Jáuregui, O.; di Lecce, G.; Andrés-Lacueva, C.; Lamuela-Raventós, R.M. Screening of the polyphenol content of tomato-based products through accurate-mass spectrometry (HPLC-ESI-QTOF). Food Chem. 2011, 129, 877–883. [Google Scholar] [CrossRef] [PubMed]
  54. McNab, H.; Ferreira, E.S.B.; Hulme, A.N.; Quye, A. Negative ion ESI-MS analysis of natural yellow dye flavonoids—An isotopic labelling study. Int. J. Mass Spectrom. 2009, 284, 57–65. [Google Scholar] [CrossRef]
  55. Simirgiotis, M.J.; Schmeda-Hirschmann, G. Determination of phenolic composition and antioxidant activity in fruits, rhizomes and leaves of the white strawberry (Fragaria chiloensis spp. chiloensis form chiloensis) using HPLC-DAD-ESI-MS and free radical quenching techniques. J. Food Comp. Anal. 2010, 23, 545–553. [Google Scholar] [CrossRef]
  56. Benzie, I.F.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure of “Antioxidant Power”: The FRAP assay. Anal. Biochem. 1996, 239, 70–76. [Google Scholar] [PubMed]
  57. Simirgiotis, M.J.; Adachi, S.; To, S.; Yang, H.; Reynertson, K.A.; Basile, M.J.; Gil, R.R.; Weinstein, I.B.; Kennelly, E.J. Cytotoxic chalcones and antioxidants from the fruits of Syzygium samarangense (Wax Jambu). Food Chem. 2008, 107, 813–819. [Google Scholar] [CrossRef] [PubMed]
  • Sample Availability: Samples of the fruits and extracts are available from the authors.

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MDPI and ACS Style

Brito, A.; Ramirez, J.E.; Areche, C.; Sepúlveda, B.; Simirgiotis, M.J. HPLC-UV-MS Profiles of Phenolic Compounds and Antioxidant Activity of Fruits from Three Citrus Species Consumed in Northern Chile. Molecules 2014, 19, 17400-17421. https://doi.org/10.3390/molecules191117400

AMA Style

Brito A, Ramirez JE, Areche C, Sepúlveda B, Simirgiotis MJ. HPLC-UV-MS Profiles of Phenolic Compounds and Antioxidant Activity of Fruits from Three Citrus Species Consumed in Northern Chile. Molecules. 2014; 19(11):17400-17421. https://doi.org/10.3390/molecules191117400

Chicago/Turabian Style

Brito, Anghel, Javier E. Ramirez, Carlos Areche, Beatriz Sepúlveda, and Mario J. Simirgiotis. 2014. "HPLC-UV-MS Profiles of Phenolic Compounds and Antioxidant Activity of Fruits from Three Citrus Species Consumed in Northern Chile" Molecules 19, no. 11: 17400-17421. https://doi.org/10.3390/molecules191117400

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