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

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.


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.

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 (R 2 = 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.

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 MS n fragmentations.HPLC fingerprints were generated (Figure 3 and Figures S6-S8) and phenolic compounds subsequently analyzed by ESI-MS n (Table 1).    1.
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 MS n 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  [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 Figures 3-6 show structures and full MS and MS n spectra of some representative compounds.The identification of all detected and tentatively characterized compounds present in citrus fruit extracts is explained below.Abreviattions: Glu: glucose; Neo: neohesperidose; Rut: rutinose.
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.

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.

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.

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, R 2 = 0.991) and quercetin (from 1.0 to 125.0 μg/mL, R 2 = 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.

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, R 2 : 0.995).Samples were analyzed in triplicate and results are expressed in μmol TE/gram dry mass.

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.

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, R 2 = 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, R 2 = 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).

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).

Conclusions
The LC-UV and ESI-MS n 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 MS n 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.

Figure 4 .
Figure 4. Structures of main flavonoids identified in Citrus species from Northern Chile.

Table 1 .
Identification of phenolic compounds in Citrus fruits and aerial parts by LC-UV, LC-MS and MS/MS data.