Characterization of Extra Early Spanish Clementine Varieties (Citrus clementina Hort ex Tan) as a Relevant Source of Bioactive Compounds with Antioxidant Activity

The most relevant nutrients and bioactive compounds (soluble sugars, dietary fiber, ascorbic acid and organic acids, individual phenolic compounds, fatty acids, and tocopherols) as well as antioxidant activity have been characterized in three extra early varieties of clementine (Citrus clementina Hort ex Tan. Basol, Clemensoon and Clemenrubí) cultivated in Valencia (Spain). Clementines are a relevant source of bioactive compounds, such as vitamin C (values around 80 mg/100 g), allowing to satisfy the recommended daily intake with the consumption of a normal portion. Sucrose was the most abundant sugar, and potassium the main mineral while manganese was the least. Fat content was very low (<0.5 mg/100 g), with palmitic acid and α-tocopherol the most abundant fatty acid and vitamin E form, respectively. Flavonoids were the predominant phenolic compounds, with narirutin/naringin and (neo)hesperidin the best represented ones. The antioxidant capacity evaluated by reducing power, DPPH, and β-carotene bleaching inhibition assays was satisfactory with values similar to those reported in other citrus fruits. Thus, this fruit is a relevant source of bioactive compounds with antioxidant properties of interest for consumers and the food industry.

pieces, and whole fresh pulp was homogenized with an Ultra-Turrax ® homogenizer (IKA ® Works, Inc., Wilmington, NC, USA). Aliquots of homogenized fresh pulp were taken to perform physico-chemical analysis, and organic acids and vitamin C determination. One portion was immediately freeze-dried (Telstar LyoQuest freeze-dryer, Madrid, Spain) and preserved in hermetic containers at −20 • C, in a dark and dry ambient until used for the analysis of proximate composition, soluble sugars, dietary fiber, mineral composition, individual polyphenols, tocopherols, fatty acids, and antioxidant activity [15].

Physico-Chemical Analysis
Physico-chemical analysis in the analyzed samples were performed following AOAC procedures [16]. Dry matter (DM) was determined by desiccation, pH was measured by potentiometry (MicropH-2000, Crison Instrument, Spain), titratable acidity (TA) was quantified by titration with NaOH (0.1 N) until a pH value of 8.1, • Brix was determined according to the official methods and Ripeness index (RI) was calculated by the relation of • Brix/TA ratio (TA expressed as mg of citric acid/100 g of edible portion) [15].

Total, Soluble and Insoluble Dietary Fiber Quantification
AOAC enzymatic-gravimetric methods (993. 19 and 991.42) were used for soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) analysis [16]. Total dietary fiber was calculated as the sum of insoluble and soluble dietary fiber fractions. Values were expressed as g/100 g fw of fruit edible portion.

Total Proteins
Total nitrogen content was determined by the Kjeldahl method and further converted to protein content using a conversion factor 6.25. Values were expressed as g/100 g fw of fruit edible portion [15].

Total Fat and Fatty Acids
Total fat was obtained after Soxhlet extraction with petroleum ether [16], and fatty acids were determined by gas-liquid chromatography with flame ionization detection (GC-FID, DANI model GC 1000 instrument, Milan, Italy) as described previously by the authors [18]. The results were processed using Clarity Software (DataApex, Prague, Czech Republic) and expressed in relative percentage (%) of each fatty acid.

Ash Content and Mineral Composition (Macro and Microelements)
Total ash content was determined following the method 930.05 of the AOAC [16]. Briefly, 0.5 g of dried and homogenized sample was incinerated in a microwave oven with high pressure (Muffle Furnace mls1200, Monroe, LA, USA) for 24 h at 550 • C. Ashes were gravimetrically quantified, and extracted with HNO 3 (50% v/v) and HCl (50% v/v). All measurements were performed by atomic absorption spectroscopy (Analyst 200 Perkin Elmer equipment, Waltham, USA). Microelements (Fe, Cu, Mn, and Zn) were directly measured. An additional 1/10 (v/v) dilution was performed for macroelements: LaCl 2 (99% purity; 1.8%, w/v) for Ca and Mg determination, and CsCl 2 (99% purity; 0.2%, w/v) for Na and K analysis.
In brief, for all macroelement standards, the stock solutions (containing of 1 g/L of each element) were prepared in distilled water with a final standard solutions of 0.1-250 mg/L. La 2 O 3 and CsCl, for Ca and Mg measurement (addition to samples and standards a solution of 58 g/L La 2 O 3 and 87.5 g/L HCl, leading to LaCl 2 ,), or for Na and K measurement (addition to samples and standards a solution of 10 g/L of CsCl). Macro and microelements were expressed as mg/100 g fw of fruit edible portion and µg/100 g fw of fruit edible portion, respectively [15].

Vitamin C and Organic Acids Characterization
Both vitamin C vitamers (ascorbic and dehydroascorbic acids), as well as other individual organic acids (oxalic, tartaric, isocitric, malic, citric, fumaric, and succinic acids) were quantified by HPLC-UV after extraction with m-phosphoric acid (4.5%, prepared in distilled water) [5]. After m-phosphoric sample extraction, to an aliquot of the extracts were added L-cysteine (4 g/100 mL, from Sigma, St. Louis, USA) in order to transform the DHAA in AA for total vitamin C content quantification. DHAA was estimated by difference between total vitamin C and AA contents [15].
Separation and identification was performed by HPLC system equipped with an isocratic PU-II pump, an AS-1555 automatic injector (Jasco, Japan), a Sphereclone ODS (2) 250 × 4.60 mm, 5 µm column (Phenomenex), and a UV-VIS detector (Thermo Separation Spectra Series UV100, Madrid, Spain). The identification and quantification were performed using Cromanec XP software (Micronec, Madrid, Spain). Values were expressed as mg/100 g fw of fruit edible portion.

Tocopherols Characterization
Tocopherols content was determined by HPLC-FL after extraction with hexane (using tocol as internal standard, IS) [19]. In brief, samples (500 mg) were homogenized with methanol and subsequently, extracted with hexane. After that, saturated NaCl aqueous solution was added, homogenized and centrifuged (Centurion K24OR refrigerated centrifuge, 6185 rpm, 5 min). The clear upper layer was transferred to a vial. The sample was re-extracted twice with hexane and the combined extracts were taken to dryness under a nitrogen stream, redissolved in n-hexane, filtered through LC filter disk, and analyzed by HPLC-FL [15].
The separation and identification were performed by HPLC (Smartline pump 1000, Knauer, Germany) connected with a FP-2020 fluorescence detector (290 nm, 330 nm; Jasco, Japan). The chromatographic separation was performed using a normal phase Polyamide II column (250, 9, 4.6 mm) from YMC Waters (Japan) at 30 • C (7971 R Grace oven). Data were analyzed using Clarity 2.4 Software (DataApex). Quantification was based on the fluorescence signal response and values were expressed as µg/100 g fw.
Detection was carried out with a mass spectrometer (API 3200 Qtrap, Applied Biosystems, Darmstadt, Germany) couple to a diode array detector (DAD, 280 nm and 370 nm). Identification was performed using the chromatographic characteristics and UV-vis and mass spectra. Tentative identifications were performed by comparing the obtained information with our library and/or data reported in the literature. Quantification was achieved using calibration curves with individual standards. The results were expressed in mg/g of hydroalcoholic extract.

Evaluation of the Antioxidant Capacity
The freeze-dried sample was extracted with methanol (1 h, at 25 • C) and filtered through Whatman No. 4 filter paper. The residue was then extracted with an additional portion of methanol. The combined methanolic extracts were evaporated under reduced pressure (rotary evaporator Büchi R-210, Flawil, Switzerland), re-dissolved in methanol at a concentration of 5 mg/mL, and stored at 4 • C for further use. The antioxidant capacity was evaluated using three in vitro assays: DPPH radical-scavenging activity, reducing power and β-carotene bleaching inhibition [15,21]. Results were expressed as EC 50 values for reducing power, calculated by the interpolation from the graph of each assay. Trolox was used as positive control.

Statistical Analysis
All the analyses were carried out by means of Analysis of variance (ANOVA), followed by Tukey's test (95% confidence level). Values were expressed as means ± standard deviations (n = 3). The proximate composition, micro and macroelements and bioactive compounds were presented in three consecutive seasons and only one range of variation between seasons to simplify the interpretation of the results (n = 9). The discussion of the obtained results is based on range of variation. A multivariable analysis, canonical correlations and principal components analysis (PCA), was performed among the variables analyzed. Mean centering was used for normalization of original data. All statistical procedures were conducted using Statgraphics Centurion XVII.I software, v.17.1.12 (Warrenton, VA, USA).

Nutritional and Chemical Profile of Clementine Varieties
Clementine varieties showed similar and homogeneous ranges of variation values for moisture, pH and • Brix. Variety Clemenrubí stands out by its higher acidity and ripening index value (Table 1). Results corresponding to the nutritional composition, micro, and macro elements, and hydrophilic compounds of pulp samples are shown in Tables 2-7 and Figure 1.
Clementine varieties showed similar values of total dietary fiber, with a different distribution between soluble (SDF) and insoluble dietary fiber (IDF) fractions, with the highest content of SDF in the Basol variety and the highest IDF content in the Clemenrubí variety ( Table 2).
According to the Food and Nutrition Board [21], the recommended dietary fiber intake should range from 19 to 31 g per day in young children (<12 years) and from 26 to 38 g per day in adolescents and adults. From our results, eating two pieces of any of the studied clementine varieties (around 170 g) would cover 8-14% of the daily recommended intake for children and 7-10% for adolescents and adults [22,23].
Related to free sugars, sucrose was the major soluble sugar, although the contents of fructose and glucose (Table 3) were also relevant and similar between them, as previously reported by [24].   Regarding micronutrients and starting with vitamins, vitamin C (Table 4), Clemenrubí possessed the highest amount of this vitamin with around 132 mg/100 g fw. In all cases, the predominant isoform was by far ascorbic acid (AA), which, as is well known, besides vitamin activity, also bears antioxidant properties. According to the Food and Nutrition Board, the vitamin C dietary reference intake (DRI) is 90 mg/day for adults over 19 years; from this point of view, one piece of any of the studied varieties (≈ 85 g) could cover up to 93% of DRI of this vitamin [22]. Organic acids were also analyzed in clementine varieties being citric acid, as expected, the best represented one, followed by malic, isocitric, and quinic acids with similar contents among varieties (Table 4). These results agree with the abundance of organic acids reported in other clementine varieties (tangerine Citrus reticulata) [25]. The mineral composition, namely microelements Cu, Fe, Mn, and Zn, and macroelements Ca, Mg, Na, and K, is shown in Table 5. Fe and Zn were the predominant microelements in clementine pulp, Basol and Clemenrubí being the varieties that presented the highest content (0.49 mg Fe/100 g fw and 0.39 mg Zn/100 g fw, respectively). As for macroelements, potassium was the most abundant one in the three varieties (up to 169.35 mg/100 g fw). These values were similar to those reported by [14] in other varieties of clementine (i.e., Clementina Fina and Clemenules). Despite the great variability of mineral content over all the years, which is highly correlated with environmental and crop conditions, we could consider these clementine varieties as interesting sources of minerals considering their higher moiety content, especially in the case of Basol and Clemenrubí varieties. Taking into account the reference intake values published by the EFSA in 2017 [26], 100 g of the edible portion of Basol clementine could cover up to 13.85% of Cu, 4.33% of Mn, and 4.26% of Mg, while a 100 g edible portion of Clemenrubí could cover up to 46.15% of Cu, 4.67% of Mn, and 4.73% of Mg.
Despite clementine fruits presenting very low fat content (0.04-0.10 g/100 g), the authors considered it of interest to analyze the lipophilic fraction (fatty acids and tocopherols) to provide a full nutrients and bioactive compounds characterization of these non-previously studied clementine varieties. In the analyzed clementine varieties, 24 fatty acids were detected. Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1n9), linoleic acid (C18:2n6), and linolenic acid (C18:3n3) were the most abundant fatty acids (Table 6). Palmitic acid was the major SFA and α-linolenic acid (ALA) was the predominant PUFA in all varieties studied, in three seasons, and oleic acid was the main monounsaturated fatty acid (MUFA). Among the fatty acid families studied, the majority families were SFA and PUFA with similar percentages, MUFA being the family with the lowest content.
Studies in other clementine varieties (i.e., variety Elarbi, from Tunisia) reported similar profile of families of fatty acids to those obtained in the present study being SFA the majority family (SFA (47.6%) < MUFA (32.54%) < PUFA (18.9%)). The main fatty acids determined in that clementine variety (Elarbi) were stearic, palmitic, oleic, linoleic and linolenic acids (representing approximately 75% of the total fatty acids), which agrees with the results obtained in the present study [27]. A similar distribution of major fatty acids was found in other citrus fruits, such as orange (Citrus sinensis (L.) Osbeck) [28].  RV: Range of variation; nd: Non-detected; tr: traces (<0.01 mg/100 g fw). Mean ± Standard deviation (n = 9). In each column, the different letters (a,b,c) indicate statistically significant differences (p < 0.05) between seasons for each variety. Regarding vitamin E, α-, β-, γ-, and δ-tocopherol were detected (Table 7), with the prevalence of α-tocopherol, representing more than 90% of the vitamin E vitamers (beta, gamma, and delta tocopherol) in all the varieties studied. The results obtained from the α-tocopherol content in the analyzed clementines are comparable to those previously reported by other authors in different citrus varieties, such as sweet orange (Citrus sinensis (L.) Osbeck) [29]. nd-non detected. In each row different letters (a,b,c) mean significant differences (p < 0.05).

Individual Phenolic Composition and Antioxidant Capacity of Clementine Fruits
Fourteen phenolic compounds were detected and tentatively identified in the studied samples, together with three other non-phenolic phytochemicals: dihydrophaseic acid and its glucoside and nomilin-glucoside. The peak characteristics (retention time, λmax in the visible region, and mass spectral data) are shown in Supplementary Materials Table S1. Tentative identifications and quantification of phenolic compounds in the hydromethanolic extracts of the three clementine varieties are shown in Supplementary Materials Table S2.
Flavonoids (peaks 5, 6, 9, 13-15, and 17) represented the largest family of phenolic compounds in the three varieties, being most of them identified as Cand O-glycosides, as expected in citrus varieties [30][31][32]. Peak 5 presented a pseudomolecular ion [M-H] − at m/z 593, releasing [33]. MS 2 fragments at m/z 503 (−90 u), 473 (−120 u), 383 (−120-90 u), and 353 (−120-120 u), a fragmentation pattern typical of C-glycosyl-flavones [33]. A compound with similar characteristics was previously identified as vicenin II (apigenin-6,8-di-C-glucoside), as the major phenolic constituent present in the citrus fruit juices [31]. Peak 9 presented a pseudomolecular ion [M-H] − at m/z 623 and was tentatively identified as diosmetin-6,8-di-C-glucoside. The presence of this latter C-glycosyl flavone has also been reported in Citrus suhuiensis and Citrus microcarpa fruits and citrus juices [31,34] Concerning phenolic acids derivatives (peaks 1-4 and 10-12), peak 1 was tentatively identified as sinapoyl-glucoside, based on its pseudomolecular ion ([M-H] − at m/z 385) and previous identification in C. limetta peel [32]. Peak 2 presented the same pseudomolecular ion as peak 1 but with a different fragmentation pattern. A compound with similar spectral characteristics was reported by [38] in the medicinal plant Mercurialis perennis L. and identified as feruloyl glucarate; the same compound (p-feruloyl-glucaric acid) and the analogous p-feruloyl-galactaric acid were also isolated from orange peel and fully identified by [39], thus, the same assumption was made for the herein-studied fruits. Mass spectral characteristics of peak 4 ([M-H] − at m/z 355 and MS 2 fragments at m/z 295, 235 and 193) were in accordance with those described by [38] for feruloyl-6 -O-glucose found in C. limetta peel and previously identified by [40] in lemon juice, so that this identity was also assumed for the compound detected herein.  [32,41]. As for peak 7 ([M-H] − at m/z 443), mass concordance has been found with the compound dihydrophaseic acid glucoside [42], a metabolite from the abscisic acid pathway identified in the peel of Citrus kawachiensis by [36]. A compound with the same mass characteristics was also found by [32] in the peel of C. limetta, although those authors did not propose a tentatively identification for the compound. Finally, peak 8 ([M-H] − at m/z 281) could correspond to the aglycone of peak 7, dehydrophaseic acid. The three extra early clementine varieties revealed high phenolic contents, being flavonoids the most abundant group (Table 8) with the flavanones narirutin/naringin and (neo) hesperidin as the main individual compounds. Table 8. Quantification (mg/g of methanolic extract) of the main phenolic compounds in extra early clementine varieties (mean ± SD, n = 9).

Season
Clementine Varieties

Total Phenolic Acids
Total Phenolic Compounds Sinapoyl-glucoside 1 Caffeoyl-hexoside 3 Vicenin II (apigenin-6,8-di-C-glucoside) 4 Narirutin/Naringin 5 Hesperidin/Neohesperidin 9  Table 9 displays the antioxidant activity of the clementine extracts. In general terms, Basol variety showed the highest antioxidant activity measured by the reducing power assay against oxidative stress (EC 50 values 1.32-2.46 mg/mL methanolic extract) and the β-carotene bleaching inhibition (0.21-0.82 mg/mL), while Clemensoon presented greater antioxidant activity measured through the DPPH assay (3.36-10.98 mg/mL methanolic extract), suggesting greater ability to avoid the formation of and/or scavenging oxidizing substances in the hydrophilic phase. Table 9. Antioxidant activity of the methanolic extracts (EC 50 , mg/mL methanolic extract) of the three studied extra early clementine varieties (mean ± SD, n = 9). The interpretation of the data of antioxidant activity of foods is complex, due to the variety of assays available. As different antioxidant activity assays measure their activity through mechanisms, the combined analysis of samples through different assays is often needed to have an overall idea of the antioxidant properties of the samples [43]. In order to assess which bioactive compounds present in the analysed clementine varieties are responsible for their antioxidant activity, a correlation analysis has been carried out. Related to antioxidant activity assays, DPPH assay provide useful information about clementines scavenging activity. Positive correlations (p < 0.05) were observed between EC 50 values from DPPH method results and vitamin C (0.4032; 0.0370); phenolic compounds (0.6647; 0.0002), which are mainly compound by sinapoyl-glucoside and caffeoyl-hexoside; flavonoids (0.7188; 0.0002), which are mainly compound by narirutin/naringin and hesperidin/neohesperidin, and PUFA (0.5023; 0.0076) mainly compound by linoleic and α-linolenic acid.
Similarly, β-carotene bleaching inhibition assay was highly and positively correlated with vitamin C (0.5132; 0.0062), flavonoids (0.5279; 0.0047) and phenolic acids (0.4943; 0.0088), which is indicative that these compounds are also active against the lipid peroxidation process. Furthermore, vitamin C is strongly correlated with α-tocopherol (0.4120; 0.0327), which would be indicative of the protective effect that vitamin C (ascorbic acid) exerts against the oxidation of free radicals. Other relevant correlations were observed by α-tocopherol and PUFA (−0.6646; 0.0002), thus avoiding its lipid peroxidation measured by β-carotene bleaching inhibition assay. This tendency was also reported by [43].
As it can be seen in Table 9, a great variability of EC 50 values was found between seasons, this could be explained due to the great variability also observed in their bioactive compounds content. Therefore, we can affirm that these bioactive compounds are the mains responsible for the antioxidant activities of clementine varieties, being useful in the prevention of several pathologies as previously reported by [44].
Overall, the antioxidant activity of all the samples can be considered satisfactory and similar with results reported in other citrus fruits like sweet orange [29,45] or pummel (Citrus grandis Osbeck) [1,46].

PCA Analysis of Clementine Fruits
In order to reduce the multidimensional structure of the data, a principal component analysis (PCA) was performed, which provided a two-dimensional map for explaining the observed variance. All the studied samples were plotted on the reduced space of the two principal components. The two components of the PCA performed explain 99.85% of the total variance (55.33% first and 44.51% second). The first principal component is highly and positively correlated to α-tocopherol (0.312771), soluble fiber (0.2957), and DPPH assay (0.2960), and negatively and highly correlated with PUFA (−0.3010), insoluble fiber (−0.2963) and inhibition of discoloration of β-carotene assay (−0.3068). The second principal component was strongly and positively correlated to soluble sugars, mainly sucrose (0.3523) and reducing power assay (0.3018), and negatively with citric acid (−0.3161), phenolic compounds, flavonoids (−0.3359), phenolic acids (−0.3338), and Fe (−0.3068).
All the studied clementine varieties are plotted on the reduced space of the two first principal components as shown in Figure 2. As it can be seen, the Basol variety was negatively characterized by the second principal component. Thus, this variety is highlighted by higher phenolic compounds, both flavonoids and phenolic acids, Fe and citric acid content, as well as by lower soluble sugars), contrarily to Clemenrubí, which was positively correlated with the first principal component. Clemensoon was negatively characterized by the first principal component and, therefore, is characterized by higher insoluble fiber and polyunsaturated fatty acids content and higher antioxidant activity measured by the inhibition method of discoloration of β-carotene. To the contrary, this variety is characterized by a lower α-tocopherol and soluble fiber content.

Conclusions
This work reports for the first time the phytochemical composition and antioxidant activity of some extra early clementine varieties (Citrus clementina Hort ex Tan. cv. Basol, Clemensoon and Clemenrubí) natives from Spain. It is important to highlight the presence of relevant levels of vitamin C (mainly ascorbic acid) that allow meeting the dietary reference intake (DRI, 80 mg/100 g) of this vitamin through the consumption of a normal portion of any of the studied clementines (around 75 g). These varieties also contained relatively high levels of total dietary fiber, with outstanding levels of the insoluble fraction in Clemenrubí variety. Furthermore, they also presented significant phenolic contents, being flavonoids the most abundant group of phenolic compounds, particularly the flavanones narirutin/naringin and (neo) hesperidin. These clementine varieties are revealed as good sources of bioactive compounds with potential promoting health benefits that could be of great interest for the food industry in order to develop foods enriched in natural compounds as well as to improve food antioxidant capacity.

Conclusions
This work reports for the first time the phytochemical composition and antioxidant activity of some extra early clementine varieties (Citrus clementina Hort ex Tan. cv. Basol, Clemensoon and Clemenrubí) natives from Spain. It is important to highlight the presence of relevant levels of vitamin C (mainly ascorbic acid) that allow meeting the dietary reference intake (DRI, 80 mg/100 g) of this vitamin through the consumption of a normal portion of any of the studied clementines (around 75 g). These varieties also contained relatively high levels of total dietary fiber, with outstanding levels of the insoluble fraction in Clemenrubí variety. Furthermore, they also presented significant phenolic contents, being flavonoids the most abundant group of phenolic compounds, particularly the flavanones narirutin/naringin and (neo) hesperidin. These clementine varieties are revealed as good sources of bioactive compounds with potential promoting health benefits that could be of great interest for the food industry in order to develop foods enriched in natural compounds as well as to improve food antioxidant capacity.