Characterization of phenolics and antioxidant abilities of red navel orange “Cara Cara” harvested from five regions of China

ABSTRACT Red-flesh navel orange “Cara Cara” is attractive for customers with its bright red color and has been widely planted in China. In this study, phenolic compounds in “Cara Cara” harvested from five different regions were identified and quantified by ultra high performance liquid chromatography (UPLC) coupled with Quadrupole-Time of Flight -Electrospray ionization-mass spectrometry (Q-TOF-ESI-MSn). Their antioxidant abilities were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorption capacity (ORAC) assays. Twenty phenolics were detected in crude extraction, with hesperidin (29.36–44.06%) as the dominant component, followed by narirutin (13.44–17.03%) and apigenin-6,8-di-C-glucoside (8.28–12.38%). No free phenolic acid was found in crude extracts and three free phenolic acids (FPA) were determined through alkaline hydrolysis, wherein ferulic acid (86.22–90.49%) existed dominantly. Fujian “Cara Cara” showed the highest total phenolic index (TPI) and total flavonoid index (TFI) values, while the lowest TPI and TFI values were detect in Chongqing “Cara Cara”. DPPH value showed positive correlation with TPC value (r2 = 0.921), while no correlation was found between ORAC and phenolic contents.


Introduction
Phenolic compounds, a large group of secondary metabolitesin plants, have aroused great scientific interest for their significant antioxidant activity. [1,2] Citrus fruits and juices are the common sources of natural phenolic compounds, which mainly include flavanone-O-glycosides, flanvone-O-, or -C-glycosides. [1,3] The composition and antioxidant activity of phenolics have been widely investigated in several citrus varieties such as mandarin, sweet orange, lemon, grapefruit, pummel, and tangerine. [4,5] "Cara Cara", a kind of lycopene-accumulated sweet orange mutated from Washington Navel orange, was first introduced to China in the 1990s and widely planted in Hubei, Fujian, Chongqing, Jiangxi, and Hunan provinces of China. Due to the accumulation of lycopene, "Cara Cara" fruits display an attractive bright red color, which makes them appealing to customers. [6] However, previous studies concerning phenolic compounds of "Cara Cara" were only focused on the analysis of the dominated ingredients such as hesperidin, narirutin, and didymin. [7,8] The detailed phenolic profiles of "Cara Cara" fruits have not been adequately studied.
Except for their contribution to human health, phenolics are also beneficial for plant itself, acting as attractants, feeding deterrents, stress-protecting agents, and physiological active compounds. [9] The accumulation of phenolics was influenced by various environmental factors. It has been demonstrated that growing region affected the phenolic concentration and composition in fruit. [10] By comparison of phenolic content harvested from different regions, the regional differences on "Cara Cara" could be analyzed, which might be helpful to planters. However, the phenolic differences of "Cara Cara" fruits have not been reported among the growing regions of China.
Phenolic compounds are usually analyzed by HPLC coupled with MS/MS on a single quadrupole instrument or by ion-trap mass spectroscopy. These approaches needed approximately 50 min for phenolic structure analysis with a relatively low accuracy. UPLC system performs the separation of compounds on smaller column particles with higher working pressure, which may take a shorter analysis time and provide a better peak capacity. UPLC-Q-TOF-ESI-MS n is an novel equipment with high mass accuracy in both full scan and MS n stages, which can provide much structural information of glycoside compounds. [11] The objective of this study is to identify and quantify the phenolics in "Cara Cara" from five typical citrus growing regions of China by UPLC-Q-TOF-ESI-MS n , and compare their antioxidant abilities based on 2,2diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorption capacity (ORAC).  15.62, which were calculated by the ratio of°Brix/acidity. [12] "Cara Cara" fruits were peeled and milled with a tissue grinder to obtain homogeneous pulp; then, they were lyophilized (−40°C,1 Pa, 48 h) by a freeze dryer (Virtis freeze mobile, Virtis Co., Gardiner, USA). Lyophilized pulp was ground into fine powder, and stored at −80°C for further analysis. In addition, commercial authentic standards of caffeic acid, p-coumaric acid, ferulic acid, rutin, narirutin, hesperidin, didymin and chemicals of ascorbic acid, Folin-Ciocalteu reagent, DPPH, AAPH2,2ʹ-azobis-(2-methylpropionamidine) dihydrochloride, and fluorescein were obtained from Yuanye Bio-Technology Co., Ltd (Shanghai, China). 6-Hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox) was purchased from Sigma (St. Louis,USA). HPLC-gradesolvent of acetonitrile was acquired from Thermo Fisher Scientific (Leicestershire, UK). Otheranalytical grade chemicals were purchased from Sinopharm chemical reagent Co., Ltd (Shanghai, China).

Extraction of phenolics
Phenolics in lyophilized pulp of "Cara Cara" were extracted by ultrasonic treatment. Briefly, 0.5 g freezedried material was extracted by 20 mL of 84.81% ethanol with 0.1% HCl (v/v) using an ultrasonic cleaner with working frequency at 40 kHz (KQ-500E, Kun Shan Ultrasound Instrument Co., Jiangsu,China) for 5.99 min at 57.37°C, which was repeated twice. The final supernatantvolume was made up to 40 mL and filtered through a 0.22-μm polytetrafluoro ethylene (PTFE) membrane for further analysis.
For UPLC-Q-TOF-ESI-MS n analysis, a MS system (Synapt G2) equipped with an ESI source and a Q-TOF analyzer (Waters Corp., MA, USA) was used. The HPLC conditions were the same as those described earlier. Parameters for MS were as follows: negative mode (ESI -); source temperature, 120°C; cone gas flow, 50 L/h; desolvation temperature, 450°C; desolvation gas flow, 800 L/h. Voltages of sample cone and capillary were set at 30 and 2500 V, respectively.

Determination of free phenolic acids
FPA in the crude extracts were determined by the existing method with minor modifications. [4,13] Briefly, the concentrated crude extracts (5 mL) were alkali hydrolyzed with 5 mL NaOH (4 M) containing EDTA (10 mM) and 1% ascorbic acid (w/v) for 6 h at room temperature. Then, HCl (6 M) was added to acidify the reaction mixture to pH 2, followed by the addition of diethyl ether: ethyl acetate (1:1 v/v) to extract the FPA for three times from the hydrolysate. Next, the extracts were evaporated to dryness using a rotary evaporator (30°C). Finally, the samples were re-dissolved in 2 mL methanoland filtered through a 0.22-μm PTFE membrane for further UPLC analysis.
Oxygen radical absorption capacity (ORAC) assay The ORAC assay was conducted based on the existing protocols with minor modifications. [16] Briefly, 200-μL fluorescein reagent (0.0868 nM) was mixed with 25-μL blank, phenolic extracts and Trolox standard (12.5-125 μM) in a 96-well plate. The mixture was incubated at 37°C for 30 min, followed by the quick addition of 25-μL AAPH (153 mM) to generate peroxyl radical reaction. The fluorescence was monitored every minute at an excited wavelength of 485 nm and an emission wavelength of 528 nm, and the total measurement was kept for 2 h at 37°C on a multi-mode plate reader (Synergy HTX, Bio Tek instrument, Winooski, VT, USA). The area under the fluorescence decay curve (AUC) was calculated, and the net AUC along with the different values of AUC between blank and phenolic extracts or Trolox standard were used for data analysis. The final results were expressed as μmol Trolox equivalent (TE) per gram dry weight of "Cara Cara" pulp (μmol TE/g DW). Phenolic extracts were diluted 40 times for ORAC assay to ensure thefluorescence can decay in 120 min.

Statistical analysis
All the experiments were conducted in triplicate. The data were expressed as mean ± SD of triplicate independent experiments. One way analysis of variance (ANOVA) was used to compare the means, and significant differences were considered at p < 0.05 (Duncan's post-hoc test). All statistical analyses were performed with IBM SPSS Statistics version 20.0.
Peak 12 was tentatively inferred as genistein-7-O-xylosylglucoside malonylated based on previous studies. [24,25]  Peak 19 without sufficient MS information was tentatively inferred as 1,2-diferuloylgentiobiose by its UV-Vis spectrum and molecular ion. [13] No free phenolic acid was detected in crude extracts, and alkaline hydrolysis was performed to investigate the FPA present in "Cara Cara" pulps. As shown in Fig. 1b, caffeic acid, p-coumaric acid, and ferulic acid appeared after alkaline hydrolysis, and they were positively confirmed by their authentic commercial standards. During alkaline hydrolysis, flavonoids were partly converted into their chalcone forms. In present study, nariutin and hesperidin chalcone were clearly observed in alkali hydrolyzed sample and confirmed by alkali hydrolyzed mixed standards of nariutin and hesperidin (Fig. 1b). Phenolic acids, only detectable in alkali hydrolyzed extracts, suggested they might exist in bound forms in "Cara Cara".

Quantification of phenolic compounds
A total of 20 phenolic compounds were found in crude extraction and three FPA were detected in alkali hydrolyzed extraction, with their individual contents listed in Tables 2 and 3, respectively. In crude extracts, individual phenolic compounds ranged from 0 to 691 mg/100 g DW, with hesperidin accounting for 29.36-44.06% as the predominant ingredient, followed by narirutin (13.44-17.03%) and apigenin-6,8-di-Cglucoside (8.28-12.38%) ( Table 2). Total phenolic index (TPI), i.e., sum of individual phenolic contents, ranged from 1177 to 1732 mg/100 g DW, with total flavonoid index (TFI), i.e., sum of individual flavonoid contents, rainging from 962 to 1500 mg/100 g DW, indicated that TFI contributed to the most of TPI. In the detected 20 phenolic compounds (Fig. 1a), nine of them were conformed as flavanones, which took up to 64.14-73.23% of TPI. In alkali hydrolyzed samples, ferulic acid accounting for 86.22-90.49% was dominated for FPA, followed by caffeic acid and p-coumaric acid (Table 3). Similar trends were also found in other citrus species of Ponkan and Huyou. [26] Due to the same genotype, phenolic profiles in "Cara Cara" harvested from five regions were identical, and the differences of phenolic contents in those fruits might be attributed to environmental influence, including fertilization, irrigation, temperature, light, and so on. [9,27,28] In our study, Fujian "Cara Cara" had the highest TPI and TFI, while the lowest TPI and TFI were detected in Chongqing "Cara Cara". The geographical origins and morphologic characteristics of "Cara Cara" were described previously [29] , and Fujian region had relatively high temperature, moderate rainfall, and lighting time. It has been demonstrated that most of flavonoid genes were responsive to mineral nutrient and light intensity, especially UV-B [30] , and flavonoid in citrus was biosynthesized and accumulated during the early period of young fruit, then decreased in the following grow period. [31] However, the accumulation of phenolics in plant was complicated, and several stressful conditions were also reported to enhance the phenolic concentration in fruits, such as wounding, nutrient deficiency, pathogens, and radiations. [27] To investigate the environmental influence on phenolic accumulation in "Cara Cara" fruits, the planting soils and the garden trails of "Cara Cara" trees should be further studied.

Total phenolic contents and antioxidant abilities
Spectrophotometric calculation of TPC, DPPH, and ORAC values are shown in Table 4. In all the extracts, TPC, ranging from 652 to 792 mg GAE/100 g DW as calculated by the Folin-Ciocalteu method, were lower than the TPI, and this phenomenon might be attribute to the fact that some peaks were quantified as using narirutin, not their authentic standards. Previous research demonstrated that TPC were higher than TPI, which might be attributed to the incomplete calculation of all peaks determined by HPLC. [32] However, the calculation of TPC and TPI was based on different quantification methods and their results might be not comparable. The trends that TPC were lower than TPI have also been reported previously. [33] DPPH assay is based on the reception of hydrogenatom from antioxidants; whereas, ORAC assay is used to calculatethe antioxidant ability of eliminating peroxyl radical (ROO·) generated by 2,2ʹazobis-(2-methylpropionamidine) dihydrochloride (AAPH). [34] DPPH values of Hubei and Hunan samples were significantly higher than others, while no significant differences were found in ORAC values of tested samples. DPPH value showed positively correlation with TPC value (r 2 = 0.921). The strong positive correlations indicated that the phenolics in "Cara Cara" pulp were the major contributors to DPPH value. [33] DPPH radical is only dissolved in organic solvent, and this method is applicable to analyze the antioxidant ability of hydrophobic systems. Hesperidin, a hydrophobic compound, predominantly existed in the phenolic extracts of Cara Cara, and this situation might contribute to the good correlation between DPPH assay and TPC. The lack of correlation was found between ORAC and TFI or TPC, and possible explanations are presented as follows: (1) phenolic compounds were not the only potential antioxidant ingredients of the corresponding samples; (2) in ORAC assay, kinetics action of antioxidants lack essential correlation with phenolics. [16,32] Conclusion Phenolic compounds in "Cara Cara" collected from five different regions of China were adequately identified and quantified by UPLC coupled with Q-TOF-ESI-MS n , with 20 phenolics detected in crude extracts. Flavonoids were confirmed to be the main phenolics, especially flavanones. Hesperidin was proved as the predominant ingredient in all samples, followed by narirutin and apigenin-6,8-di-Cglucoside. Phenolic profiles in those "Cara Cara" pulps were similar, while phenolic contents were different, which might be attributed to the environmental differences. Phenolic in "Cara Cara" pulp was the main contributor to DPPH value. These results not only enrich the nutritional knowledge of "Cara Cara", but also provide valuable guide for "Cara Cara" planting.

Funding
This work was supported by National Natural Science Foundation of China (grant number 31571847). Values are expressed as mean ± SD, n = 3. Values followed by different letters in the same column are significantly different (p < 0.05). b TPC: total phenolic content (gallic acid equivalent, mg GAE/100 g DW).