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Orange juice is largely produced, exported and consumed in Brazil. It is an important source of bioactive compounds, such as flavonoids and phenolic acids, which are beneficial to the health of consumers. The aim of this work was to evaluate the phenolic compounds profile of commercial orange juice from Brazil using HPLC-DAD and UPLC-ESI-MS, and multivariate analysis. Forty-five phenolic compounds and one precursor were identified: cinnamic acid, 19 cinnamic acid derivatives, 8 flavanones and 18 flavones. Rutin, eriocitrin, narirutin, naringin, hesperidin, naringenin, nobiletin and tangeritin as well as caffeic, p-coumaric and ferulic acids were present in juices from all brands. Hesperidin and narirutin presented the highest levels considering all brands, while tangeretin and ferulic acid had the lowest levels in all juices. Principal component analysis of the phenolic compounds profile showed a wide variety among juices within the same brands, making it hard to perceive major differences among the brands.
Department of Food and Nutrition, School of Pharmaceutical Science, São Paulo State University–UNESP, Araraquara, SP, Brazil
Estela Mesquita
Department of Food and Nutrition, School of Pharmaceutical Science, São Paulo State University–UNESP, Araraquara, SP, Brazil
Magali Monteiro*
Department of Food and Nutrition, School of Pharmaceutical Science, São Paulo State University–UNESP, Araraquara, SP, Brazil
*Address all correspondence to: magali.monteiro@unesp.br
1. Introduction
Brazil is the largest producer and exporter of orange juice worldwide. The crop of 2017/2018 produced 370 million boxes of oranges (40.8 kg/box) and a total of 1.3 million ton of juice. In 2019/2020, the production was of 365.4 million boxes of oranges and 965 thousand ton of juice. A total juice production of 1.2 million ton is predicted for the 2020/2021 crop. Brazilian yearly exportation was of 1.1 million ton of juice from 2018 to 2020. The majority of the juice was exported to Belgium and the Netherlands, Japan, China and the US [1].
Fresh orange juice is processed to result in Not from Concentrate (NFC) and Frozen Concentrated Orange Juice (FCOJ). While the NFC juice maintains the aroma and flavor characteristic of fresh orange juice, the FCOJ is a most desirable final product in terms of stability and storage, which is a good alternative for exportation [2, 3]. The NFC orange juice is widely available in the Brazilian market and is widely preferred by consumers when compared to FCOJ. It is also being exported alongside FCOJ. On the other hand, there is still little information concerning its composition and quality.
Orange juice is an important source of ascorbic acid and contains other important bioactive compounds such as flavonoids and phenolic acids, which have been associated to health benefits [4, 5, 6].
High Performance Liquid Chromatography (HPLC) is the main technique used for phenolic compound analysis in a wide variety of matrixes, especially foods. Reverse-phase columns, diode array detectors (HPLC-DAD) and mass spectrometer (HPLC-MS and HPLC-MS-MS) is very usual, as well as acid and polar solvents. For the separation of compounds with different structures and polarities, it is necessary to use a solvent gradient. Due to the presence of the characteristic nuclei and specific radicals, the UV/Vis spectra of flavonoids are typical for each class, usually with two bands of absorption: between 240 and 280 nm (band I, maximum absorption) and between 300 and 380 nm (band II). More specifically, flavanones present maximum absorption between 275 and 290 nm (band I) and between 300 and 380 nm (band II), with a shoulder between 300 and 340 nm (band II). Flavones, in turn, have maximum absorption between 245 and 260 nm (band I) and absorption between 350 and 380 nm (band II). The structure variations within a same class will result in discreet variations in the spectra, with hypsochromic or bathocromic shifts, although this is not sufficient for structure elucidation or differentiation. The mass spectra of flavonoids are also characteristic of the common nuclei among classes. The break of bonds produces specific fragments for glycosides and aglycones, thus making mass spectrometry a good option for structure differentiation and elucidation, and compounds identity confirmation. The same fundamentals apply for phenolic acids [7, 8].
The majority of the works report high levels of the flavonoids hesperidin and narirutin as well as lower levels of other flavanones and flavones in juice made of oranges from several varieties, including Pera-Rio [7, 9, 10]. There are fewer reports on the phenolic acid contents of orange and orange juice and to the best of our knowledge there is no available data on the phenolic composition of the Brazilian commercial orange juice. Thus, the aim of this work was to evaluate the profile of phenolic compounds of Brazilian commercial orange juice from different brands using HPLC-DAD and UPLC-ESI-MS, and employing multivariate analysis. The novelty of this work lies on the growing importance of NFC juice consumption and exportation in Brazil, which affects economic and health issues. To our knowledge, this is the first study that aims to profile the phenolic composition of commercial juice and, on top of that, evaluate this profile throughout the orange harvest. To achieve that, we applied a previously developed and validated HPLC-DAD method, alongside UPLC-ESI-MS use for identification of the compounds.
Standards (95–99%) of gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, trans-cinnamic acid, rutin, eriocitrin, quercitrin, narirutin, naringin, hesperidin, quercetin, naringenin, hesperitin, nobiletin and tangeretin were obtained from Sigma Aldrich (Missouri, USA) and ChromaDex (California, USA). Standards were weighted and diluted in methanol in order to obtain standard solutions. Formic acid of analytical grade was from Synth (São Paulo, Brazil). Acetonitrile, methanol and ethyl acetate of HPLC grade were from J.T. Baker (Pennsylvania, USA), and ultrapure water was obtained from a direct Q-3 UV System from Millipore (Massachusetts, USA).
2.2 Orange juice samples
Commercial NFC orange juice from eight brands (A, B, C, D, E. F, G and H) was purchased from retail stores in the region of Araraquara, SP, Brazil. Three bottles (1 L) from each brand with distinct production dates (1, 2 and 3) between January and November of the year 2017 were acquired (n = 24). Samples were frozen and lyophilized in a Moduloyd Freeze Dryer RV8 A65413906 (Massachusetts, USA) before extraction.
2.3 Extraction of phenolic compounds
The phenolic compounds were extracted from the lyophilized juice matrix (3 g) with aqueous methanol solution (90%, v/v; 5 mL) in ultrasonic bath (20 min). Extracts were centrifuged (9000 g/20 min), supernatants were collected and the extraction was repeated once. The clean-up step using solid-phase C18 (500 mg, 6 mL) cartridges (Bond Elut, Agilent Technologies) was performed as described in a work previously published by our research group [9]. Briefly: cartridges were conditioned with ethyl acetate and aqueous formic acid (18 mL each). Juice extracts were eluted and then cartridges were washed with ethyl acetate (30 mL). Extracts were then dried under N2 flow and suspended in aqueous formic acid (0.1% v/v), filtered through 0.22 μm cellulose disk filters and stored at −20°C until analysis. Juices were extracted in duplicate, and analyzed within a maximum of 15 days.
2.4 HPLC-DAD and UPLC-ESI-MS profiling of phenolic compounds
Profiling of phenolic compounds of Brazilian commercial orange juice was performed in two steps. At first, HPLC-DAD was used for separation, UV-Vis spectra assessment and comparison with standards (spectra and retention time). An Acquity ARC system with a diode array detector (Waters, USA) equipped with a BEH X-Bridge C18 column (250 × 4.6 mm, 5 μm) and a guard column (20 × 4.6 mm, 5 μm) was employed. Conditions were as developed and validated in the previous work of Mesquita and Monteiro [9], which were as follows: mobile phase of water:formic acid (99.9:0.1, v/v) and acetonitrile, column temperature of 50°C, flow rate of 1.0 mL.min−1, and injection volume of 20 μL. The gradient was 6–10% of acetonitrile (0–16 min), 10–22% (16–36 min), 22–100% (36–38 min), and then maintained for 5 min. Column was equilibrated for 10 min between runs. The spectra were acquired from 210 to 400 nm. Orange juice extracts and standard solutions were injected in duplicate.
On the second step of the profiling an UPLC-ESI-MS was used for identification and/or confirmation of phenolic compounds identity. The system employed was the Acquity UPLC H-Class with ESI-QtoF (Xevo G2-XS QTOF Waters, USA). Conditions were adapted to the UPLC system using a BEH C18 column (2.1 × 100 mm, 1.7 μm): flow rate was of 0.4 mL min−1 and injection volume was of 2.0 μL. The gradient was 6–10% acetonitrile (0–10 min), 10–22% (10–24 min), 22–100% (24–24.5 min), maintained for 2.5 min. Column was equilibrated for 3 min between runs. Capillary voltage used was of 3.0 kV (ESI+) and 2.7 kV (ESI−), source temperature was 120°C and gas temperature was 400°C. The mass spectra (m/z 50–1200) were obtained in positive and negative modes. Orange juice extracts and standard solutions were injected in duplicate: once in positive and once in negative modes.
2.5 Quantification of phenolic compounds
The quantification of phenolic compounds was also performed in the HPLC-DAD system, with the conditions described in Section 2.4. Calibration curves of chlorogenic, caffeic, p-coumaric and ferulic acids, rutin, eriocitrin, narirutin, naringin, hesperidin, naringenin, nobiletin and tangeritin were prepared and injected in triplicate. Curve parameters are presented in Table 1. Orange juice extracts were injected in duplicate. Chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, trans-cinnamic acid, rutin, eriocitrin, quercitrin, narirutin, naringin, hesperidin, naringenin, nobiletin and tangeretin were quantified using the respective standards. The phenolic compounds identified by the UV-vis spectra characteristic of chemical class and by ESI-MS were quantified and expressed as the major compound of each chemical class present in the matrix; flavanones were expressed as hesperidin, cinnamic acid derivatives as ferulic and p-coumaric acids; and flavones as tangeretin and rutin.
Regression equations, r values and concentration range of the calibration curves employed in the quantification of the phenolic compounds of Brazilian commercial orange juice, with limits of detection and quantification.
LoD (limit of detection) and LoQ (limit of quantification) values from Mesquita and Monteiro [9].
Chlorogenic acid was not included in the validation step.
2.6 Statistical analysis
Principal component analysis (PCA) was performed based on the correlation matrix of phenolic compounds area values of all juices from each brand; 21 variables and 24 cases, respectively. Compounds were represented by name or peak number according to Table 2. Only the variables with a correlation ≥0.65 (rotate component matrix) were included. The PCA was performed using STATISTICA 10.0 (StatSoft, Oklahoma, USA).
The typical phenolic compounds profile of Brazilian commercial orange juice obtained with HPLC-DAD is presented in Figure 1. The chromatographic profile was similar among juices from all brands, considering all production dates.
Figure 1.
Typical HPLC-DAD chromatogram of Brazilian commercial orange juice. Conditions as described in Section 2.4. Peaks numbered according to Table 1.
Table 2 presents the compounds identified in the juices; peaks were numbered according to retention time (1–46). The identification was based on: (1) retention time from HPLC-DAD separation and UV-vis spectra of compounds compared to pure analytical standards, (2) UV-vis spectra characteristic of flavonoids or phenolic acid chemical classes, and (3) molar mass obtained by UPLC-ESI-MS. Forty six compounds were identified: 26 flavonoids and 19 phenolic acids, as well as the phenolic acid precursor cinnamic acid (Table 2). The majority of the compounds were identified in all brands evaluated, considering all production dates.
All phenolic acids identified in Brazilian commercial orange juice were cinnamic acid derivatives, as verified by the UV-vis spectra characteristic of this chemical class. The spectra showed absorption from 270 to 370 nm, with a band of high intensity from 300 to 370 nm, a band of lower intensity from 270 to 300 nm and maximum absorption between 325 and 330 nm [9, 11]. Sinapinic acid (peak 13), hydroumbellic acid (peak 15), dihydroxycinnamic acid (peak 16), and p-coumaric acid (peak 21), as well as 11 unnamed derivatives (peaks 2–7, 9, 10, 32, 36 and 39) were present in juices from all brands, in varying levels. Chlorogenic acid (peak 8), caffeic acid (peak 11), ferulic acid (peak 26), and one unnamed derivative (peak 1) were also identified, but not in all brands.
Quantified levels of all compounds are shown in Tables 3 and 4. Table 3 shows the range of concentration and Table 4 shows the concentration of all the compounds in the brands evaluated, considering all production dates. The highest level was of the derivative represented by peak 7 (7.98 mg 100 g−1), whereas the lowest was of ferulic acid (0.04 mg 100 g−1). The precursor cinnamic acid was also found in low levels (0.54–0.94 mg 100 g−1) in two of the brands evaluated, C and E (Table 3). Chlorogenic acid (0.3–2 mg 100 g−1), caffeic acid (0.2–1 mg 100 g−1) and ferulic acid (0.04–0.7 mg 100 g−1) were present only in some of the brands evaluated, whereas p-coumaric acid (0.1–0.8 mg 100 g−1) was in all of them.
Peak
Compound/chemical class
Range of concentration (mg 100 g−1 of lyophilized juice)
A
B
C
D
E
F
G
H
1
Cinnamic acid derivative
0.16–0.24
nd
nd
0.17–0.27
0.10–0.23
0.13–0.21
0.16–0.51
nd
2
Cinnamic acid derivative
0.24–0.33
0.27–0.75
0.13–0.46
0.20–0.39
0.11–0.25
0.14–0.39
0.32–0.45
0.14–0.61
3
Cinnamic acid derivative
0.18–0.29
0.26–0.52
0.11–0.51
0.27–0.35
0.15–0.44
0.13–0.31
0.27–0.51
0.25–0.46
4
Cinnamic acid derivative
0.36–0.47
0.39–1.17
0.17–0.54
0.32–0.59
0.21–0.51
0.37–0.60
0.42–0.64
0.21–0.81
5
Cinnamic acid derivative
1.11–1.88
0.76–1.17
0.43–2.71
0.65–0.95
0.15–0.86
1.61–1.79
0.92–1.19
0.52–0.89
6
Cinnamic acid derivative
nd–0.66
0.13–0.73
nd–0.06
nd–0.30
0.07–3.21
nd–0.59
nd–1.03
0.58–0.99
7
Cinnamic acid derivative
3.28–4.34
1.15–1.56
1.76–4.37
1.47–2.52
2.71–4.25
2.61–7.98
1.97–3.78
1.06–2.11
8
Chlorogenic acid
nd–0.35
nd–1.31
nd
0.53–0.75
nd
nd
nd–1.95
0.56–1.29
9
Cinnamic acid derivative
0.56–0.65
0.31–0.70
0.22–0.53
0.33–0.74
0.25–0.52
0.26–0.74
0.45–0.57
0.36–0.69
10
Cinnamic acid derivative
0.08–0.21
0.09–0.12
0.06–0.18
0.07–0.19
0.06–0.08
0.13–0.22
nd–0.17
nd–0.08
11
Caffeic acid
nd–0.43
nd
nd–1.40
0.18–0.41
nd
nd - 0.34
nd–0.79
nd–0.44
12
Flavone
nd–1.48
0.90–2.24
0.44–2.08
0.15–1.82
1.00–3.27
0.08–2.91
0.27–2.89
0.94–1.64
13
Sinapinic acid
0.22–0.72
0.34–0.61
0.20–0.86
nd–0.64
0.22–0.47
0.27–0.59
nd–0.57
0.31–0.54
14
Lucenin 2
0.73–0.95
0.36–1.33
0.31–0.90
0.07–0.57
0.54–1.73
0.37–1.99
nd–1.13
0.66–1.63
15
Hydroumbellic acid
1.77–1.92
1.15–1.83
0.78–3.13
1.47–2.45
0.92–2.05
0.41–2.95
1.46–1.83
1.39–4.77
16
Dihydroxycinnamic acid
0.11–0.28
0.17–0.25
0.11–0.35
0.06–0.28
0.13–0.30
0.15–0.43
0.13–0.38
0.14–0.33
17
Vicenin 2
9.87–17.02
8.33–15.72
3.61–19.18
6.24–16.24
5.26–21.00
4.86–24.20
13.09–19.10
7.27–10.80
18
Diosmetin
1.52–1.65
0.87–1.20
0.47–2.02
0.94–1.59
0.64–2.14
1.24–1.90
1.89–1.94
0.73–1.50
19
Astragalin
0.31–1.20
0.24–0.35
0.11–0.47
0.13–0.29
0.16–0.42
0.19–0.53
0.31–0.34
0.15–0.37
20
Apigenol
0.26–0.33
0.17–0.34
0.07–0.46
0.02–0.14
0.19–0.77
0.10–0.46
0.40–0.88
0.16–0.46
21
p-coumaric acid
0.30–0.80
0.13–0.30
0.12–0.21
0.19–0.36
0.10–0.19
0.47–0.84
0.05–0.19
0.08–0.19
22
Flavanone
2.19–3.56
2.01–3.72
0.92–4.40
0.98–1.92
1.39–4.42
2.45–4.69
3.34–4.24
2.02–3.50
23
Rhoifolin
0.38–0.54
0.25–0.60
0.11–0.60
0.44–2.59
0.15–0.44
0.37–0.50
0.58–0.88
0.16–0.37
24
Flavanone
2.62–5.05
3.56–28.35
1.46–7.20
2.74–3.86
2.35–10.25
3.46–6.74
6.64–9.69
2.79–5.56
25
Natsudaidain
0.19–0.28
0.12–0.32
0.06–0.37
0.15–0.20
0.15–0.46
0.25–0.40
0.25–0.33
0.17–0.31
26
Ferulic acid
nd–0.74
nd- 0.12
nd–0.07
0.05–0.10
nd–0.48
0.09–0.10
nd
nd–0.04
27
Isovitexin
0.87–1.58
0.66–1.75
0.28–1.92
0.78–1.21
0.57–2.35
0.96–1.93
1.92–2.33
0.88–1.74
28
Rutin
0.27–0.99
0.14–0.68
0.15–0.58
0.25–0.28
0.35–1.00
0.37–1.47
0.61–0.76
0.45–0.89
29
Isoquercitrin
0.68–1.14
0.66–1.54
0.26–1.65
0.66–0.77
0.51–1.94
0.65–1.60
1.52–1.85
0.68–1.40
30
Vicenin 1/3
0.45–0.52
0.25–0.42
0.11–0.84
0.25–0.46
0.20–0.54
0.34–0.52
0.42–0.48
0.14–0.30
31
Eriocitrin
3.72–6.11
3.14–5.18
1.51–7.36
2.62–3.90
2.89–10.47
3.72–8.25
5.43–7.39
1.90–5.21
32
Cinnamic acid derivative
0.80–1.34
1.07–2.18
0.37–3.62
0.84–1.29
0.76–2.65
0.97–1.83
2.07–2.41
0.94–1.91
33
Flavanone
0.38–1.35
0.37–0.53
0.19–1.12
0.22–0.56
0.08–0.77
0.61–1.70
0.77–1.21
0.35–0.93
34
Narirutin
8.22–17.60
8.15–19.80
4.45–24.91
8.76–15.82
7.62–36.76
5.21–30.11
19.86–28.56
8.02–13.76
35
Naringin
1.75–1.84
0.92–1.54
0.50–2.17
0.51–1.18
0.88–2.09
1.61–3.01
1.53–2.10
0.69–5.39
36
Cinnamic acid derivative
0.19–0.38
0.17–0.27
0.09–0.56
0.14–0.21
0.14–0.43
0.18–0.43
0.28–0.44
0.15–0.25
37
Hesperidin
60.94–99.89
41.80–62.51
29.20–113.17
46.27–122.00
23.21–88.85
49.79–97.45
42.25–72.44
30.14–44.21
38
Cinnamic acid
nd
nd
nd–0.94
nd
nd–0.54
nd
nd
nd
39
Cinnamic acid derivative
0.24–0.38
0.25–0.51
0.10–0.79
0.20–0.34
0.17–0.53
0.19–0.44
0.53–0.57
0.26–0.47
40
Naringenin
0.54–1.09
0.65–2.15
0.34–2.00
1.32–1.73
0.44–2.22
0.64–1.19
1.98–2.73
0.80–1.84
41
Diosmin
0.15–0.37
0.21–0.51
0.05–0.39
0.24–0.63
0.13–0.51
0.09–0.32
0.34–0.50
0.07–0.31
42
3.5.6-trihydroxy-3′.4′.7-trimethoxyflavone
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
43
Nobiletin
0.26–0.38
0.26–0.72
0.10–0.54
0.42–0.75
0.16–0.65
0.23–0.36
0.52–0.68
0.28–0.65
44
Heptamethoxyflavone
0.12–0.18
0.13–0.38
0.06–0.22
0.13–0.39
0.05–0.21
0.13–0.17
0.16–0.24
0.11–0.25
45
Tangeritin
<LoQ
<LoQ–0.02
<LoQ
0.01–0.03
<LoQ–0.02
<LoQ–0.02
0.01
<LoQ–0.01
46
3.5.8-trihydroxy-3′.4′-dimethoxyflavone
0.02–0.04
nd–0.02
nd
nd–0.07
nd
nd–0.05
nd
nd
Table 3.
Phenolic compounds (mg 100 g−1) of Brazilian commercial orange juice.
Results expressed as mean value.
nd: not detected; LoQ: limit of quantification [9].
Peak
Compound/chemical class
Concentration (mg 100 g−1)
A
B
C
D
E
F
G
H
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
Cinnamic acid derivative
0.24
0.19
0.16
nd
nd
nd
nd
nd
nd
0.17
0.19
0.27
0.14
0.23
0.10
0.13
0.18
0.21
0.16
0.28
0.51
0.16
0.28
0.51
2
Cinnamic acid derivative
0.24
0.27
0.33
0.75
0.31
0.27
0.13
0.46
0.34
0.20
0.22
0.39
0.25
0.21
0.11
0.14
0.27
0.39
0.32
0.39
0.45
0.32
0.39
0.45
3
Cinnamic acid derivative
0.29
0.22
0.18
0.52
0.27
0.26
0.11
0.33
0.51
0.30
0.27
0.35
0.44
0.33
0.15
0.13
0.22
0.31
0.27
0.51
0.51
0.27
0.51
0.51
4
Cinnamic acid derivative
0.47
0.36
0.38
1.17
0.49
0.39
0.17
0.54
0.35
0.32
0.36
0.59
0.51
0.36
0.21
0.37
0.48
0.60
0.42
0.56
0.64
0.42
0.56
0.64
5
Cinnamic acid derivative
1.88
1.11
1.19
1.17
1.05
0.76
0.43
1.85
2.71
0.95
0.65
0.86
0.15
0.86
0.56
1.61
1.79
1.66
1.10
1.19
0.92
1.10
1.19
0.92
6
Cinnamic acid derivative
0.66
nd
0.58
0.73
0.16
0.13
0.06
nd
nd
0.14
nd
0.30
0.17
3.21
0.07
0.54
0.59
nd
nd
0.54
1.03
nd
0.54
1.03
7
Cinnamic acid derivative
4.34
3.94
3.28
1.56
1.56
1.15
1.76
4.37
3.03
2.52
1.72
1.47
4.25
2.71
2.71
2.61
4.93
7.98
3.78
2.90
1.97
3.78
2.90
1.97
8
Chlorogenic acid
0.35
nd
nd
1.31
nd
nd
nd
nd
nd
0.61
0.53
0.75
nd
nd
nd
nd
nd
nd
nd
1.29
1.95
0.63
0.56
1.29
9
Cinnamic acid derivative
0.56
0.60
0.65
0.70
0.55
0.31
0.23
0.53
0.22
0.34
0.33
0.74
0.52
0.38
0.25
0.26
0.59
0.74
0.57
0.57
0.45
0.57
0.57
0.45
10
Cinnamic acid derivative
0.08
0.17
0.21
0.12
0.11
0.09
0.06
0.18
0.08
0.12
0.07
0.19
0.06
0.08
0.07
0.13
0.21
0.22
0.17
0.11
nd
0.17
0.11
nd
11
Caffeic acid
0.43
nd
nd
nd
nd
nd
nd
nd
1.40
0.18
0.41
nd
nd
nd
nd
0.34
0.29
nd
nd
0.79
nd
0.44
nd
nd
12
Flavone
nd
1.48
1.18
2.24
1.27
0.90
0.44
1.83
2.08
1.69
0.15
1.82
3.27
1.55
1.00
0.08
0.35
2.91
2.89
0.27
2.36
2.89
0.27
2.36
13
Sinapinic acid
0.22
0.72
0.51
0.61
0.41
0.34
0.20
0.66
0.86
0.46
nd
0.64
0.47
0.28
0.22
0.27
0.46
0.59
0.57
nd
0.53
0.57
nd
0.53
14
Lucenin 2
0.73
0.80
0.95
1.33
0.64
0.36
0.31
0.77
0.90
0.07
0.11
0.57
1.73
1.02
0.54
0.37
1.19
1.99
1.13
nd
0.94
1.13
nd
0.94
15
Hydroumbellic acid
1.92
1.83
1.77
1.71
1.83
1.15
0.78
2.03
3.13
1.55
1.47
2.45
2.05
1.52
0.92
0.41
1.89
2.95
1.83
1.75
1.46
1.83
1.75
1.46
16
Dihydroxycinnamic acid
0.11
0.28
0.27
0.25
0.20
0.17
0.11
0.35
0.32
0.10
0.06
0.28
0.30
0.15
0.13
0.15
0.20
0.43
0.38
0.13
0.27
0.38
0.13
0.27
17
Vicenin 2
17.02
9.87
11.93
15.72
8.33
10.68
3.61
16.08
19.18
6.24
6.34
16.24
21.00
14.92
5.26
4.86
11.85
24.20
19.10
18.75
13.09
19.10
18.75
13.09
18
Diosmetin
1.52
1.54
1.65
1.20
0.97
0.87
0.47
1.35
2.02
1.00
0.94
1.59
2.14
1.16
0.64
1.24
1.64
1.90
1.94
1.89
1.91
1.94
1.89
1.91
19
Astragalin
1.20
0.34
0.31±
0.35
0.24
0.26
0.11
0.38
0.47
0.13
0.13
0.29
0.42
0.27
0.16
0.19
0.37
0.53
0.32
0.31
0.34
0.32
0.31
0.34
20
Apigenol
0.32
0.33
0.26
0.17
0.21
0.34
0.07
0.46
0.46
0.02
0.06
0.14
0.77
0.56
0.19
0.10
0.34
0.46
0.40
0.49
0.88
0.40
0.49
0.88
21
p-coumaric acid
0.80
0.31
0.30
0.13
0.30
0.13
0.12
0.21
0.16
0.25
0.19
0.36
0.19
0.11
0.10
0.84
0.57
0.47
0.19
0.10
0.05
0.19
0.08
0.08
22
Flavanone
3.56
2.96
2.19
3.72
2.35
2.01
0.92
3.47
4.40
1.92
1.48
0.98
4.42
2.46
1.39
2.45
2.96
4.69
4.24
3.92
3.34
4.24
3.92
3.34
23
Rhoifolin
0.54
0.44
0.38
0.60
0.31
0.25
0.11
0.42
0.60
0.44
0.48
2.59
0.44
0.26
0.15
0.37
0.48
0.50
0.58
0.73
0.88
0.58
0.73
0.88
24
Flavanone
5.05
3.30
2.62
5.03
28.35
3.56
1.46
5.51
7.20
3.86
2.84
2.74
10.25
4.99
2.35
3.46
4.04
6.74
9.69
8.39
6.64
9.69
8.39
6.64
25
Natsudaidain
0.28
0.25
0.19
0.32
0.12
0.13
0.06
0.20
0.37
0.20
0.15
0.15
0.46
0.24
0.15
0.25
0.31
0.40
0.33
0.30
0.25
0.33
0.30
0.25
26
Ferulic acid
0.74
0.51
nd
nd
0.12
nd
0.04
0.07
nd
0.06
0.05
0.10
0.48
nd
nd
0.09
0.10
0.09
nd
nd
nd
0.04
nd
nd
27
Isovitexin
1.58
0.93
0.87
1.75
1.01
0.66
0.28
1.22
1.92
0.78
1.02
1.21
2.35
1.13
0.57
0.96
1.21
1.93
1.92
2.14
2.33
1.92
2.14
2.33
28
Rutin
0.99
0.37
0.27
0.68
0.40
0.14
0.15
0.23
0.58
0.28
0.27
0.25
1.00
0.69
0.35
0.37
1.14
1.47
0.61
0.76
0.69
0.45
0.47
0.89
29
Isoquercitrin
1.14
0.74
0.68
1.54
0.75
0.66
0.26
1.07
1.65
0.66
0.70
0.77
1.94
1.16
0.51
0.65
1.02
1.60
1.52
1.73
1.85
1.52
1.73
1.85
30
Vicenin 1/3
0.45
0.52
0.50
0.42
0.28
0.25
0.11
0.44
0.84
0.25
0.25
0.46
0.54
0.27
0.20
0.34
0.46
0.52
0.42
0.46
0.48
0.42
0.46
0.48
31
Eriocitrin
6.11
4.04
3.72
5.18
4.27
3.14
1.51
5.23
7.36
2.62
2.69
3.90
10.47
4.65
2.89
3.72
5.19
8.25
7.39
6.33
5.43
2.86
1.90
5.21
32
Cinnamic acid derivative
1.34
1.02
0.80
2.18
1.07
1.18
0.37
1.81
3.62
1.29
0.84
0.84
2.65
1.40
0.76
0.97
1.19
1.83
2.07
2.41
2.18
2.07
2.41
2.18
33
Flavanone
1.35
0.49
0.38
0.41
0.37
0.53
0.19
1.12
1.11
0.56
0.37
0.22
0.08
0.77
0.43
0.61
0.71
1.70
1.21
0.86
0.77
1.21
0.86
0.77
34
Narirutin
17.60
10.33
8.22
19.80
8.15
12.99
4.45
13.97
24.91
8.76
13.48
15.82
36.75
22.61
7.62
5.21
9.71
30.11
28.56
27.02
19.86
8.68
13.76
8.02
35
Naringin
1.78
1.84
1.75
1.54
0.92
1.32
0.50
2.17
1.97
0.51
0.64
1.18
2.09
1.58
0.88
1.61
2.48
3.01
2.10
1.76
1.53
0.69
0.69
5.39
36
Cinnamic acid derivative
0.38
0.25
0.19
0.27
0.19
0.17
0.09
0.27
0.56
0.21
0.14
0.16
0.43
0.29
0.14
0.18
0.29
0.43
0.44
0.36
0.28
0.44
0.36
0.28
37
Hesperidin
99.89
78.04
60.94
62.51
41.80
47.12
29.20
65.45
113.17
46.27
53.98
122.00
88.85
46.11
23.21
49.79
71.80
97.45
72.44
64.45
42.25
30.14
44.21
38.84
38
Cinnamic acid
nd
nd
nd
nd
nd
nd
nd
nd
0.94
nd
nd
nd
0.54
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
39
Cinnamic acid derivative
0.38
0.28
0.24
0.51
0.27
0.25
0.10
0.40
0.79
0.25
0.20
0.34
0.53
0.32
0.17
0.19
0.32
0.44
0.53
0.57
0.54
0.53
0.57
0.54
40
Naringenin
1.09
0.74
0.54
2.15
0.65
0.80
0.34
1.67
2.00
1.32
1.47
1.73
2.22
0.99
0.44
0.64
0.86
1.19
1.98
2.01
2.73
1.39
0.80
1.84
41
Diosmin
0.37
0.15
0.16
0.51
0.26
0.21
0.05
0.32
0.39
0.47
0.24
0.63
0.51
0.25
0.13
0.09
0.14
0.32
0.36
0.50
0.34
0.36
0.50
0.34
42
3.5.6-trihydroxy-3′.4′.7-trimethoxyflavone
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
<LoQ
43
Nobiletin
0.38
0.30
0.26
0.72
0.40
0.26
0.10
0.39
0.54
0.67
0.42
0.75
0.65
0.36
0.16
0.23
0.36
0.36
0.52
0.67
0.68
0.42
0.28
0.65
44
Heptamethoxyflavone
0.18
0.16
0.12
0.38
0.21
0.13
0.06
0.22
0.18
0.13
0.14
0.39
0.21
0.12
0.05
0.13
0.17
0.16
0.16
0.20
0.24
0.16
0.20
0.24
45
Tangeritin
<LoQ
<LoQ
<LoQ
0.02
0.02
<LoQ
<LoQ
<LoQ
<LoQ
0.03
0.01
0.01
0.02
0.01
<LoQ
<LoQ
0.02
0.02
0.01
0.01
0.01
0.01
<LoQ
<LoQ
46
3.5.8-trihydroxy-3′.4′-dimethoxyflavone
0.04
0.03
0.02
nd
0.02
nd
nd
nd
nd
nd
nd
0.07
nd
nd
nd
0.05
0.04
nd
nd
nd
nd
0.16
0.28
0.51
Table 4.
Concentration of phenolic compounds (mg 100 g−1) of Brazilian commercial orange juice.
Results expressed as mean value; standard deviations varied from ±0.00 to ±2.19.
LoQ (limit of quantification) according to Table 3.
nd: not detected.
These results were compared with those reported in the work of Mesquita and Monteiro [9], which was developed previously. The authors evaluated organic and conventional orange juice throughout the 2016 harvest. All of the phenolic acids identified by them were cinnamic acid derivatives like in this work, although we could not confirm if they were the same ones. Also, a greater number of phenolic acids (19 compounds, and the precursor cinnamic acid) were identified in the present work, whereas only 7 was previously reported [9].
Among the flavonoids identified in Brazilian commercial orange juice were 8 flavanones: eriocitrin (peak 31), narirutin (peak 34), naringin (peak 35), hesperidin (peak 37), naringenin (40) and 3 compounds with no matches in MS library (peaks 22, 24 and 33). These compounds showed UV-vis spectra characteristic of this class: absorption from 250 to 360 nm with a band of high intensity (250–300 nm), maximum absorption between 280 and 285 nm and a shoulder from 300 to 360 nm [8, 9]. Regarding the flavones, 18 compounds were identified: lucenin-2 (peak 14), vicenin-2 (peak 17), diosmetin (peak 18), astragalin (peak 19), apigenol (20), rhoifolin (peak 23), natsudaidain (peak 25), isovitexin (peak 27), rutin (peak 28), isoquercitrin (peak 29), vicenin 1 or 3 (peak 30), diosmin (peak 41), 3.5.6-trihydroxy-3′.4′.7-trimethoxyflavone (peak 42), nobiletin (peak 43), heptamethoxyflavone (peak 44), tangeretin (peak 45) and 3.5.8-trihydroxy-3′.4′-dimethoxyflavone (peak 46). Only the compound represented by peak 12 had no match in the MS library. It showed UV-vis spectra corresponding to the flavones class (200–400 nm, band from 230 to 280 nm and maximum absorption at 255 nm) [9, 12].
The majority of the flavonoids were identified in all of the brands evaluated, with the exception only of naringenin (not identified in brand B), lucenin 2 (identified only in brands D, E and H), heptamethoxyflavone (identified in brands A, C, D, E and G) and 3,5,8-trihydroxy-3′,4′-dimethoxyflavone (identified in A, B, C, F, G and H).
The most expressive compounds of the juice were hesperidin (23–122 mg 100 g−1) and narirutin (4–37 mg 100 g−1), respectively. Intermediate levels of eriocitrin (1–10 mg 100 g−1) and naringin (0.5–5 mg 100 g−1) were also observed. Naringenin (0.3–3 mg 100 g−1), rutin (0.1–1 mg 100 g−1) and nobiletin (0.1–0.8 mg 100 g−1) levels were lower. Tangeretin was present in even lower levels (0.01–0.03 mg 100 g−1), sometimes below the limit of quantification (0.91 μg) [9] (Table 3).
Orange juice is widely consumed worldwide; however, there are few works that report the profile of phenolic compounds of this matrix. The most reported flavonoids in orange and orange juice are the flavanones hesperidin and narirutin [7, 10]. Mesquita and Monteiro [9] identified rutin, hesperidin, narirutin, eriocitrin, nobiletin and tangeritin in organic and conventional Pêra-Rio orange juices from Brazil throughout the 2016 harvest. All of the compounds identified and confirmed by them were also identified in juices from all brands evaluated in this work. Besides those compounds, the authors also found 12 other flavonoids they could not identify (6 flavones and 6 flavanones). In this work, the majority of the flavonoids were identified by MS, even when standards were not available. Brazilian commercial orange juice showed levels lower than NFC, FCOJ and freshly-squeezed organic and conventional orange juices as reported by Mesquita and Monteiro [9], considering the compounds evaluated.
3.2 Principal component analysis
For principal component analysis (PCA), phenolic compounds were represented by name and/or peak number according to Table 2. Only the variables with a correlation ≥0.65 were used; chlorogenic acid, nobiletin, naringenin, isovitexin, isoquercitrin, apigenol, narirutin, diosmetin, natsudaidain, vicenin 2, vicenin 1/3, rutin, hesperidin, astragalin, eriocitrin, and compounds represented by peaks 3, 7, 22, 32, 36 and 39.
The PCA model was calculated on the autoscaled data (21 variables, 24 samples). The first two principal components (PC 1 and PC 2) explained a variation of 80.36% in the levels of phenolic compounds of the orange juices. PCA enabled the discrimination of the juice from different brands and production dates according to spatial distribution. As seen on Figure 2a, all compounds were loaded negatively in PC 1. Chlorogenic acid, nobiletin, naringenin, isovitexin, isoquercitrin and apigenol, and compounds correspondent to peaks 3, 32 and 39 were loaded positively in PC 2. Narirutin was loaded on zero in PC 2. Diosmetin, vicenin 2, vicenin 1/3, natsudaidain, rutin, hesperidin, eriocitrin, astragalin and compounds correspondent to peaks 7, 22 and 36 were loaded negatively in PC2 (Figure 2a).
Figure 2.
Principal component analysis of phenolic compounds (named according to Table 1) (a) from Brazilian commercial orange juices from 8 brands (A, B, C, D, E, F, G and H), and from different production dates (1, 2 and 3) (b).
Figure 2b shows the plot distribution of juice samples from different brands (A, B, C, D, E, F, G and H) and production dates (1, 2 and 3). Overall, there was a difference in the levels of phenolic compounds of juices within a same brand for most of the brands evaluated. All juices from G were loaded negatively in PC 1; G1 was loaded negatively in PC2 as well, whereas G2 and G3 were loaded positively. Juices from B, D and H were all loaded positively in PC 2: D1, D2 and D3 were loaded together, showing that this brand was the one with the least difference among production dates. Juices from brand B (B2 and B3) were loaded positively in PC 1 and also close together, whereas B1 was loaded negatively in PC1. Juices from brand A (A1, A2, A3) showed a similar profile and were loaded closer. Juices from brands C, E and F showed the greatest differences among production dates. Overall, G1, G2, G3, C3, E1 and F3 were the juices with the highest levels of expressive phenolic compounds.
The presented results showcase that PCA allowed the differentiation of the Brazilian commercial orange juice according to differences and similarities in the phenolic compounds profile. A wide variety was observed among the juices within the same brands, as opposed to among the brands. This great variation was observed in the majority of the brands, making it difficult to verify any major differences among brands.
Considering that juices were acquired from January to November 2017, we can assume that the oranges used were in different maturity degrees, since the orange crop harvest is from July to October. It is well known that the levels of secondary metabolites (such as the phenolic compounds) vary as the fruit matures throughout the harvest. Thus, it is understandable that juices show this difference in the profile.
The chromatographic profile of phenolic compounds in Brazilian commercial orange juice from 8 brands was evaluated. Forty-six compounds were identified and quantified: 26 flavonoids, 19 phenolic acids, and the precursor cinnamic acid. All phenolic acids identified were cinnamic acid derivatives, as confirmed by MS and/or UV-vis spectra. Regarding the flavonoids, 8 of the compounds were flavanones, and 18 were flavones. The most expressive levels in all juices were of hesperidin and narirutin, respectively. The lowest levels were of ferulic acid and tangeretin, which sometimes was even lower than the limit of quantification. PCA was able to showcase the differences and similarities of commercial orange juice from different brands according to the influence of phenolic compounds, considering different production dates.
This work showcases the importance of the phenolic compounds profile and how significantly it can influence the juice. We observed that even within a same brand, there were great differences among juices produced. This can be related to the maturity of the fruits and harvest period. Finally, this work assessed the quality of the NFC juice, which is widely consumed in Brazil and exported worldwide. To our knowledge, there are no other works that evaluate the profile of these juices. It is an important work to base new research aiming to further evaluate the contents of orange juice.
This work was funded by São Paulo Research Foundation (FAPESP) (Processes n° 2013/10138-0 and 2014/23303-1) and National Council for Scientific and Technological Development—CNPq (scholarship grant of author M.R. Estevam). Authors would also like to thank Waters Technologies (São Paulo, SP, Brazil) for the support with UPLC-ESI-MS analysis.
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Written By
Maria Rita Estevam, Estela Mesquita and Magali Monteiro
Submitted: 07 October 2022Reviewed: 03 January 2023Published: 03 February 2023
Open access peer-reviewed chapter
