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Available from: Jacqueline Silveira Retrieved on: 20 January 2016 https://www.researchgate.net/publication/281034367_Fresh_and_Commercially_Pasteurized_Orange_Juice_An_Analysis_of_the_Metabolism_of_Flavonoid_Compounds?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_2 https://www.researchgate.net/publication/281034367_Fresh_and_Commercially_Pasteurized_Orange_Juice_An_Analysis_of_the_Metabolism_of_Flavonoid_Compounds?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_3 https://www.researchgate.net/?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_1 https://www.researchgate.net/profile/Jacqueline_Silveira2?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_4 https://www.researchgate.net/profile/Jacqueline_Silveira2?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_5 https://www.researchgate.net/institution/Sao_Paulo_State_University?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_6 https://www.researchgate.net/profile/Jacqueline_Silveira2?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_7 https://www.researchgate.net/profile/Thais_Cesar?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_4 https://www.researchgate.net/profile/Thais_Cesar?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_5 https://www.researchgate.net/institution/Sao_Paulo_State_University?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_6 https://www.researchgate.net/profile/Thais_Cesar?enrichId=rgreq-70832b1b-7cce-4246-bb85-0c894151b67b&enrichSource=Y292ZXJQYWdlOzI4MTAzNDM2NztBUzoyNjM0ODkzODYxODQ3MDRAMTQzOTgzMjE3MzUwOQ%3D%3D&el=1_x_7 229Proc. Fla. State Hort. Soc. 126: 2013. Proc. Fla. State Hort. Soc. 126:229–231. 2013. Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the U.S. Department of Ag- riculture. The U.S. Department of Agriculture prohibits discrimination in all its programs and activities on the basis of race, color, national origin, gender, religion, age, disability, political beliefs, sexual orientation, and marital or family status. *Corresponding author; email: tcesar@fcfar.unesp.br Handling & Processing Section Fresh and Commercially Pasteurized Orange Juice: An Analysis of the Metabolism of Flavonoid Compounds Jacqueline q. Silveira1, ThaiS B. ceSar1*, John a. ManThey2, and elizaBeTh a. Baldwin2 1Department of Nutrition, São Paulo State University, São Paulo, Brazil, 14801-902 2Citrus and Subtropical Products Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Ft. Pierce, FL 34945 AdditionAl index words. orange juice, processing methods, flavonoids, metabolites Orange juice is a rich source of flavonoids, mainly the flavanones hesperidin and narirutin, associated with health benefits in humans. The objective of this study was to analyze the uptake of flavonoids in humans after the consumption of two types of orange juice, fresh squeezed (fresh juice, FJ) and commercially extracted and pasteurized (processed juice, PJ). Preliminary measurements showed that the main flavanones in PJ were approximately three-fold higher than in FJ. This study involved healthy volunteers including 12 men and 12 women, aged 27 ± 6, with a BMI of 24 ± 3 kg/m2. Volunteers drank 11.5 mL/kg body weight of fresh orange juice, and after an interval of 30 days they drank the same quantity of pasteurized orange juice. Urine was collected from each volunteer during 24 hours following juice consumption. Urine metabolites were recovered by solid phase extraction, and measured by HPLC–ESI–MS. Analyses of the urine samples showed high concentrations of glucuronic acid and sulfate conjugates of hesperetin and naringenin. The data indicate that the concentrations of the flavanone metabolites following consumption of PJ were approximately three times higher than for FJ, thus matching the relative doses of these compounds in the juices provided to the volunteers. Orange juice is a dietary source of citrus flavonoids such as hesperidin (hesperetin-7-rutinoside) and narirutin (naringenin-7- rutinoside), which have been linked to health benefits in several epidemiologiocal studies (Kimmons et al., 2009; O’Neil et al., 2009, 2011). Evidence shows that the intake of these compounds is associated with a reduction of chronic disorders such as cancer, hypercholesterolemy, inflammation, and heart disease (Asgary and Keshvari, 2013; Devaraj et al., 2011; Ghorbani et al., 2012). Among certain consumers, there is a belief that fresh orange juice may exert higher levels of health benefits than what is obtained from commercially produced, pasteurized orange juice. Although debatable, it is a fact that orange juice process- ing methods influence the concentrations of many of the juice components. Fresh juice was previously shown to contain higher levels of the orange fruit polymethoxylated flavones compared to commercially extracted and pasteurized juice (Bai et al., 2012). On the other hand, flavonoid glycosides, alkaloids, and limonoids were present at higher concentrations in commercially extracted and pasteurized juice (Bai et al., 2012). The objective of this study was to determine if there were differences in the uptake of flavonoids in humans after the consumption of fresh and commercially processed juices. Materials and Methods subjects. Participants included 24 volunteers, 12 men and 12 women, aged 27 ± 6, healthy, with BMIs of 24 ± 3 kg/m2. All volunteers were nonsmoking and did not use medication, hor- mones, or dietary supplements. The study was approved by the Ethics Committee of the Faculty of Pharmaceutical Sciences at São Paulo State University (UNESP), Araraquara, Brazil (CAAE 00558712.5.0000.5426). study design. Each subject was submitted to two treatments. They drank 11.5 mL/kg body weight of fresh squeezed orange juice and the same quantity of commercially extracted and pas- teurized orange juice. There was a month interval between the two treatments and collection of the samples. This protocol was conducted at the Laboratory of Nutrition, Faculty of Pharmaceuti- cal Sciences, UNESP. orAnge juices. Two standard boxes of fresh oranges (cultivar Pera Rio) and 20 L of commercially extracted and pasteurized orange juice (PJ), made from the same batch of fruit, were pro- vided by Fisher Group (Matao, Brazil). The processed orange juice was stored in 1-L bottles at −20 °C for 4 weeks. The fresh juice, prepared with a commercial fresh juicer (FJ), was produced with the same fruit on the morning before starting the procedure for each subject. urine sAmples. Urine was collected from each volunteer dur- ing 24 h after ingestion of juices. The total volume collected was homogenized and 200-mL samples were passed through a C18 Sep Pac. The metabolites were eluted with methanol and dried under vacuum. Measurements of citrus metabolites were moni- 230 Proc. Fla. State Hort. Soc. 126: 2013. tored by high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS). stAtisticAl AnAlysis. Results from each experimental group were pooled and presented as mean ± standard deviation. Statisti- cal differences of the urinary excretion of flavonoids were tested comparing FJ and PJ orange juice data sets, using the software Sigma Stat 3.0. Results and Discussion Table 1 shows the anthropometric, hemodynamic, and bio- chemical characteristics of the participants of this study. The data showed that all measurements were within normal standards, which are compatible with healthy subjects. Analyses of the flavonoid metabolites in the urine samples obtained from the participants after the consumption of both types of juice showed high concentrations of glucuronic acid and sulfate conjugates of hesperetin and naringenin (Table 2). The chromatographic peaks of flavanones 557 amu, 381 amu and 477 amu, 447 amu, identified in the urine samples, are shown in Figures 1 and 2, respectively. The flavanone metabolites were detected during the 24-h period following ingestion. Vallejo et al. (2010) reported that no further excretion of flavonoid metabolites following orange juice inges- tion occurs after 24 h. We observed that during the 24-h urine collection, the excretion of metabolites after ingestion of PJ was greater than after ingestion of the FJ, and these differences were highly significant (Table 2). Flavanones and their metabolites exhibit distinctive ultraviolet spectra with wavelength maxima between 280 and 286 nm. Peaks that exhibited such spectra, as well as exhibiting mass ions antici- pated for flavanone conjugates were assigned as metabolites. Mass spectra of these peaks frequently exhibited neutral mass losses of 176 atomic mass units which were consistent with glucuronic acid conjugates. Neutral losses of 80 atomic mass units were also common, and were attributed to sulfate conjugates. Verification of these assignments awaits compound isolations and further spectroscopic analyses. Peaks A–C tentatively assigned to sulfate conjugates of hesper- etin glucoronides appeared at 10.2, 10.9, and 11.1 min, respectively. Metabolite A occurred in the urine of participants following the consumption of PJ, but not in the urine of participants consuming FJ. Metabolites B and C exhibited levels 11 and 2.4 times higher in the urine of participants consuming PJ compared to the levels in the urine of participants consuming FJ. Metabolite D with a retention time of 14.8 min is tentatively assigned to a sulfate conjugate of hesperitin, and the difference in the amount of this metabolite was 2.5 times greater after ingestion of PJ than after ingestion of FJ. Peaks E (rt 9.6) , F (rt 10.7), G (rt12.5) and H (rt 12.8), tentatively assigned to different naringin glucuronides, were present, respectively, at 1.7, 1.7, 2.3, and 2.7 times higher after ingestion of PJ compared to the levels following the inges- tion of FJ. HPLC–ESI–MS analysis at m/z 477 for the peak with a retention time of 11.6 min revealed that this metabolite was not found in the urine 24 h after ingestion of FJ, but was found in the urine after intake of PJ. Similarly, for the tentatively assigned peaks for hesperetin glucuronides at 13.2 min (J), 13.6 min (K), and 15.7 min (L) the urine levels following the consumption of PJ were 2.29, 2.48, and 1.9 times higher, respectively, than fol- lowing consumption of FJ. These values are consistent with the initial measurements showing that the flavonoid concentrations in the PJ were approximately three-fold higher than in the FJ. Table 1. Anthropometric, hemodynamic, and biochemical characteristics of the volunteers participating in the study. Parametersz Men (12) Women (12) Anthropometric BMI (%) 24.5 ± 3y 23.6 ± 3 Body fat (%) 23.4 ± 5 30 ± 3 Waist circumference (cm) 85.5 ± 10 73.7 ± 7 Blood pressure Systolic (mmHg) 122 ± 10 112 ± 11 Diastolic (mmHg) 67 ± 8 72 ± 10 Biochemical Glucose (mg/dL) 84.8 ± 7 81 ± 4 Insulin (mg/dL) 8.3 ± 2 10.8 ± 7 Total cholesterol (mg/dL) 185.6 ± 30 166.8 ± 22 HDL-cholesterol (mg/dL) 45.7 ± 8 63.2 ± 9 White blood cell (µg/dL) 6.6 ± 1 6.7 ± 1 zAll parameters were within normal references. yValues are expressed as mean ± standard deviation. Table 2. Metabolite urinary excretion after consumption of commercially extracted and pasteurized (PJ) and fresh squeezed orange juice (FJ). Metabolite excretion (µg/ 200 mL urine) Flavonone metabolite Molecular ion Retention time (min) Peak PJ FP P Hesperetin glucoronide sulfate 557 10.2 A 1.75 ± 0.8z,y --- --- 10.9 B 40.16 ± 13.5 3.59 ± 0.8 <0.001 11.1 C 40.64 ± 6.5 17.30 ± 13.7 <0.001 Hesperetin sulfate 381 14.8 D 153.9 ± 28.6 61.6 ± 11.2 <0.001 Naringenin glucuronide 447 9.6 E 3.98 ± 0.46 2.3 ± 0.26 <0.001 10.7 F 16.75 ± 2.12 9.66 ± 0.9 <0.001 12.5 G 23.19 ± 3.9 9.95 ± 3.6 <0.001 12.8 H 25.06 ± 4.0 9.26 ± 3.0 <0.001 Hesperetin glucuronide 477 11.6 I 3.38 ± 1.12 --- --- 13.2 J 31.44 ± 4.41 13.72 ± 5.1 <0.001 13.6 K 107.66 ± 14.43 43.46 ± 12.9 <0.001 15.7 L 3.68 ± 0.8 1.93 ± 0.34 <0.001 zSignificantly different from between PJ and FJ (P < 0.001). yValues are expressed as mean ± standard deviation. 231Proc. Fla. State Hort. Soc. 126: 2013. Literature Cited Asgary S. and M. Keshvari. 2013. Effects of Citrus sinensis juice on blood pressure. ARYA Atheroscler. 9(1):98–101. Bai, J., J.A. Manthey, B.L. Ford, G. Luzio, R.G. Cameron, J.N. Narciso, and E.A. Baldwin. 2013. Effect of extraction, pasteurization and cold storage on flavonoids and other secondary metabolites in fresh orange juice. J. Sci. Food Agr. 93:2771–2781. Devaraj, S., I. Jialal, J. Rockwood, and D. Zak. 2011. Effect of orange juice and beverage with phytosterols on cytokines and PAI-1 activity. Clin. Nutr. 30:668–671. Ghorbani, A., M. Nazari, M. Jeddi-Tehrani, and H. Zand. 2012. The citrus flavonoid hesperidin induces p53 and inhibits NF-κB activation in order to trigger apoptosis in NALM-6 cells: involvement of PPARγ- dependent mechanism. Eur. J. Nutr. 51:39–46. Kimmons, J., C. Gillespie, J. Seymour, M. Serdula, and H.M. Blanck. Fig. 1. Representative chromatograms of urine metabolites detected. Hesperitin sulfate 381 m/z (top) and hesperitin glucuronide sulfate 557 m/z (bottom). Fig. 2. Representative chromatograms of urine metabolites detected. Hesperitin glucuronide 477 m/z (top) and naringenin glucuronide 447 m/z (bottom). 11 Figure 1. Representative Chromatograms of Urine Metabolites Detected. Hesperitin sulfate 381 m/z (top) and Hesperitin glucuronide sulfate 557 m/z (bottom). 12 Figure 2. Representative Chromatograms of Urine Metabolites Detected. Hesperitin glucuronide 477 m/z (top) and Naringenin glucuronide 447 m/z (bottom). 2009. Fruit and vegetable intake among adolescents and adults in the United States: Percentage meeting individualized recommendations. Medscape J. Med. 11:26. O’Neil, C.E., T.A. Nicklas, G.C. Rampersaud, and V.L. Fulgoni, 3rd. 2011. One hundred percent orange juice consumption is associated with better diet quality, improved nutrient adequacy, and no increased risk for overweight/obesity in children. Nutr. Res. 31:673–682. O’Neil, C.E., T.A. Nicklas, G.C. Rampersaud, and V.L. Fulgoni, 3rd. 2012. One hundred percent orange juice consumption is associated with better diet quality, improved nutrient adequacy, decreased risk for obesity, and improved biomarkers of health in adults. National Health and Nutrition Examination Survey, 2003–2006. Nutr. J. Vol. 11:107. Vallejo, F., M. Larrosa, E. Escudero, M.P. Zafrilla, B. Cerdá, J. Boza, M.T. García-Conesa, J.C. Espín, and F.A. Tomás-Barberán. 2010. Concentration and solubility of flavanones in orange beverages affect their bioavailability in humans. J. Agr. Food Chem. 58:6516–6524.