Food Research International 53 (2013) 882–890 Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres Antioxidant activity of aqueous extract of passion fruit (Passiflora edulis) leaves: In vitro and in vivo study Juliana Kelly da Silva a, Cinthia Baú Betim Cazarin a, Talita C. Colomeu b, Ângela Giovana Batista a, Laura M.M. Meletti c, Jonas Augusto Rizzato Paschoal a, Stanislau Bogusz Júnior d, Mayra Fontes Furlan d, Felix Guillermo Reyes Reyes a, Fábio Augusto d, Mário Roberto Maróstica Júnior a,⁎, Ricardo de Lima Zollner b,⁎⁎ a University of Campinas, School of Food Engineering, Rua Monteiro Lobato, 80, Cidade Universitária Zeferino Vaz, 13083-862, Campinas, SP, Brazil University of Campinas, Department of Internal Medicine, Faculty of Medical Science, Campinas, SP 13083-887, Brazil c Center of Research and Development on Plant Genetic Resources, Agronomic Institute of Campinas, Campinas, SP, Brazil d University of Campinas, Institute of Chemistry, Instituto Nacional de Ciência e Tecnologia em Bioanalítica (INCTBio), Campinas, SP, Brazil b a r t i c l e i n f o Article history: Received 16 October 2012 Received in revised form 14 December 2012 Accepted 20 December 2012 Keywords: Passiflora edulis leaves Antioxidant potential Oxidative stress Bioactive compounds Polyphenol a b s t r a c t Passion fruit (Passiflora edulis), including its leaves, is a rich source of bioactive compounds, as polyphenols. This study investigated the in vitro and in vivo antioxidant potential of aqueous extract of P. edulis leaves and identification of phenolic compounds by HPLC-PDA and ESI-MS/MS analysis. Male Wistar rats were divided in two groups (n = 6 per group). Control group received water and experimental group received P. edulis leaf extract (Tea) (1.1 mg dry leaves mL−1), both ad libitum. Total phenols and antioxidant potential (DPPH, FRAP, ABTS and ORAC assay) were determined in aqueous extract. Antioxidant status was analyzed by FRAP, ORAC in serum and by SOD, GR and GPx activities, GSH contents and TBARS in liver, brain and kidneys. Gut microbiota profile and short-chain fatty acids were determined in feces. Vitexin, isovitexin and isoorientin were analyzed in the extract of P. edulis leaves. The animals which received tea showed a decrease of 20% of TBARS in liver. GSH contents in kidneys increased 40% relative to control group. The GR activity was 2 times higher and GPx 3.2 times lower in liver than control group. Animals from TEA group showed a 45% reduction on SOD activities in liver and brain. Serum antioxidant potential was not altered. Tea intake also promoted colonic bacteria growth, although there was a decreasing in the SCFA production. Therefore, P. edulis leaf extract could be an option to enhance the supply of antioxidants and to safeguard against oxidative stress. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction The genus Passiflora encompasses approximately 450 species, usually called passion fruits. The best known and more used is Passiflora edulis, that is popular, not only because of its pulp, but also because of the infusions made with the leaves. It has been largely used in American and European countries as sedative or tranquilizer (Coleta et al., 2006; Ferreres et al., 2007; Petry et al., 2001), and currently, the tea prepared by the infusion of the leaves has been recognized for its antiinflammatory potential (Montanher, Zucolotto, Schenkel, & Frode, 2007). Both Passiflora alata and P. edulis have been used in many ⁎ Corresponding author at: Departamento de Alimentos e Nutrição, Rua Monteiro Lobato, 80, Cidade Universitaria Zeferino Vaz, Campinas, SP, Brazil. Tel./fax: +55 19 3521 4059. ⁎⁎ Corresponding author at: Departamento de Clínica Médica, Faculdade de Ciências Médicas, Rua Tessalia Vieira de Camargo, 126, Cidade Universitaria Zeferino Vaz, Campinas, SP, Brazil. Tel./fax: + 55 19 32893709. E-mail addresses: mario@fea.unicamp.br (M.R. Maróstica Júnior), zollner@unicamp.br (R. de Lima Zollner). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2012.12.043 pharmaceutical preparations and it is widely employed in the food industry (Petry et al., 2001). P. edulis is native from Brazil, but now it is cultivated in all parts of the world. It is commonly called yellow passion fruit, maracuja, yellow granadilia, and pomme liane jaune (Sunitha & Devaki, 2009). The interest in P. edulis has been increased because of its antioxidant compounds. Nowadays, studies have identified several phenolic compounds in the leaves of Passiflora species, like orientin 2″-O-rhamnoside and luteolin 7-O-(2-rhamnosylglucoside) (Coleta et al., 2006), apigenin and luteolin derivatives (Ferreres et al., 2007). The polyphenols have antioxidant properties as they could neutralize or quench oxidants (Pietta, 2000). Nevertheless, before absorption, most dietary polyphenols are transformed in the colon by microbiota. Gut bacteria can hydrolyze glycosides, glucuronides, sulfates, amides, esters, and lactones. This conversion may influence absorption and modulates the biological activity of these dietary compounds (Selma, Espin, & Tomas-Barberan, 2009). On the other hand, unabsorbed phenolic compounds remain in the lumen modulating the microbial population in gastrointestinal tract and affecting short-chain fatty acid (SCFA) production by bacterial fermentation. In J.K. da Silva et al. / Food Research International 53 (2013) 882–890 this way, phenolic compounds may play a role on intestinal health (Lee, Jenner, Low, & Lee, 2006). Currently, the search for natural sources of antioxidants is a strong tendency (Ferreres et al., 2007). The cause of the oxidative stress is an imbalance between protector systems and production of free radicals (McCord, 2000). The excess of reactive species can damage cell lipids, proteins and DNA by oxidative action, which might result in loss of function and even cellular death (Habib & Ibrahim, 2011), which has linked the oxidative stress to some diseases (Durackova, 2010). The organisms use endogenous and exogenous antioxidant defenses to protect against harms of oxygen and nitrogen reactive species. They are classified in enzymatic: catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD); and non-enzymatic systems: thiol reduced (GSH), vitamins, minerals and polyphenols (Rezaie, Parker, & Abdollahi, 2007). Sometimes endogenous antioxidants are not able to prevent oxidative damages, requiring the supply of exogenous antiradicals. Thus, consumption of dietary sources may support the prevention of oxidative stress (Habib & Ibrahim, 2011). Despite of whole fruits and vegetables intake, typically non-edible parts could be a good alternative to add dietary bioactive compounds (Leite-Legatti et al., 2012). The literature data indicates that the P. edulis leaf extracts possess in vitro and ex vivo antioxidant activity against protein oxidative damage, being considered as new sources of natural antioxidants (Rudnicki et al., 2007). The literature data indicates that the P. edulis leaf extracts possess in vitro and ex vivo antioxidant activity against protein oxidative damage, being considered as new sources of natural antioxidants (Rudnicki et al., 2007). This work tries to shed some light on the functional properties of tea of P. edulis leaves. To the best of our known, this is the first report about the in vitro and in vivo antioxidant effects of P. edulis aqueous extract. 883 total phenolic, antioxidant potential, and phenolic compound identification by HPLC-PDA and MS/MS. Aqueous extracts for animal experiment were prepared according to the results of IC50 of DDPH assay and just before giving to animals. 2.4. Total phenolic content The total phenolic content of the P. edulis aqueous extract was determined according to Folin–Ciocalteu's method (Swain & Hillis, 1959), with some modifications. In a vial, 50 μL of extract, 800 μL distilled water and 25 μL (0.25 N) Folin–Ciocalteu's reagent were mixed and incubated at room temperature for 3 min. Then, 100 μL sodium carbonate solution (1 N) was added and further incubated for 2 h at room temperature. The absorbance was read at 725 nm in a microplate reader Synergy HT, Biotek (Winooski, USA); with Gen5™ 2.0 data analysis software spectrophotometer. Gallic acid was used in a standard curve and the results were expressed in terms of gallic acid equivalent (GAE g−1). 2.5. Determination of antioxidant potential in P. edulis leaf aqueous extract The readings of absorbance and fluorescence were done in a microplate reader Synergy HT, Biotek (Winooski, USA); with Gen5™ 2.0 data analysis software spectrophotometer. 2.5.1. DPPH free radical scavenging activity The free radical scavenging activity of the extracts was determined based on the DPPH method (Braca et al., 2001). The extract (33 μL) was added in 1.3 mL DPPH solution diluted in methanol (0.024 mg mL−1), shaking and incubated for 30 min in dark, and absorbance was measured at 515 nm. The percentage scavenging radical was calculated from [(A0 − A1) / A0] × 100, where A0 is the absorbance of the control, and A1 is the absorbance of the extract. 2. Material and methods 2.1. Chemicals L-Glutathione reduced, glutathione reductase, glutathione oxidized form disodium salt, β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), hypoxanthine, xanthine oxidase, nitrotetrazolium blue chloride (NTB), 5′5′-dithio-bis-2-nitrobenzoic acid (DTNB), albumin from bovine serum (BSA), 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS); (±)6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox); 2,2′-azobis(2-methylpropionamidine) dihydrochloride (APPH), 2,4,6tris(2-pyridyl)-s-triazine (TPTZ), 2-thiobarbituric acid (TBA), 2,2diphenyl-1-picrylhydrazyl (DPPH), phenolic compound standards (vitexin, isovitexin and isoorientin), SCFA standards (Acetic, propionic and butyric acids), L-cysteine a and dicloxacillin were all obtained from Sigma-Aldrich (São Paulo — Brazil). Fluorescein sodium salt and metaphosphoric acid were purchased from Vetec Química Fina (Sao Paulo — Brazil). Malondialdehyde standard (MDA) was purchased from Cayman Chemical Company (#10009202). MRS agar and peptone water were purchased from Oxoid (UK). 2.2. P. edulis leaves Leaves of P. edulis were obtained from the Agronomic Institute of Campinas, dried in an oven with circulating air (MARCONI, PiracicabaSP, Brazil) at 50 °C, 48 h, after grinding to a fine homogeneous powder and storing in amber glass bottles at 8 °C. 2.3. Extracts of P. edulis leaves The aqueous extract was prepared by boiling 1 g of sample and 25 mL of water at 100 °C. This extract was used to the analysis of 2.5.2. FRAP assay (ferric reducing antioxidant power) The ferric reducing ability of tissues was determined by FRAP method (Benzie & Strain, 1996), with adaptations. In the dark, FRAP reagent was made with 300 mmol L −1 acetate buffer (pH 3.6), 10 mmol TPTZ in a 40 mmol L −1 HCl solution and 20 mmol L −1 FeCl3. Sample or standard solutions, ultrapure water and FRAP reagent were mixed and kept in a water bath for 30 min at 37 °C. After cooling to room temperature, samples and standard were read at 595 nm. The Trolox standard curve was made (10–800 μmol TE). Results were expressed in μmol Trolox equivalent (TE) mL−1. 2.5.3. TEAC (Trolox equivalent antioxidant capacity) assay Tissues' TEAC levels were determined based on the reports from Rufino et al. (2010) with modifications. The ABTS solution was prepared by mixing 5 mL of 7.0 mmol ABTS and 88 μL of 145 mmol potassium persulfate solution, which was left to react for 12–16 h, at room temperature in the dark. Ethanol (99.5%) was added to the solution until absorbance reached 0.700±0.05 at 734 nm. Trolox (10–800 μmol TE) was used as reference antioxidant. The ABTS solution was added to the sample or standard solutions and reacted for 6 min before reading at 734 nm, room temperature. Results expressed in μmol Trolox equivalent (TE) mL−1. 2.5.4. ORAC assay (hydrophilic oxygen radical absorbance capacity) In the dark, 20 μL of sample (prepared as described above), 120 μL of fluorescein in phosphate buffer — PB (pH 7.4), and 60 μL of AAPH were added in black microplates. The microplate reader was adjusted as previously described (fluorescent filters, excitation wavelength, 485 nm; emission wavelength, 520 nm) (Prior et al., 2003). ORAC values were expressed in μmol Trolox equivalent (TE) per mL serum by using the standard curves (2.5–50.0 μM TE) (Davalos, Gomez-Cordoves, & Bartolome, 2004; Prior et al., 2003). 884 J.K. da Silva et al. / Food Research International 53 (2013) 882–890 2.6. HPLC-PDA and MS/MS analysis 2.9. Biochemical analyses Major phenolic compounds from the leaves were determined in an HPLC system equipped with a degasser, quaternary pump, autosampler, photodiode detector (Agilent Technologies 1200 series — Englewood, CO, USA). The separation of polyphenols was performed on a C18 column (250× 4.6 mm, 5 μm Luna, Phenomenex, Torrance, CA, USA) at a controlled temperature (30 °C). The mobile phase used was A: deionized water: acetic acid (90.5:0.5 v/v), B: deionized water: acetonitrile (90:10 v v − 1) and C: deionized water:methanol (85:15 v v − 1). The gradient program was used as follows: 0 min 75% A, 10% B and 15% C; 25 min 55% A, 20% B and 25% C; 30 min 75% A, 10% B and 15% C. The flow rate and injection volume were 1.0 mL min − 1 and 20 μL, respectively. The peak identification of each polyphenol was based on the comparison of the relative retention time (RT), peak area percentage, and spectra data with polyphenol standards (vitexin, isovitexin and isoorientin). The standard curves for all polyphenols were determined using the same chromatographic conditions described above. The extracts were diluted in deionized water: methanol (20:80, v:v), and filtered through 0.45 μm filters (Chromafil® Xtra RC 45/25 membrane; MACHEREY-NAGEL, Bethlehem, PA, USA), before injecting in the HPLC. The same extracts used in the HPLC-UV/PAD analysis were also used for MS analysis. The equipment used was a Q-ToF Micro® (Micromass, UK) mass spectrometer with a hybrid system of analyzers composed by quadrupole (Q) and time-of-flight (ToF) analyzers and a hexapole collision cell (with helium as collision gas), operating to get full scan and mass spectra in tandem (MS/MS), with m/z resolution of 5000. Samples were introduced in the mass spectrometer through an electrospray ionization source (ESI-MS/MS). The ESI-MS/MS system control and data acquisition was performed by MassLynx 4.0 software (Micromass, UK). In order to improve the signal-to-noise ratios, the quadrupole analyzer was used as a filter for ion collection. For the identity confirmation analysis, the molecular ions and their product ions were considered. The ionization conditions selected were: cone gas flow (50 L h−1), desolvation gas flow (400 L h−1), polarity (ESI+), capillary energy (3000 V), sample cone energy (50 V), extraction cone energy (2 V), desolvation temperature (250 °C), source temperature (100 °C), ionization energy (2 V), collision energy (15 V), and multi-channel plate detector energy (2780 V). The method was suitable for the determination of the analytes and the QToF technique made it possible to obtain m/z ratios with less than 10 ppm of error for each analyte. 2.9.1. Lipid peroxidation by thiobarbituric acid reactive substances (TBARS) assay TBARS determinations were done in liver and serum according to Ohkawa, Ohishi, and Yagi (1979), with adaptations. The organ samples were macerated in liquid nitrogen. Samples (organs or serum) were mixed with 8.1% sodium dodecyl sulfate (SDS) and working reagent (2-thiobarbituric acid — TBA, 5% acetic acid and 20% sodium hydroxide). After heating at 95 °C for 60 min, the samples remained in ice bath for 10 min and centrifuged at 10,000 g, 10 min. The supernatant was read at 532 nm, using a clear 96-well microplate. Standard curve (0.625–50 nmol MDA mL−1) was obtained using malondialdehyde standard (MDA). Results were expressed in nmol MDA mg−1 tissue (or nmol MDA mL−1 serum). 2.7. In vivo experimental design The study was approved by the Institutional Animal Care and Use Committee. The animals were cared for in accordance with the institutional ethical guideline. Seventy seven-day-old male Wistar rats were used in this study. Animals were allocated under controlled conditions of temperature (22 °C±2), humidity (60–70%), and a light–dark cycle (12/12 h). They were divided into 2 groups (n=6). Rats were fed semi-purified experimental diet AIN-93M (Reeves, Nielsen, & Fahey, 1993); control group received water and experimental group (TEA) aqueous extract of P. edulis leaves. There was free access to water/extract and food. The extract was prepared and changed every 48 h. After 15 days animals were anesthetized with ketamine and xylazine and died by exsanguination by cardiac puncture. 2.9.2. Enzymatic and non-enzymatic endogenous antioxidant system 2.9.2.1. Thiol group content (GSH). GSH levels were determined in the PB homogenates of liver, kidneys and brain using Ellman's reagent (DTNB) (Ellman, 1959), with modifications. GSH solution (2.5– 500 nmol GSH mL −1) was used as standard and absorbance was read at 412 nm. Reduced thiol contents were expressed in nmol GSH mg −1 protein. The protein concentration of tissue homogenates was done by Bradford method (Bradford, 1976). 2.9.2.2. Glutathione peroxidase activity (GPx). GPx activity was quantified in PB homogenates of liver. This assay is based on the oxidation of 10 mmol reduced glutathione by glutathione peroxidase coupled to the oxidation of 4 mmol NADPH by 1 U enzymatic activity of GR in the presence of 0.25mmol H2O2. The rate of NADPH oxidation was monitored by the decrease in absorbance at 365nm (Flohe & Gunzler, 1984). Results were expressed in nmol NADPH consumed min−1 mg−1 protein. 2.9.2.3. Glutathione reductase activity (GR). GR activity in liver was measured in PB homogenates, following the decrease in absorbance at 340nm induced by 1 mmol oxidized glutathione in the presence of 0.1 mmol NADPH in phosphate buffer (Carlberg & Mannervik, 1985). The results were expressed in nmol NADPH consumed min−1 mg−1 protein. 2.9.2.4. Superoxide dismutase activity. SOD activity was analyzed in liver, kidneys and brain. One hundred microliters of appropriately diluted PB homogenates was added in 96-well microplate and 150 μL of the working solution (0.1 mmol hypoxanthine, 0.07 U xanthine oxidase, and 0.6 mmol NTB in PB in 1:1:1 proportions) and the kinetic reaction was monitored at 560 nm (Winterbourn, Hawkins, Brian, & Carrell, 1975). The area under the curve (AUC) was calculated and the SOD activity was expressed as U mg −1 protein. 2.9.3. Antioxidant potential in serum Serum was treated with ethanol:ultrapure water and 0.75 mol L −1 metaphosphoric acid (Leite et al., 2011). These extracts were used in ORAC (Davalos et al., 2004; Prior et al., 2003) and FRAP (Benzie & Strain, 1996) assays, carried out as described above. 2.8. Blood samples and carcasses 2.9.4. Fecal pH The feces samples were collected in cecum and diluted with deionized water (1 mg mL −1) and homogenized and it was measured the fecal pH in a pH meter (Tecnal model TEC-5, Piracicaba, SP — Brazil) (Asvarujanon, Ishizuka, & Hara, 2005). Blood samples were collected in appropriated tubes and centrifuged at 2000 g for 20 min. Serum was separated and stored at −80 °C until analyses. Liver, kidneys and whole brain were removed, weighed and frozen in liquid nitrogen, after kept at −80 °C. Carcasses were weighed and stored at 4 °C or less. 2.9.5. Cecal microbiota (Bifidobacterium, Lactobacillus, Enterobacteriaceae, total aerobics) Feces samples were homogenized in peptone water (100 mg mL−1) and then ten-fold serial dilutions were made in the same medium. Aliquots of 0.1 mL of the appropriate dilution were spread onto the J.K. da Silva et al. / Food Research International 53 (2013) 882–890 MRS agar media for Lactobacillus count and supplemented MRS agar (0.5 mg L−1 dicloxacillin, 1 g L−1 LiCl and 0.5 g L−1 L-cysteine hydrochloride) for Bifidobacterium. Culture plates were incubated in anaerobic condition at 37 °C for 24–48 h. Similarly, 1 mL of the diluted sample was spread onto specific count plates Petrifilm (3M®, São Paulo, MN) for Enterobacteriaceae and total aerobic. Plates were incubated at 37 °C for 24–48 h. After the incubation, the specific colonies grown on the selective culture media were counted and the number of viable microorganism g−1 feces (CFU g−1) was calculated. The mean and standard error were calculated from the log 10 values of the CFU g−1. 2.9.6. Short-chain fatty acids (SCFA) Short chain fatty acids were analyzed by gas chromatography (Zhao, Nyman, & Jonsson, 2006) using Agilent 6890N equipment with flame ionization detector (FID) and autosampler N10149 (Agilent, EUA). A 30 m × 0.25 mm i.d. × 0.25 μm NukolTM capillary column (Supelco, Bellefonte, PA, US) was used. Chromatographic conditions were: injector and detector temperatures set at 250 °C, injected volume 1 μL with split ratio set to 1:10; carrier gas was hydrogen at 1.0 mL min−1. The column oven was programmed as follows: kept at 100 °C for 0.5 min, then heated at 8 °C min−1 until 180 °C, kept for 1 min, heated at 20 °C min−1 until 200 °C and kept for 5 min. 2.10. Statistical analyses Parametric data were expressed as means ± standard error (SEM). The statistical analyses were carried out using GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA) software. Analyses were based on Student's t-test and the limit of significance was set at P b 0.05. 3. Results and discussion 3.1. Total phenol and antioxidant potential in extract of P. edulis leaves Tea is the second most popular beverage in the world and it contains several flavonoids, substance that are among the polyphenolic compounds (El-Beshbishy, 2005). The results of total polyphenols, DPPH IC50, FRAP assay, ABTS and ORAC assays for the Passiflora aqueous extract are shown in Table 1. A concentration of 1100 μg mL−1 of P. edulis aqueous extract was able to scavenge 50% of DPPH radical (Table 1). This concentration was used to prepare the aqueous extracts to the rats. DPPHIC50 is in agreement with those shown by Sunitha and Devaki (2009), 875 μg mL−1, in ethanol extract of P. edulis leaves. The concentration of bioactive compounds in plants (as well as their biological effects) could depend on geography, photoperiod and temperature. These differences have been reported to influence the biosynthesis of many secondary metabolites as flavonoids and it could be followed by variations in antioxidant potential (Jaakola & Hohtola, 2010; Kumazawa, Hamasaka, & Nakayama, 2004). Phenolic contents in P. edulis leaves (Table 1) were similar to Curcuma zeoderia leaves (Prakash, Suri, Upadhyay, & Singh, 2007) 885 but lower than found in commercial samples of black and green tea (Khokhar & Magnusdottir, 2002), other leaves (Prakash et al., 2007) and other byproducts of passion fruit (Oliveira et al., 2009). Studies using Passiflora leaves extract showed phenolics' concentration of 10 times higher than the ones described here; however they used tannic acid as standard or they were calculated on a dry basis (Pabón et al., 2011; Rudnicki et al., 2007). Additionally, it is important to clarify that the Folin–Ciocalteu phenol analysis estimates the total phenolic content, but chromatographic techniques are more precise to quantify them (Rudnicki et al., 2007). Regarding the antioxidant potential, literature data are consistent with our results. The ABTS assay was carried out in the methanol extract from the leaves of 5 Passiflora species. The results showed values between 5 and 15 μmol TE g −1 in four of samples (Bendini et al., 2006), which is lower than in the present study (19.2 μmol TE g −1). Pabón et al. (2011) verified that leaves of some passion fruit species have higher antioxidant potential than the pulp by ABTS, DPPH and FRAP assays. They found ABTS values from 3.4 to 163 μmol TE g −1 (Pabón et al., 2011). Our aqueous extract, obtained by heating and positive pressure, showed higher ABTS and FRAP results (Table 1). A study showed that the leaves of berries exhibited higher antioxidant potential (determined by ORAC) compared to the fruits (Wang & Lin, 2000). Table 1 shows the results of ORAC of P. edulis leaf extract (373.0 ± 1.63 μmol TE g −1). Our results suggest that the aqueous extract of the leaves of P. edulis could be a good source of phenolic compounds and an ingredient with high antioxidant potential. 3.2. Determination of major phenolic compounds P. edulis leaf extract by HPLC-PDA and ESI-MS/MS Three flavonoids were found in the aqueous extracts of P. edulis leaves: vitexin, isovitexin and isoorientin (concentrations of 0.40 ± 0.05; 0.50 ± 0.04 and 1.05 ± 0.03 mg g −1, respectively), which is in accordance with other authors (Ferreres et al., 2007; Zucolotto et al., 2012). The analyses were carried out based on the RT and using UV/PDA spectra for comparison to standards (Fig. 1). In addition, ESI-MS/MS was performed to confirm the presence of these compounds and peaks were observed with the same m/z (433.113 — vitexin and 449.108 — isovitexin, isoorientin). 3.3. Weight gain and intake parameters There were no differences (P > 0.05) in weight gain (320.9 ± 4.1 g), diet (21.0 ± 1.0 g day −1) and hydric (41.4 ± 2.7 mL day −1) consumptions between group experimental and control. The consumption of tea was similar to water intake, showing that P. edulis leaf extract was well accepted by rats in the concentration used. There were also no differences in diet intake and weight gain of tea group relative to control group. Nevertheless, many research studies have pointed possible effects of leaf extracts against obesity or diabetes using animal models (Jaiswal, Kumar Rai, Kumar, Mehta, & Watal, 2009; Wu et al., 2010). However, P. alata leaf extract orally administered did not show to be effective on the reduction of rats' weight gain (Doyama, Rodrigues, Novelli, Cereda, & Vilegas, 2005). 3.4. Serum antioxidant potential Table 1 Antioxidant activity in extract aqueous of Passiflora edulis leaves. Antioxidant activity Results Total phenolic content DPPH (IC50) FRAP ABTS ORAC 8.3 ± 0.22 mg GAE g−1 1100 μg mL−1 205.7 ± 4.12 μmol TE g−1 19.2 ± 0.50 μmol TE g−1 373.0 ± 1.63 μmol TE g−1 The groups did not show variations on antioxidant status according to FRAP and ORAC assays (P= 0.133 and 0.566, respectively) (Fig. 2a, b). Serum antioxidant potential was not affected by P. edulis' extract aqueous in experimental group possibly because the experimental period was short to promote such modifications. In humans the black tea consumption did not change the plasmatic antioxidant status (by ORAC and FRAP assay) even after chronic experimental period (Widlansky et al., 2005). Literature's data about antioxidant response after short-term treatment with phenolic compounds are conflicting. Studies have 886 J.K. da Silva et al. / Food Research International 53 (2013) 882–890 Fig. 1. Typical HPLC-UV/PDA chromatogram (λ = 350 nm) from boiled water P. edulis leaf extract; (1) UV/PDA spectra of isoorientin; (2) UV/PDA spectra of vitexin and (3) UV/PDA spectra of isovitexin. reported no change in the antioxidant response after consumption of Pinnus maritima extract (Silliman, Parry, Kirk, & Prior, 2003), anthocyanins from Myrciaria jaboticaba (Leite et al., 2011), controlled diets high in fruits and vegetables (Paterson et al., 2006) or a spray-dried extract made from fruits and vegetables (Record, Dreosti, & McInerney, 2001). However, other studies with berry administration have shown an increase in in vivo antioxidant activity (Hassimotto, Pinto, & Lajolo, 2008; Leite et al., 2011). 3.5. Lipid peroxidation ROS damage of tissues through lipid peroxidation could result in changes in cellular biomembranes (Skrzydlewska, Ostrowska, Farbiszewski, & Michalak, 2002). According to TBARS assay, the treatment with Passiflora extract did not modify the serum lipid peroxidation (P= 0.551) (Fig. 3a). Differently, another study reported a reduction in plasma lipid peroxidation in Wistar rats that received Fig. 2. Ferric reducing power assay (a) and Oxigen Radical Absorbance Capacity (ORAC) (b) in serum. TEA = animals fed commercial diet plus extract aqueous of Passiflora edulis leaves; Control = fed commercial diet. Data expressed in mean ± SE. 887 J.K. da Silva et al. / Food Research International 53 (2013) 882–890 b Liver TBARS (nmol MDA mg-1 tissue) 3 2 1 0 1.5 1.0 * 0.5 l A TE C C on TE tr o l A 0.0 tr o Serum TBARS (nmol MDA mL-1) 4 on a Fig. 3. Lipid peroxidation by TBARS assay in serum (a) and liver (b). TEA = animals fed commercial diet plus aqueous extract of Passiflora edulis leaves; Control = fed commercial diet. Data expressed in mean ± SE. *Indicates statistical differences from AIN group according with Student's t-test (1 code = P b 0.05; 2 codes = P b 0.01; and 3 = P b 0.001). passion fruit juice twice a day for 28 days (Souza et al., 2012). Green tea was effective in avoiding lipid oxidative damage in serum and liver of rats drinking tea for 5 weeks (Skrzydlewska et al., 2002). On the other hand, despite of a reduced ingestion period, the P. edulis leaf extract was able to reduce liver TBARS in 20% (P b 0.05) (Fig. 3b). This indicates that P. edulis leaves could prevent formation of reactive species and, consequently, damage the liver cells in rats. The green tea also reduced MDA level in rats evaluated in different ages on normal conditions (Augustyniak, Waszkiewicz, & Skrzydlewska, 2005). Most studies that show antioxidant action of tea in the liver, especially green tea, are oftentimes associated with some kind of local damage (Augustyniak et al., 2005; El-Beshbishy, 2005). The increased oxidative stress is related to an overproduction of free radicals or deficiency in the antioxidant defense system. GSH values in liver were similar for both groups (P= 0.638) (Fig. 4a). However, TEA group showed a 40% increase in GSH level in kidneys relative to control group (Fig. 4b). GR was 2 times increased and GPx 3.5 times reduced in the liver of animals from TEA group compared to control (Fig. 5a, b, respectively). Glutathione is a major source of reducing power and it is maintained in the reduced form by GR, acting together with NADPH (Skrzydlewska et al., 2002). Therefore, the increment or maintenance of total glutathione and rise in GR could be an indication b 50 Kidneys GSH (nmol GSH mg-1 protein) 40 30 20 10 80 * 60 40 20 on C d Kidneys SOD (U mg-1 protein) 20 15 ** 5 8 6 4 2 TE A Brain SOD (U mg-1 protein) C C on tr o TE A e ol 0 l 0 10 tr 10 tr o A C on TE tr o A TE Liver SOD (U mg-1 protein) c l 0 l 0 on Liver GSH (nmol GSH mg-1 protein) a 3.6. Thiol group content and antioxidant enzymes 1.5 1.0 ** 0.5 ro l on t C TE A 0.0 Fig. 4. Thiol group content (GSH) in liver (a), kidneys (b) and superoxide dismutase activity (SOD) in liver (c), kidneys (d) and brain (e). TEA = animals fed commercial diet plus aqueous extract of Passiflora edulis leaves; Control = fed commercial diet. Data expressed in mean± SE. *Indicates statistical differences from AIN group according with Student's t-test (1 code = P b 0.05; 2 codes = P b 0.01; and 3 = P b 0.001). 888 J.K. da Silva et al. / Food Research International 53 (2013) 882–890 b 10 5 C on tr ol 0 10 5 *** 0 l 15 tr o 20 15 C on *** TE A Liver GPx (nmol NADPH consumed min-1 mg-1 protein) 25 TE A Liver GR (nmol NADPH consumed min-1 mg-1 protein) a Fig. 5. Antioxidant enzymes: glutathione reductase (GR) (a) and glutathione peroxidase (GPx) (b) in liver. TEA = animals fed commercial diet plus aqueous extract of Passiflora edulis leaves; Control = fed commercial diet. Data expressed in mean ± SE. *Indicates statistical differences from AIN group according with Student's t-test (1 code = P b 0.05; 2 codes = P b 0.01; and 3 = P b 0.001). of antioxidant status improvement after P. edulis extract intake. SOD activity in liver, kidneys and brain was reduced in TEA group compared to standard (Fig. 4c, d, e). A decrease in GPx activity could be due to reduced generation of H2O2, corroborating SOD results. These evidences suggest that bioactive compounds of tea may avoid formation of ROS and consequently antioxidant enzymes. Antioxidants may chelate metal ions and prevent lipid peroxidation leading to an increase on H2O2 and O2− concentrations as a consequence of reduced SOD and GPx activities (Skrzydlewska et al., 2002). Fig. 6 illustrates these hypotheses. Nevertheless, more analyses like GSSH/GSH levels and GPx, GR in kidneys would be necessary to corroborate the results. 3.7. Cecal microbiota Phenolic compounds can interact with the gut microbiota, modulating the microbial population. Substantial levels of unabsorbed and metabolites of dietary phenolics remain in the gut, exerting significant effects on the intestinal environment (Selma et al., 2009). Study with polyphenols from tea detected growth inhibition of potentially pathogenic bacterial species like Clostridium ssp. and Bacteroides spp., but benefic species as Bifidobacterium ssp. and Lactobacillus ssp. were less affected (Lee et al., 2006). Other authors found a positive association between phenolic compounds or source foods and growth or adhesion of Bifidobacterium and Lactobacillus ssp. (Larrosa et al., 2009; Pozuelo et al., 2012). In our study the count of bacteria in feces was significantly higher (increasing from 6 to 25%, as presented in Fig. 7a), in TEA group for all species analyzed (Bifidobacterium, Lactobacillus, Total Aerobic Bacteria and Enterobacteriaceae). However, it should be emphasized that the aqueous extract was not selective for one or other species. These results are supported by a study using rats fed with diet plus grape antioxidant fiber. The authors found higher proliferation in Lactobacillus, E. coli and Bacteroides in the animals fed with the ingredients (Pozuelo et al., 2012). The mechanisms by which phenolic compound could modulate microbiota are not well-known (Selma et al., 2009). Some authors state that the bacterial rise is linked to the capacity for metabolizing these flavonoid compounds (Landete, 2012). On the other hand, SCFA concentrations were reduced in TEA group compared to control, with significantly lower levels of acetic and butyric acids (21% and 66% smaller, respectively) (Fig. 7b). The ingestion of P. edulis aqueous extract was not able to promote changes on fecal pH, which ranged between 8.39 and 8.81. Gut bacteria cleaves non-digestible polysaccharides using saccharidases. Smaller oligomers can be assimilated and metabolized by the colonic bacteria to produce short-chain fatty acids (Macfarlane & Macfarlane, 2012). There is increasing evidence that individual polyphenols or classes of polyphenols may directly influence on the activities of key enzymes in the gut (Boath, Stewart, & McDougall, 2012; McDougall et al., 2005). Fruit extracts, green and black teas have shown inhibitory effect on α-amylase activity in vitro and in vivo (McDougall et al., 2005). The same mechanisms could be used to inhibit bacterial proteins and Fig. 6. Possible mechanisms involved in enzymatic antioxidant status. ROS=reactive oxygen species; AOX=antioxidant compounds; M+x =metallic ions; SOD=superoxide dismutase; GPx=glutathione peroxidase. Possible actions of the bioactive compounds: they may avoid formation of ROS (green arrow) or chelate metals ions (red arrow) and, thus, antioxidant enzymes (SOD and GPx) may be spared. J.K. da Silva et al. / Food Research International 53 (2013) 882–890 a b 15 Acetic Acid Propionic Acid Butyric Acid ** 10 5 TE A on tr ol C TE A on tr ol C TE A on tr ol *** 0 C SCFA (mmol g-1 wet feces) 20 Fig. 7. Microbiota of cecal contents (Bifidobacterium, Lactobacillus, total aerobic bacteria and Enterobacteriaceae) and short-chain fatty acid (SCFA — acetic, propionic and butyric). TEA = animals fed commercial diet plus aqueous extract of Passiflora edulis leaves; Control = fed commercial diet. Data expressed in mean ± SE. *Indicates statistical differences from AIN group according with Student's t-test (1 code = P b 0.05; 2 codes = P b 0.01; and 3 = P b 0.001). enzymes to hamper colonic fermentation with decreased SCFA production. 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