Food Chemistry 133 (2012) 671 677 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Bioactivities of açaí (Euterpe precatoria Mart.) fruit pulp, superior antioxidant and anti-inflammatory properties to Euterpe oleracea Mart. q Jie Kang a, Keshari M. Thakali a, Chenghui Xie a, Miwako Kondo b, Yudong Tong a, Boxin Ou b, Gitte Jensen c, Marjorie B. Medina d, Alexander G. Schauss e,, Xianli Wu a, a USDA Arkansas Children s Nutrition Center, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 15 Children s Way, Little Rock, AR 72202, USA b Brunswick Laboratories, 200 Turnpike Rd., Southborough, MA 01772, USA c NIS Labs, 1437 Esplanade, Klamath Falls, OR 97601, USA d USDA, ARS, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA e AIBMR Life Science, 4117 S Meridian, Puyallup, WA 98373, USA article info abstract Article history: Received 12 September 2011 Received in revised form 5 December 2011 Accepted 17 January 2012 Available online 25 January 2012 Keywords: Açaí Euterpe precatoria Mart. Antioxidant Anti-inflammation Carotenoid There are two predominant palm tree species producing edible fruit known as açaí found widely dispersed through the Amazon: Euterpe oleracea Mart. and Euterpe precatoria Mart. They differ from each other in terms of how the plants grow and their phytochemical composition. E. oleracea (EO) has received considerable attention as a super fruit because of its high antioxidant capacity, while studies on E. precatoria (EP) remain rare. In this study, the antioxidant and anti-inflammatory activities of EP fruit pulps were evaluated by different assays including a series of oxygen radical absorbance capacity (ORAC) based assays, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the cell-based antioxidant protection in erythrocyte (CAP-e) assay, as well as the nuclear factor-kappa B (NF-jB) secreted embryonic alkaline phosphatase (SEAP) assay. Total phenolics were also measured as an indication of the total phenol content. For comparative purposes, the EO fruit pulp was included. The antioxidant capacity of the EP fruit pulp was determined to be superior to the EO fruit pulp in every chemical based assay. In the cell-based CAP-e assay, the EP fruit pulp showed a dose-dependent inhibition against oxidative damage with an IC 50 of 0.167 g/l. In the SEAP reporter assay, the EP fruit pulp polyphenol-rich extracts inhibited lipopolysaccharide (LPS)-induced NF-jB activation by 23% (p < 0.05) at 20 lg/ml, whereas the extract of the EO fruit pulp did not show a significant inhibitory effect at comparable doses. In addition, carotenoids were quantified for the first time in EP, since EP has high scavenging capacity against singlet oxygen. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Euterpe is a genus of native tropical palm trees found growing indigenously in various areas of the Amazon area in South America. There are three predominant species producing edible fruits found widely dispersed through the Amazon: Euterpe edulis Mart., Euterpe precatoria Mart. and Euterpe oleracea Mart. (Schauss, 2010). The former species produces a fruit called either juçara or açaì, while the latter two heterotypic species produce a fruit commonly called açaì, although other common name synonyms exist. E. edulis is a primary source of heart-of-palm, rather than for its fruit, which is similar in appearance to E. precatoria and E. oleracea. The q Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. Corresponding authors. Tel.: +1 253 286 2888 (A.G. Schauss), tel.: +1 501 364 2813; fax: +1 501 364 3161 (X. Wu). E-mail addresses: alex@aibmr.com (A.G. Schauss), wuxianli@uams.edu (X. Wu). major difference between the two remaining species harvested for their fruit is how the palms grow. E. precatoria (EP) is a slender single-stem, pinnate-leaved palm that reaches heights up to 20 m, with a diameter reaching 25 cm. By comparison, E. oleracea (EO) is multi-stemmed with up to 25 stems growing up from 18 to 33 m from one clump (Fig. 1). The two species also differ from each other in their phytochemical composition (Pacheco-Palencia, Duncan, & Talcott, 2009). In the last decade, EO has received considerable attention as a new super fruit because of its high antioxidant capacity and potential anti-inflammatory effects (Heinrich, Dhanji, & Casselman, 2011; Honzel et al., 2008; Schauss, Wu, Prior, Ou, Huang et al., 2006; Schauss, Wu, Prior, Ou, Patel et al., 2006). However, studies on EP remain limited. To our knowledge, only two papers have reported the nutrient and phytochemical composition or bioactivities of EP (Galotta, Boaventura, & Lima, 2008; Pacheco-Palencia, Duncan, & Talcott, 2009). The major phytochemicals in EP fruit pulps were reported in a recent paper (Pacheco-Palencia, Duncan, & Talcott, 2009). The EP fruit pulp contains higher levels of polyphenols than those in EO 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.048
672 J. Kang et al. / Food Chemistry 133 (2012) 671 677 Fig. 1. Açaí palm trees: the single-stem, Eutepe precatoria (A) and, a cluster of multiple-stemmed Euterpe oleracea (B). fruit pulp, but the antioxidant activities and anti-inflammatory effects of the EP fruit pulp remain unknown. Based on a consensus that polyphenols are major contributors to the antioxidant activities of various foods, particularly, fruits and vegetables, the EP fruit pulp would be expected to show high antioxidant activities. In the present study, the antioxidant activities of the EP fruit pulp were evaluated by multiple assays, including: oxygen radical absorbance capacity (ORAC) based assays; 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay; and cell-based antioxidant protection in erythrocyte (CAP-e) assay. The first two assays are chemical assays while the third is a cell-based assay. As indicated in our recent paper (Kang et al., 2010), due to the complexity of the anti-oxidant defense system in the body, a single antioxidant assay cannot reflect all aspects of activities of a given food or its food components. In evaluating the anti-oxidant capacities of a given food or natural product, combining both chemical and cell-based assays provides a useful approach towards understanding the anti-oxidant effects of antioxidants found in food and their biological relevance to any health benefits that may be observed in vivo. The first objective of this study was to evaluate the antioxidant capacities of EP by both chemical and cell-based methods. Total phenolics (TP) were also measured in the EP fruit pulp as an indication of total phenol content. The potential anti-inflammatory activity was evaluated by the lipopolysaccharide (LPS)-induced secreted embryonic alkaline phosphatase (SEAP) reporter assay. The SEAP reporter assay was designed to measure nuclear factor-kappa B (NF-jB) activation (Berger, Hauber, Hauber, Geiger, & Cullen, 1988; Moon, Hahn, Lee, & Kim, 2001). As a major transcription factor, NF-jB plays a key role in regulating genes responsible for innate and adaptive immune responses (Brasier, 2006; Hoffmann, Natoli, & Ghosh, 2006). This assay was employed in evaluating the anti-inflammatory activities of pure flavonoids isolated from EO in our previous study (Kang et al., 2011). The results of our current study suggest that certain flavonoids isolated from EO are potential NF-jB inhibitors. The second objective of this study was to evaluate the anti-inflammatory activities of fruit pulp polyphenol-rich extracts of both the EP and EO pulp by the SEAP reporter assay. In addition, the EP showed extremely high scavenging capacity against singlet oxygen when assessed by the ORAC-based SOAC assay. Carotenoids, which are important dietary singlet oxygen scavengers, were analysed and quantified in EP and EO fruit pulps for the first time. 2. Materials and methods 2.1. Chemicals and reagents 2,2 0 -Azobis(2-amidinopropane)dihydrochloride (AAPH) was purchased from Wako Chemicals (Richmond, VA). Hydroethidine fluorescent stain (HE) was obtained from Polysciences (Warrington, PA). Randomly methylated b-cyclodextrin (RMCD) was obtained from Cyclodextrin Technologies Development (High Springs, FL). Potassium phosphate dibasic (K 2 HPO 4 ) and potassium phosphate monobasic (KH 2 PO 4 ) were obtained from VWR (West Chester, PA). Fetal bovine serum (FBS) was bought from Hyclone (Logan, UT). Dulbecco s Modified Eagle s Medium (DMEM), 2 0,7 0 - dichlorofluorescein diacetate (DCFDA), were obtained from Invitrogen (Carlsbad, CA). Zeocineosin and RAW-Blue cells were purchased from Invivogen (San Diego, CA). Xanthine oxidase, xanthine, hydrogen peroxide (H 2 O 2 ) solution, 3-morpholinosydnonimine (SIN-1), lithium molybdate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), dihydrorhodamine-123 (DHR-123), fluorescein (sodium salt) (FL), hexane, methanol (MeOH), ethanol (EtOH), methyl tertiary butyl ether (MTBE), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), formic acid, phosphate-buffered saline (PBS), RPMI-1640 culture medium, Histopaque 1077. Histopaque 1119 Fast Blue BB (4-benzoylamino- 2,5-dimethoxybenzenediazonium chloride hemi-[zinc chloride]) salt, Folin Ciocalteu s Phenol Reagent (2 M) and gallic acid were all obtained from Sigma Aldrich (Milwaukee, WI). 2.2. Plant materials Freeze-dried EO and EP fruit pulps were obtained from Eco- Fruits (South Jordan, UT) and Saborama (Santa Cruz, Bolivia),
J. Kang et al. / Food Chemistry 133 (2012) 671 677 673 respectively. The fruits of EO and EP were collected in Para State, Brazil and Santa Cruz Department, Bolivia, respectively. The fruits were processed to pure pulp within hours of harvesting, and stored at 20 C, until lyophilisation. 2.3. ORAC-based assays Freeze-dried EP (1 g) and EO (1 g) fruit pulp were extracted according to the method published previously by our group (Wu, Beecher et al., 2004; Wu, Gu et al., 2004). Hexane/dichloromethane (50:50, v/v) was used to extract lipophilic fractions, and followed by acetone/water/acetic acid (AWA) (70:29.5:0.5, v/v/v) for extracting hydrophilic fractions. The hydrophilic fractions were used for measuring H-ORAC, HORAC, NORAC, SORAC, and SOAC. The lipophilic fraction was used for L-ORAC analysis. ORAC-based assays, included antioxidant capacity against peroxyl radicals containing hydrophilic (H-ORAC) and lipophilic (L-ORAC), hydroxyl radicals (HORAC), peroxynitrite anion (NOR- AC), superoxide anion radical (SORAC), and singlet oxygen (SOAC), and were conducted in accordance with several recent reports (Huang, Ou, Hampsch-Woodill, Flanagan, & Deemer, 2002; Mullen et al., 2011; Ou et al., 2002; Wu, Beecher et al., 2004; Wu, Gu et al., 2004; Zhang et al., 2009). All assays were carried out on a Synergy HT microplate reader (Bio-Tek Instruments, Winooski, VT). Trolox was used as the standard, and results expressed as lmol trolox equivalent (TE) per gram of dry weight (DW). FL was used as a fluorescent probe in H-ORAC, L-ORAC, and HORAC assays. DHR-123 was used in the NORAC assay as a fluorescent probe, whereas HE fluorescent stain solution was used in the SORAC and SOAC assays. Fluorescence filters with an excitation wavelength of 485 ± 20 nm and emission wavelength of 520 ± 20 nm were used in all assays. The incubators were kept at room temperature except for H-ORAC, L-ORAC and SOAC assays, in which the temperature was set to 37 C due to the heat-sensitive property of AAPH and the reaction temperature of H 2 O 2 disproportionation. In the H- and L-ORAC assays, AAPH was used as a peroxyl radical generator (Prior et al., 2003). H 2 O 2 was used as a hydroxyl radical generator in the HORAC assay (Ou et al., 2002). The peroxynitrite anion generator, SIN-1, was used in the NORAC assay (Dubost, Ou, & Beelman, 2007). Xanthine and xanthine oxidase were used to produce superoxide anion radicals in the SORAC assay (Zhang et al., 2009). Singlet oxygen was generated by the molybdate-catalysed disproportionation of H 2 O 2 at 37 C in the SOAC assay. 2.4. Total phenolic analysis The hydrophilic fractions used in the ORAC assay were subjected to TP analysis by two methods. For the first method, Folin Ciocalteu reagent was used according to the method reported before (Wu, Beecher et al., 2004; Wu, Gu et al., 2004), and the result was expressed as milligrams of gallic acid equivalents (GAE) per gram of DW. TP was also measured by a recently developed Fast Blue BB (FBBB) method (Medina, 2011a). Briefly, açaí powders (0.2 g) were extracted three times with 5 ml 70% ethanol and the extracts were further diluted with water. The TP analysis consisted of adding 0. 1 ml of 0.1% Fast Blue BB diazonium dye to 1 ml gallic acid standards (0, 25 500 lg/ml) and samples followed by the addition of 0.1 ml of 5% NaOH. After 90 min reaction time, the optical density was measured at 420 nm with Synergy HT micro-plate reader (Bio-Tek Instrument, Winooski, VT). Total phenolics were also reported as GAE per gram. 2.5. DPPH assay The hydrophilic fraction (AWA) of EP or EO fruit pulps used in the ORAC assay was subjected to DPPH assay according to the reported method (Ozgen, Reese, Tulio, Scheerens, & Miller, 2006). Briefly, the samples (50 ll) were added to 150 ll of DPPH/ DMSO solution (0.25 mg/ml in DMSO), and the absorbance was measured at 525 nm on a Synergy HT microplate reader (Bio-Tek Instruments, Winooski, VT) after 1 h of incubation. The results were expressed in lmol TE/g. 2.6. CAP-e assay Freeze-dried EP (0.5 g) fruit pulp was extracted with 5 ml of phosphate buffered saline (PBS) for 1 h at room temperature on an orbital shaker. Then the extract was centrifuged and the supernatant was ready for CAP-e assay after filtration. CAP-e assay was conducted following the method published by Honzel et al. (2008), but using an accelerated and more sensitive microplate-based protocol. Briefly, a red blood cell suspension was prepared for the CAP-e assay by adding 0.1 ml packed red blood cells to 10 ml physiological saline (ph 7.4) in a V-bottom 96-well microplate. In twelve control wells, no antioxidants were added to the cells, such that six wells were negative controls (no oxidative damage was induced) and six wells were positive controls (maximum oxidative damage). To another twelve wells, serial dilutions of the reference compound gallic acid were added in duplicate. To an additional set of twelve wells, serial dilutions of EO versus EP were tested in duplicate. The cells were incubated for 20 min to allow antioxidants to enter into the cells. Unabsorbed compounds were removed from the cells by two washes in physiological saline. Cell pellets were re-suspended and lysed in water, and the precursor dye DCFDA was added to the wells for 15 min. Oxidative damage was induced using AAPH for 1 h. The green fluorescence intensity, as a measurement of oxidative damage, was recorded at 488 nm using a Tecan Spectrafluor plate reader (Durham NC). The inhibition of oxidative damage was calculated as the reduced fluorescence intensity in the wells, where the cells were pretreated with test products, compared to the baseline (negative controls) and maximum oxidative damage (positive controls). The CAP-e value reflects the IC 50 dose (g/l) of the test products. 2.7. SEAP reporter assay Freeze-dried EP (20 g) or EO (20 g) fruit pulps were extracted with 50 ml of methanol/water (85:15, v/v) for 1 h at room temperature on an orbital shaker. After centrifugation, the supernatant was taken and dried under N 2 flow. The residue was then suspended in water and extracted with hexane and EtOAc successively. The EtOAc extracts of EP and EO were dried under N 2 flow prior to being dissolved in pure DMSO for the SEAP reporter assay. The SEAP reporter assay was conducted in RAW-Blue cells according to the procedure described previously by our group (Kang et al., 2011). Briefly, RAW-Blue cells (1 10 5 cells/well) were pretreated with EP and EO extracts dissolved in DMSO (0.5% in culture media) for 2 h, and then stimulated by LPS (100 ng/ml) for 18 h. The supernatants were collected for the SEAP reporter assay. QUANTI-Blue powder was dissolved in endotoxin-free water and sterile filtered (QuantiQuanta-blue substrate). RAW-Blue cell supernatant (40 ll/well) was added to Quanti- Quanta-blue substrate (160 ll/well) and incubated at 37 C for 0.5 1 h. Absorbance was measured at 620 nm in a Polarstar microplate reader (BMG Labtech, Durham, NC). 2.8. Analysis of carotenoids by HPLC MS/MS Analyses of carotenoids were carried out in an HPLC MS/MS system. Freeze-dried fruit pulps of EP and EO were extracted with a mixture of MTBE/EtOH (1:4) containing 2 lg/ml of vitamin E. Then, the supernatant after centrifugation was used for
674 J. Kang et al. / Food Chemistry 133 (2012) 671 677 HPLC MS/MS analysis. Analysis was carried out using the Shimadzu HPLC system LC-20AT and SHIL-HTC (Shimadzu, Kyoto, Japan) coupled with a 4000 Q TRAP mass spectrometer (Applied Biosystems, Forest City, CA). Separation was performed on a MAC-MOD HydroBond PS-C18 column (50 2.1 mm, 3 lm) using a flow rate of 1.2 ml/min. The solvent consisted of (A) 0.4% of formic acid (v/v) in water, (B) acetonitrile containing 1% formic acid (v/v), (C) methanol containing 1% formic acid (v/v). The 8.5 min gradient was as follows: A:B:C = 20:60:20 (0 0.1 min), A:B:C = 4:60:36 (0.1 2 min), A:B:C = 28:36:36 (2 8.5 min). Multi-reaction monitoring (MRM) mode scan was used for quantitation. The transitions monitored were mass to charge ratio (m/z): m/z 536.8? 444.5 for both b-carotene and lycopene; m/z 597.4? 147.2 for astaxanthin; m/z 568.8? 338.1 for lutein; m/z 568.9? 476.3 for zeaxanthin. The mass spectrometer equipped with an ESI-Turbo V source was operated in the positive ion mode. Major parameters were optimised as follows: ion spray voltage, 4.5 kv; 50 for curtain gas (CUR), 400 C for source temperature; 30 and 50 for nebulising (GS1) and turbo spray gas (GS2). The entrance potential (EP), declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) were optimised individually with each standard. The analysis was controlled by Analyst v1.4.2 (Applied Biosystems, Forest City, CA). 2.9. Statistical analysis The results were expressed as mean ± SD (n = 3). Data were subjected to t-test for statistical analyses and a value of p < 0.05 was considered as a significant difference. Statistical analyses were performed using SigmaStat statistical software (SigmaStat 3.5). 3. Results and discussion 3.1. Antioxidant activities of EP fruit pulp measured by chemical assays The antioxidant activities of EP fruit pulp were initially measured by two widely used chemical assays, the ORAC-based assay and the DPPH assay. The TP was also measured as an indication of total phenolic content in the EP fruit pulp. The EO fruit pulp was included for comparative purposes. ORAC is one of the most widely used chemical assays (Prior, Wu, & Schaich, 2005) to measure antioxidant capacities of food extracts, pure compounds and biological samples (Kang et al., 2011; Prior et al., 2007; Wu, Beecher et al., 2004; Wu, Gu et al., 2004). ORAC was originally developed to measure antioxidant inhibition of hydrophilic components against peroxyl radical induced oxidations (H-ORAC) (Ou et al., 2002; Prior, Wu, & Schaich, 2005). Later, a method was developed to measure lipophilic components (L-ORAC) (Huang, Ou, Hampsch-Woodill, Flanagan, & Deemer, 2002). However, in addition to peroxyl radical, there are other reactive oxygen or nitrogen species (ROS or RNS) such as hydroxyl radical ( OH), superoxide anion radical O 2 and peroxyni- trite anion (ONOO ) involved in the pathophysiology of human diseases (Valko et al., 2007). To gain a full understanding of the antioxidant activity towards different free radicals of a given food, a series of assays were developed based on the classic ORAC assay in recent years to measure antioxidant inhibition against other ROS/RNS moieties, including OH, O 2, ONOO and 1 O 2 (Ou et al., 2002; Zhang et al., 2009). The assays were thus given the names HORAC, SORAC, NORAC, and SOAC, accordingly. The sum of the results from these six assays is referred as the Total ORAC, which provides a more global view of the potential antioxidant activity of a given food sample. The DPPH assay is another commonly used chemical assay to measure antioxidant capacity (Prior, Wu, & Schaich, 2005). This assay is based on the measurement of the reducing ability of antioxidants toward a stable free radical DPPH. The TP is also measured as an indication of the total phenolic content in EP fruit pulp. The Folin Ciocalteu reagent has been widely used for many years in measuring total phenolics. The basic mechanism is an oxidation/reduction reaction, and is thus regarded as another antioxidant capacity assay (Prior, Wu, & Schaich, 2005). However, this method has been criticised for its lack of specificity since several non-phenolic substances such as ascorbic acid, glucose, and fructose in juices, fruits and vegetables can interfere with the total phenols measurement (Medina, 2011b). FBBB, a recently developed, simple, rapid, and direct detection of phenolics in foods, beverages, and agricultural byproducts, was also used to measure TP (Medina, 2011a, 2011b). The FBBB is based on the coupling of phenolic compounds with the Fast Blue BB diazonium salt resulting in the formation of azo complexes where coupling mostly occurs in the para position to the phenolic activating group. Coupling or substitution may also occur in the ortho position to the activating group. Phenolics in slightly alkaline solution are converted to the more active phenoxide ions, allowing the coupling to occur. The values of all ORAC based assays, including the H-ORAC, L- ORAC, HORAC, NORAC, SORAC, SOAC, and Total ORAC, for both EP and EO are presented in Table 1. The results of the DPPH assay and TP are reported in Table 2. Remarkably, the antioxidant capacity of the EP fruit pulp was found to be superior to the EO fruit pulp in every single assay conducted. Among the six ORAC-based assays, the SOAC and SORAC values of EP fruit pulp were nearly nine times and six times higher, respectively, than that of the EO fruit pulp. These data suggest that the EP fruit pulp may have greater potential in scavenging 1 O 2 and O 2, which could translate to stronger meaningful bioactivities than EO, though in vivo confirmation is still needed. For the other ORAC-based assays, the values of the EP fruit pulp were from 20% higher in the L-ORAC assay to over 300% higher in the HORAC assay than that of the EO fruit pulp. Taken together, the total ORAC of EP fruit pulp resulted in a three times higher value than that of the EO fruit pulp (7698.6 lmol TE/g vs. 2649.1 lmol TE/g). The DPPH value of the EP fruit pulp was 320.3 lmol TE/g, approximately 2.4 times higher than that of EO (133.4 lmol TE/g) (Table 2). Total phenolics by FBBB were about three times higher in EP than in EO (Table 2). The TP ratios of FBBB:FC were higher in EP and the lower ratio in EO suggests presence of non-phenolic reducing compounds such as carotenoids and ascorbic acid that are detected by FC but not with FBBB methods. The ph of the extracts suggests higher acidic components in EO (4.53) than in EP (5.73). FBBB method may also detect the presence of carotenoids as background absorbance in the extracts when measured directly at 420 nm. This background absorbance is subtracted from the total optical density of the phenolic-azp complex. In this analysis EP Table 1 Values from the ORAC based assays on the EP and the EO fruit pulp. ORAC based assays ORAC value (lmol TE/g, DW a ) EP EO ORAC (against peroxyl radical) b 1828.4 1014.0 H-ORAC (hydrophilic fraction) 1792.3 ± 89.7 985.9 ± 57.4 L-ORAC (lipophilic fraction) 36.1 ± 2.6 28.1 ± 2.1 HORAC (against hydroxyl radical) 4113.8 ± 312.9 1357.3 ± 67.7 SORAC (against superoxide anion radical) 1040.4 ± 55.1 169.0 ± 12.3 NORAC (against peroxynitrite anion) 86.7 ± 5.9 37.2 ± 2.6 SOAC (against singlet oxygen) 629.3 ± 33.4 71.6 ± 8.8 ORAC (total) 7698.6 2649.1 a b DW, dry weight. The values are calculated as sum of H-ORAC and L-ORAC.
J. Kang et al. / Food Chemistry 133 (2012) 671 677 675 Table 2 Antioxidant capacities and total phenolics of the EP and EO fruit pulps obtained by the DPPH assay, Folin Ciocalteu and FBBB methods. Assay EP EO DPPH (lmol TE/g, DW a ) 320.3 ± 23.8 133.4 ± 11.2 Total phenolics (mg GAE/g, DW) Folin Ciocalteu 73.0 ± 4.8 31.2 ± 2.6 FBBB 247.7 ± 16.8 92.9 ± 8.9 a DW, dry weight. shows ten times higher absorbance than EO at 420 nm and this observation indicates a correlation with the carotenoid content (Section 3.4). As being demonstrated in a recent discovery by our group, the ORAC values vary distinctly according to different structurally similar flavonoid compounds based on the numbers and positions of the hydroxyl groups and/or other substitute groups (Kang et al., 2010, 2011). To fully understand why the EP fruit pulp showed such high antioxidant capacity and to isolate the constituents and bioactive antioxidants, a systematic isolation and evaluation of purified compounds would be necessary. Also, additional compounds other than polyphenols may contribute to scavenging activity of free radicals. For instance, complex sugars present plentiful hydroxyl groups and may contribute to radical quenching activities. Another group of compounds, the carotenoids, are poor at scavenging peroxyl radicals (ROO ), but are excellent for quenching 1 O 2. Furthermore, it is worthwhile mentioning that both the ORAC values and the DPPH value of EP fruit pulp are much higher than all other dark colour berries that have been studied to date, including black raspberry, blackberry, blueberries, red raspberry, strawberry, and purple grapes (Ozgen, Reese, Tulio, Scheerens, & Miller, 2006; Wu, Beecher et al., 2004; Wu, Gu et al., 2004). 3.2. Antioxidant activities of the EP fruit pulp measured by CAP-e assay The antioxidant capacities of the EP were also assessed by the recently developed cell-based CAP-e assay (Honzel et al., 2008). Since an aqueous extract was used, the data from this CAP-e assay reflects whether water soluble antioxidants can enter into and protect live cells from oxidative damage by peroxyl radicals (ROO ). The CAP-e assay was conducted using five concentrations of fruit pulp extract: 0.01, 0.05. 0.27, 1.33, and 6.67 g/l. The EP fruit pulp extract was able to enter into the live cells and dosedependently inhibit AAPH generated peroxyl radical formation (Fig. 2). The IC 50 of the EP extract was estimated to be 0.167 g/l. However, compared to EP, inhibition of the EO fruit pulp extract was no more than 20% from 0.1 to 10 g/l (Fig. 2), as was observed in a previous study (Honzel et al., 2008). This striking difference suggests that the EP fruit pulp contains much higher levels and/ or strong water soluble antioxidants that can enter live human cells and effectively inhibit ROS formation. Unfortunately, the active compounds are not yet identified. In our previous studies, we found that only flavonoid aglycones can get into living cells, but their glycosides cannot penetrate the living cells. Of the aglycones that can get into the living cells, their antioxidant activities varied distinctively based on their structures (Kang et al., 2010). The bioactive compounds responsible for high antioxidant activity in the CAP-e assay and their mechanisms are interesting research topics for future investigation. Moreover, since açaí is usually consumed as juice, the much higher water soluble antioxidants make EP more attractive than EO. 3.3. Anti-inflammatory effects of EP fruit pulp NF-jB is one of the principal inducible transcription factors in mammals and has been shown to play a pivotal role in the mammalian innate immune response and in chronic inflammatory conditions (Bremner & Heinrich, 2002; Li & Stark, 2002). From our previous reports, certain flavonoids isolated from EO fruit pulp, such as luteolin, apigenin and velutin, were found to be potential NF-jB inhibitors as assessed by the SEAP reporter assay in RAW- Blue cells (Kang et al., 2011). In this study, the activity and potency of polyphenol-rich extracts of the EP or EO fruit pulps in inhibiting NF-jB activation were evaluated using the same assay. In the SEAP reporter assay, four different doses, from 2.5 to 20 lg/ml of EtOAc extract from EP or EO, were tested. The EtOAc extract of EP inhibited LPS-induced NF-jB activation by 23% at 20 lg/ ml (p < 0.05), whereas the EO extract did not show significant inhibitory effects at any doses (Fig. 3). The SEAP reporter assay results suggest that the EtOAc extract of EP contains potential NF-jB inhibitors. However, because of the low concentrations tested, an inhibitory effect was only observed at 20 lg/ml. Further studies are required to identify the compounds inhibiting NF-jB activation. Fig. 2. CAP-e values of EP fruit pulp (solid line) and EO fruit pulp ( # dotted line, value of EO fruit pulp was cited from our previous paper (Honzel et al., 2008). Fig. 3. Ability of açaí fruit pulp polyphenol-rich extracts (2.5 20 lg/ml) to inhibit NF-jB activation by the SEAP reporter assay ( p < 0.05).
676 J. Kang et al. / Food Chemistry 133 (2012) 671 677 Table 3 Carotenoid concentrations in the EP pulp. Carotenoid EP EO Concentration (lg/g, DW a ) b-carotene 221.5 ± 25.1 10.8 ± 1.2 Lycopene 186.5 ± 14.2 BDL b Astaxanthin 18.7 ± 1.5 BDL Lutein 483.0 ± 37.8 BDL Zeaxanthin 54.0 ± 3.1 BDL Total 963.7 10.8 a b DW, dry weight. BDL: below detection limit. 3.4. Carotenoids in EP and EO Carotenoids are widely present in fruits and have been shown to exhibit activity for quenching singlet oxygen (Di Mascio, Devasagayam, Kaiser, & Sies, 1990; Di Mascio, Murphy, & Sies, 1991). Considering the extremely high SOAC value, the carotenoids were analysed in EP fruit pulp. Five carotenoids, b-carotene, lycopene, astaxanthin, lutein and zeaxanthin, were detected and quantified in EP fruit pulps (Table 3). Their levels ranged from 18.7 lg/g DW of astaxanthin to 483.0 lg/g DW of lutein for EP. The total carotenoids in EP was calculated as 963.7 lg/g DW. Only b-carotene reached a detectable level and quantified at 10.8 lg/g DW in EO (Table 3). This difference in the carotenoid content may partly explain the dramatic difference in the SOAC values between the EP and EO fruit pulp. 4. Conclusion The antioxidant and anti-inflammatory activities of the less known species of açaì (Euterpe precatoria Mart.) were evaluated by various chemical and cell-based assays for the first time. By comparing the data with that of the other well-known açaì species (Euterpe oleracea Mart.), we found that the antioxidant activities of the EP fruit pulp were superior to that of the EO fruit pulp in all assays reported herein. Remarkably, the EP contained much stronger water soluble antioxidants that can enter the live cells and effectively inhibit ROS formation compared to those from EO. The TP did not correlate with the observed antioxidant capacities, suggesting that other compounds yet to be identified may contribute considerably to the wide free radical scavenging capacities of EP. However, since the literature about EP remains limited, the actual antioxidants in EP remain to be characterised. We also observed that the EP fruit pulp polyphenol-rich extract inhibited NF-jB activation as assessed by the SEAP reporter assay, suggesting that the EP fruit pulp has potential anti-inflammatory effects. Further investigation is warranted to identify the actual anti-inflammatory compounds and investigate the mechanisms of action. Finally, because of the high SOAC value showing very strong scavenging ability against 1 O 2 in the EP fruit pulp compared to the EO fruit pulp, the carotenoids were detected and quantified in the EP fruit pulp. 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