Food Chemistry 170 (2015) 16–21

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Food Chemistry

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Beverages of and exotic noni and papaya with potential for anticholinergic effects

Amadeo Gironés-Vilaplana a, Patrícia Valentão b, Paula B. Andrade b, Federico Ferreres a, ⇑ Diego A. Moreno a, , Cristina García-Viguera a a Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, E-30100, Espinardo, Murcia, Spain b REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, no 228, 4050-313 Porto, Portugal article info abstract

Article history: Lemon (Citrus limon (L.) Burm. f.) juice beverages enriched either with noni ( L.) (LN) or Received 13 May 2013 papaya (Carica papaya L.) (LP), were characterized by HPLC-DAD-ESI/MSn, the antioxidant capacity was Received in revised form 11 June 2014 Å Å Å evaluated by (DPPH ), superoxide (O2 ), hydroxyl radicals ( OH) and hypochlorous acid (HOCl) assays, Accepted 7 August 2014 and their potential as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors was also Available online 17 August 2014 assessed. The fruits are rich in a wide range of bioactive phenolics. Regarding DPPHÅ, ÅOH and HOCl assays, Å the LP displayed strong activity, and LN was the most active against O2 . Concerning cholinesterases, LP Keywords: was the most active, mainly due to lemon juice contribution. The effect on the cholinesterases was not as Exotic fruits strong as in previous reports on purified extracts, but the bioactive-rich beverages offer the possibility of Bioactive Antioxidant dietary coadjutants for daily consumption of health-promoting substances by adults with aging-related Anticholinergic cognitive or physical disorders. Noni Ó 2014 Elsevier Ltd. All rights reserved. Papaya Lemon juice

1. Introduction play a determinant role in the cognitive impairment associated with Alzheimer’s disease (AD), senile dementia, ataxia, myasthenia Nowadays it is widely accepted the fact that diets rich in fruits gravis and Parkinson’s disease (Mukherjee et al., 2007). The drugs and vegetables have a positive impact on health and wellbeing. used as cholinesterase inhibitors have adverse side-effects, like Therefore, the current trend is to demonstrate the relationship gastrointestinal disturbances, nausea, vomiting, and diarrhoea, as between food consumption and reduction of diseases risk or pre- well as problems of bioavailability. For this reason, scientific vention of different health conditions. The growing interest in research is seeking natural alternatives of AChE and BuChE new added-value foods and beverages with health-promoting inhibitors with a safer profile. A range of plant compounds with properties has prompted to develop new beverages based on dif- this inhibitory activity have been found, including alkaloids ferent type of waters, , and non-alcoholic drinks enriched (Mukherjee et al., 2007), or xanthones (Brühlmann et al., 2004), with fruits, as natural sources of nutrients, colours, and bioactive but natural foods or juices for this purpose may be a better option phytochemicals, being the evaluation of their bioactivity necessary. in treatment of aging adults under nutrition because of mastica- In the regulation of cognitive functions, the central cholinergic tion, improper deglutition, or swallowing, usual problems in age- system is considered to be the most important neurotransmitter related cognitive decline disorders. involved (Mukherjee, Kumar, Mal, & Houghton, 2007). Further- Noni (Morinda citrifolia L.) and papaya (Carica papaya L.) fruits more, cholinesterases, such as acetylcholinesterase (AChE) and are grown in tropical and sub-tropical regions, with demonstrated butyrylcholinesterase (BuChE), are key enzymes that play signifi- positive implications in the cholinergic system (Essa et al., 2012; cant roles in cholinergic transmission, hydrolysing the neurotrans- Pachauri et al., 2012). However no previous works about noni or mitter acetylcholine (Brühlmann, Marston, Hostettmann, Carrupt, papaya fruits used as inhibitors of cholinesterases has been per- & Testa, 2004). The loss of basal forebrain cholinergic cells results formed to the best of our knowledge. Other positive effects against in an important reduction of acetylcholine, which is believed to (Brown, 2012; Nguyen, Shaw, Parat, & Hewavitharana, 2013), diabetes (Juárez-Rojop et al., 2012; Sabitha, Adhikari Prabha, Shetty Rukmini, Anupama, & Asha, 2009), and obesity ⇑ Corresponding author. Tel.: +34 968 396304; fax: +34 968 396213. E-mail address: [email protected] (D.A. Moreno). (Athesh, Karthiga, & Brindha, 2012; Lin et al., 2012) have been http://dx.doi.org/10.1016/j.foodchem.2014.08.021 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved. A. Gironés-Vilaplana et al. / Food Chemistry 170 (2015) 16–21 17 ascribed to these exotic fruits. In this sense, previous research 2.4. HPLC-DAD-ESI/MSn designing beverages by combining lemon juice (Citrus limon (L.) Burm. f.) with some exotic fruits or berries (Gironés-Vilaplana, Chromatographic analyses were carried out on a Luna C18 Valentão, Andrade, et al., 2012; Gironés-Vilaplana, Valentão, column (250 4.6 mm, 5 mm particle size; Phenomenex, Maccles- Moreno, et al., 2012) revealed an effective increase of the field, UK). Water:formic acid (99:1, v/v) and acetonitrile were used antioxidant properties of the lemon juice and great anticholines- as the mobile phases A and B, respectively, with a flow rate of terases activity in the blends, complementary to the neurodegener- 1 mL/min. The linear gradient started with 8% of solvent B, reach- ative, cardiovascular and metabolic reported for lemon fruits ing 15% at 25 min, 22% at 55 min, and 40% at 60 min, which was (González-Molina, Domínguez-Perles, Moreno, & García-Viguera, maintained up to 70 min. The injection volume was 30 lL. Chro- 2010). matograms were recorded at 280 and 360 nm for all flavonoids. With all this in mind, the aim of this work was to design new The HPLC-DAD-ESI/MSn analyses were carried out in an Agilent blends of lemon juice enriched with noni and papaya fruits looking HPLC 1100 series machine equipped with a photodiode array for potential benefits on cognitive function as cholinesterases’ detector and a mass detector in series (Agilent Technologies, inhibitors (acetylcholinesterase (AChE) and butyrylcholinesterase Waldbronn, Germany). The equipment consisted of a binary pump (BuChE)) for food product developments in the area of nutrition (model G1312A), an autosampler (model G1313A), a degasser for aging adults. First, the phytochemical characterization of (model G1322A), and a photodiode array detector (model polyphenols from beverages by HPLC-DAD-ESI-MSn, and the deter- G1315B). The HPLC system was controlled by ChemStation soft- mination of their antioxidant capacity by different assays (2, ware (Agilent, version 08.03). The mass detector was an ion trap Å Å 2-diphenyl-1-picrylhydrazyl (DPPH ), superoxide (O2 ) and hydro- spectrometer (model G2445A), equipped with an electrospray xyl (ÅOH) radicals and hypochlorous acid (HOCl)) was carried out. ionization interface and controlled by LCMSD software (Agilent, version 4.1). The ionization conditions were 350 °C and 4 kV, for 2. Methods and materials capillary temperature and voltage, respectively. The nebulizer pressure and nitrogen flow rate were 65.0 psi and 11 L/min, 2.1. Chemicals respectively. The full-scan mass covered the range from m/z 100 to m/z 1200. Collision induced fragmentation experiments were The reagents used were commercially available: DPPHÅ, b-nico- performed in the ion trap using helium as the collision gas, with tinamide adenine dinucleotide (NADH), phenazine methosulfate voltage ramping cycles from 0.3 up to 2 V. The mass spectrometry (PMS), nitrotetrazolium blue chloride (NBT), trizina hydrochloride, data were acquired in the positive ionization mode for anthocya- bovine albumin, sodium chloride, AChE from electric eel, BuChE nins and in the negative ionization mode for other flavonoids. n from equine serum, acetylthiocholine iodide, S-butyrylthiocholine The MS was carried out in the automatic mode on the more abun- (n1) chloride, 5,50-dithiobis(2-nitrobenzoic acid) (DTNB), sodium boro- dant fragment ion in MS . Prior to injection, the samples were hydride, sodium hypochlorite solution, ferrium chloride (45% solu- centrifuged (12000 rpm, 5 min) and filtered through a PVDF Filter tion), 2-deoxy-D-ribose, and 2-thiobarbituric acid were obtained (0.22 lm). Flavonols and flavones were quantified as quercetin from Sigma–Aldrich (Steinheim, Germany); potassium dihydrogen 3-O-rutinoside (rutin) at 360 nm, and flavanones as hesperidin at phosphate, ethylenediaminetetraacetic acid (EDTA), hydrogen per- 280 nm. oxide (30%), and trichloroacetic acid were purchased from Merck Å (Darmstadt, Germany); magnesium chloride hexahydrate and 2.5. DPPH scavenging activity ascorbic acid were bought from Fluka Chemika (Neu-Ulm, Switzer- land). Ultrapure water was produced using a Millipore water puri- The antiradical capacity was estimated spectrophotometrically fication system. in a Multiskan Ascent plate reader (Thermo Electron Corporation), by monitoring the disappearance of DPPHÅ at 515 nm, according to 2.2. Samples Ferreres et al. (2009). Three experiments were performed in triplicate. Papaya and noni lyophilized fruits were provided by Ecuadorian Å Rainforest, LLC. (USA). Lemon juice was obtained, from ‘Fino’ 2.6. Superoxide radical (O2 ) scavenging activity freshly collected from the CEBAS-CSIC experimental farm (‘La Matanza’, Santomera, Murcia, SE Spain; 38°601400N, This antiradical activity was determined spectrophotometri- 1°105900W), using a domestic squeezer (‘Citromatic’, Braun Española cally, in a 96-well plate reader, by monitoring the effect of the Å S.A., Barcelona, Spain). The juice was stored frozen (20 °C) until controls and the new drink on the O2 induced reduction of NBT use. at 560 nm. Superoxide radicals were generated by the NADH/ PMS system according to a described procedure (Ferreres et al., 2.3. Experimental design 2009). Three experiments were performed in triplicate.

Lyophilized and powdered fruits were added to lemon juice 2.7. Hypochlorous acid (HOCl) scavenging activity separately to obtain final concentrations of 5% w/v (5 g of pow- dered fruit in 100 ml of lemon juice). In addition, a control solution The inhibition of the hypochlorous acid-induced oxidation of using the same proportion in 0.18 M citric acid buffer (pH 2.46) 5-thio-2-nitrobenzoic acid (TNB) to 5,50-dithiobis(2-nitrobenzoic was prepared, to study the activities of the fruits without lemon. acid) was studied according to a described procedure (Valentão Lemon juice alone was also assayed (pH 2.14). The homogenized et al., 2002), in a double beam spectrophotometer (Hekios a, Uni- mixtures and control solutions were centrifuged (7 min at cam, Leeds, UK) at 412 nm. Three experiments were performed in 4000 rpm), and the supernatant was separated from the solid. triplicate. The juices were stored frozen (20 °C) until use. The samples were labelled as follows: L (lemon juice control), LP (5 g of papaya fruit 2.8. Hydroxyl radical (ÅOH) scavenging activity in 100 ml of lemon juice), AP (5 g of papaya fruit in 100 ml of citric acid buffer), LN (5 g of noni fruit in 100 ml of lemon juice), and AN The deoxyribose method for determining the scavenging effect (5 g of noni fruit in 100 ml of citric acid buffer). of samples on hydroxyl radicals was performed as described before 18 A. Gironés-Vilaplana et al. / Food Chemistry 170 (2015) 16–21

(Valentão et al., 2002), in a double beam spectrophotometer erin in papaya samples (Fig. 1, Table 1) is of interest, since these (Hekios a, Unicam, Leeds, UK) programmed in photometric func- xanthone glycosides have been described as potent hypolipidemic tion, with the wavelength fixed at 532 nm. Three experiments and antidiabetic agents (Sellamuthu, Muniappan, Perumal, & were performed in triplicate. Kandasamy, 2009). Mangiferin was the major flavonoid present in papaya control (0.28 ± 0.01 mg/100 ml); higher concentrations 2.9. AChE and BuChE inhibitory activity of quercetin 3-O-rutinoside were found in LP (0.60 ± 0.01 mg/ 100 ml) due to lemon juice contribution, since this compound is The inhibition of AChE activity was determined based on also present in lemon samples. Two quercetin hexosides were also Ellman’s method, as reported previously (Ferreres, Taveira, identified, but in lower levels. It is important to note that both the Pereira, Valentão, & Andrade, 2010). The absorbance was measured number and the concentration of flavonols and xanthone glyco- at 405 nm and the rates of reaction were calculated using Ascent sides of the blends were notably higher than in the controls. Software version 2.6 (Thermo Labsystems Oy). The BuChE inhibi- Noni beverages displayed some flavonols in small quantities: tion assay was performed in a similar way, using 25 lL of substrate quercetin 3-O-rutinoside-7-O-pentoside, quercetin 3-O-rutinoside (15 mM butyrylthiocholine) and 25 lL of enzyme (0.1 U/mL). and kaempferol 3-O-rutinoside, (Fig. 1, Table 1). Previous works Three experiments were performed in triplicate. have described the rutinosides of quercetin and kaempferol (Dussossoy et al., 2011), but not quercetin 3-O-rutinoside-7-O- 2.10. Statistical analyses pentoside, identified and quantified for the first time in this work. Furthermore, lucidin (a noni characteristic anthraquinone (Deng, The data shown correspond to mean values (n = 3). All data West, Jensen, Basar, & Westendorf, 2009)) was also detected and were subjected to analysis of variance (ANOVA) and a Multiple quantified in significant amounts (2.06 ± 0.08 and 2.04 ± 0.13 mg/ Range Test (Tukey’s test), using PASW Statistics 18 software 100 ml in AN, and in LN, respectively). As it happened with the (Somers, New York, USA). Pearson correlation analysis was per- papaya beverages, the addition to the lemon juice increased the formed to corroborate relationships between the selected flavonols content in LN with respect to AN, probably due to the parameters. addition of compounds from the two sources, or by improving the solubility/extractability of the flavonoids in the food matrix 3. Results and discussion (beverage) as also found in other beverages (i.e. green tea enriched in lyophilized vegetable powder), where the concentration of 3.1. Phenolic compounds active compounds in the new beverage was higher than in the extract of the vegetable alone (Domínguez-Perles, Moreno, The HPLC-DAD-ESI/MSn analysis of controls of noni and papaya Carvajal, & García-Viguera, 2011). revealed the presence of flavonols and xanthones, the new bever- Flavones and flavanones, typically present in Citrus fruits, were ages also showing the typical flavone and flavanone profiles of found in lemon juice control and new blends (Table 1, Fig. 1). lemon (Fig. 1, Table 1). The identification of mangiferin (a xanthone Flavones were mainly represented by diosmetin 6,8-di-C-gluco- glucoside) and the galloylated forms of mangiferin and isomangif- side, followed by diosmetin 7-O-rutinoside, and apigenin 6,

Fig. 1. Chromatograms of lemon-noni (LN) and lemon-papaya (LP) beverages recorded at 360 nm and 280 nm. The identities of the compounds associated with the peaks shown here are given in Table 1. A. Gironés-Vilaplana et al. / Food Chemistry 170 (2015) 16–21 19

Table 1 Phenolic compounds (mg/100 ml) in newly designed blends.

Compounds Rt (min) [MH] MS2 Blends LAPANLPLN Xanthones glycosides F1 Mangiferin 23.3 421 366, 241 – 0.28 ± 0.01 – 0.28 ± 0.01 – F3 Mangiferin gallate 32.9 573 366, 241 – 0.08 ± 0.00 – 0.11 ± 0.01 – F5 Isomangiferin gallate 35.3 573 366, 241 – 0.25 ± 0.02 – 0.34 ± 0.02 – Total 0.61 ± 0.04 0.73 ± 0.02 Flavonols glycosides F2 Quercetin 3-O-rutinoside-7-O-hexoside 24.1 771 609, 301 0.19 ± 0.01 – – 0.19 ± 0.00 0.18 ± 0.02 F6 Quercetin 3-O-rutinoside-7-O-pentoside 36.8 741 609, 301 – – 0.13 ± 0.00 – 0.15 ± 0.00 F7 Quercetin 3-O-rutinoside 40.7 609 301 0.61 ± 0.01 0.18 ± 0.01 0.98 ± 0.02 0.60 ± 0.01 1.59 ± 0.09 F8 Quercetin 3-O-galactoside 42.4 463 301 – 0.06 ± 0.00 – 0.08 ± 0.00 + F9 Quercetin 3-O-glucoside 45.2 463 301 – 0.07 ± 0.00 – 0.15 ± 0.02 – F10 Kaempferol 3-O-rutinoside 51.7 593 285 – – 0.06 ± 0.01 – 0.11 ± 0.03 Total 0.80 ± 0.02 0.31 ± 0.02 1.18 ± 0.03 1.03 ± 0.02 2.03 ± 0.10 Anthraquinones F4 Lucidin 34.0 269 – – – 2.06 ± 0.08 – 2.04 ± 0.13 Flavones glycosides L1 Apigenin 6,8-di-C-glucoside 24.2 593 503, 473 0.64 ± 0.01 – – 0.65 ± 0.01 0.69 ± 0.01 L2 Diosmetin 6,8-di-C-glucoside 28.8 623 503, 413, 383 5.35 ± 0.09 – – 4.94 ± 0.13 4.95 ± 0.27 L3 Diosmetin 7-O-rutinoside 54.3 607 299 2.39 ± 0.04 – – 2.10 ± 0.05 1.92 ± 0.03 Total 3.02 ± 0.05 2.75 ± 0.04 2.62 ± 0.06 Flavanones glycosides L4 Eriodictyol 7-O-rutinoside 35.3 595 287 2.91 ± 0.05 – – 2.76 ± 0.10 2.73 ± 0.18 L5 Hesperetin 7-O-rutinoside 51.9 609 301 3.11 ± 0.06 – – 2.92 ± 0.08 2.91 ± 0.27 Total 6.03 ± 0.10 5.67 ± 0.18 5.63 ± 0.45

L = lemon juice, AP = papaya 5% in citric acid solution, AN = noni 5% in citric acid solution, LP = lemon juice + papaya fruit 5%, LN = lemon juice + noni fruit 5%, n =3.

8-di-C-glucoside. Regarding to flavanones, eriodictyol 7-O-rutino- (Dussossoy et al., 2011) and papaya (Lim, Lim, & Tee, 2007). The side and hesperetin 7-O-rutinoside were found in similar amounts, antioxidant capacity of the blends was notably increased when characteristic composition of ‘‘Fino’’ lemon fruits (Gironés- adding the fruits, LP being the most effective. This increased anti- Vilaplana, Valentão, Andrade, et al., 2012). radical activity of the blends with respect to controls was probably due to the effect of the higher richness of phytochemicals in the 3.2. Antioxidant capacity new beverages, as described above. Also, the high correlation found Å between DPPH and total flavones (r = 0.943, P < 0.001) and total The scavenging ability of lemon, noni and papaya beverages flavanones (r = 0.945, P < 0.001) supports the results. Å Å was tested against different reactive species: DPPH ,O2 , HOCl Å Å Å and OH. DPPH is a non-biological hydrophobic radical extensively 3.2.2. Superoxide radical (O2 ) scavenging used to test the antioxidant capacity of vegetal samples. Free rad- Superoxide radicals have been observed to kill cells, inactivate Å Å icals like O2 and OH and other reactive oxygen species (HOCl) are enzymes and degrade DNA, cell membranes and polysaccharides. produced in the body as a result of aerobic metabolism, playing an Therefore, the study of the superoxide scavenging effects is one important role in the formation of other reactive species that result of the most important ways of illustrating the mechanism of in a wide array of biological damages in living cells (Brand- antioxidant activity (Sakanaka, Tachibana, & Okada, 2005). All bev-

Williams, Cuvelier, & Berset, 1995). erages exhibited low IC50 values (Table 2), indicative of high activ- ity against this reactive oxygen species. The values ranged between 3.2.1. DPPHÅ 2.22 mg/ml (in LN) and 6.72 mg/ml (AP), being noni fruit particu- The model of scavenging the stable DPPHÅ allows evaluating larly effective: displaying the lowest value among controls antioxidative activities in a relatively short time compared to other (2.36 ± 0.11 mg/ml for AN), and being LN the most antioxidant methods (Brand-Williams et al., 1995). The IC50 values were calcu- blend. The addition of papaya to lemon juice (LP) resulted in the lated, in order to compare the different samples and methods highest increase when compared to its control (AP). A strong corre- Å (Table 2). Regarding to controls, lemon juice displayed the highest lation between O2 scavenging and total flavonols glycosides was activity, and AP and AN showed high IC50 values (low activity). A found (r = 0.800, P < 0.001), confirming previous results with noni good DPPHÅ scavenging activity was already cited for noni extracts (Calzuola, Luigi Gianfranceschi, & Marsili, 2006) or lemon

Table 2

Antioxidant and anticholinesterases inhibitory activity (IC50 in mg/ml) of newly designed blends.

Å Å Å Blends DPPH O2 OH HOCl AChE BuChE L 13.26 ± 0.27 b 5.18 ± 0.48 b 6.92 ± 0.22 c 25.56 ± 0.62 b 13.18 ± 0.08 c 12.82 ± 0.21 b AP 29.38 ± 1.43 d 6.72 ± 0.44 c 3.96 ± 0.27 a 40.85 ± 0.57 c 20.47 ± 0.76 d 19.80 ± 0.84 c AN 25.00 ± 0.97 c 2.36 ± 0.11 a 4.98 ± 0.06 b 43.61 ± 0.89 d 19.97 ± 0.39 d 20.82 ± 0.27 c LP 7.73 ± 0.39 a 2.39 ± 0.19 a 3.32 ± 0.09 a 19.58 ± 0.17 a 7.64 ± 0.14 a 7.54 ± 0.16 a LN 10.92 ± 1.20 b 2.22 ± 0.21 a 3.90 ± 0.43 a 28.22 ± 0.91 b 9.07 ± 0.64 b 11.92 ± 0.72 b LSD P < 0.05 0.784 0.264 0.206 0.831 0.419 0.428

Means (n = 3) in the same columns followed by different letters are significantly different at P<0.05 according to Tukey’s test. L = lemon juice, AP = Papaya 5% in citric acid solution, AN = noni 5% in citric acid solution, LP = lemon juice + papaya fruit 5%, LN = lemon juice + noni fruit 5%. 20 A. Gironés-Vilaplana et al. / Food Chemistry 170 (2015) 16–21 juice (Gironés-Vilaplana, Valentão, Andrade, et al., 2012; Gironés- results obtained can be attributed, at least partially, to the presence Vilaplana, Valentão, Moreno, et al., 2012), and milder activity of in the new beverages of bioactive flavonols, flavones, and flavanon- papaya extracts (Yang et al., 2009). es, as supported by Pearson’s correlations between the compounds and the activity and cited in literature (González & Nazareno, Å 3.2.3. Hydroxyl radical ( OH) 2011; Vinholes et al., 2011). Among the oxygen radicals, hydroxyl radical is the most reac- tive and can induce severe damage to the adjacent biomolecules 3.3. AChE and BuChE inhibitory activity (Sakanaka et al., 2005). IC50 scavenging activities reported for all beverages are high (Table 2). Noni (AN) and papaya (AP) controls In the last years, the challenging search of natural inhibitors of were more active than lemon juice control (L), and also the new cholinesterases is increasing, since these enzymes are associated blends were more effective (3.32 mg/ml for LP and 3.90 mg/ml with neurodegenerative disorders like Alzheimer’s disease, senile for LN). Good hydroxyl radical scavenging activity was reported dementia, ataxia, myasthenia gravis, and Parkinson’s disease, for papaya extracts (Yang et al., 2009), but not so high for noni among others (Mukherjee et al., 2007). Cholinesterases inhibitory juice (Ikeda, Wada, Nishigaki, & Nakashima, 2009), in contrast to activity of the beverages was tested against acetylcholinesterase Å our results. Moreover, correlation was found between OH scaveng- and butyrylcholinesterase (Table 2, Fig. 2). The results obtained ing activity and total xanthones glycosides (r = 0.635, P = 0.01), against both enzymes were similar, displaying strong correlations supporting the activity of LP. Nevertheless, that does not mean that between them (r = 0.972, P < 0.001). Lemon juice exhibited the other components not identified or quantified in these matrices, strongest inhibition among controls. This inhibitory activity can also contribute to the observed activity. increased when both noni and papaya were added (LP and LN), although the effect of noni on BuChE was not significant. This high 3.2.4. Hypochlorous acid (HOCl) anticholinesterases effect of lemon blends can be explained by the HOCl is a powerful oxidant which reacts readily with many bio- Pearson’s correlation found between its components and the logically important molecules. It is produced in the organism by activities: r = 0.905, P < 0.001 for AChE and flavones, r = 0.915, oxidation of Cl ions at sites of inflammation by the neutrophil P < 0.001 for AChE and flavanones, r = 0.912, P < 0.001 for BChE enzyme myeloperoxidase. The oxidizing properties of HOCl induce and flavones and r = 0.919, P < 0.001 for BChE and flavanones. the conversion of TNB (kmax = 412 nm) to DTNB (kmax = 325 nm). The LP blend was the most potent among all the drinks against A HOCl scavenger inhibits the conversion of TNB to DTNB by this both enzymes (Table 2, Fig. 2), reporting highest anticholinergic species (Valentão et al., 2002). The activity observed against HOCl effects than juices prepared with lemon and berries (Gironés- was the lowest among all tested reactive specie. The IC50 values Vilaplana, Valentão, Andrade, et al., 2012; Gironés-Vilaplana, varied significantly, between 19.58 and 43.61 mg/mL (Table 2). Valentão, Moreno, et al., 2012). Although this effect is significantly Among controls, lemon juice showed the highest activity, suggest- lower than pharmacological drugs used in neurological diseases, 4 ing that lemon components play a crucial role. Furthermore, to such as huperzine A (IC50 =10 lM) (Mukherjee et al., 2007), or support this hypothesis, strong correlations were found between galantamine (IC50 for AChE = 0.14 lg/mL) (Wszelaki, Kuciun, & HOCl scavenging activity and lemon phytochemicals (r = 0.943, Kiss, 2010), this is a natural food matrix (juice) with potential for P < 0.001, and r = 0.946, P < 0.001, for flavones and flavanones, daily consumption, without any side effects. Some natural prod- respectively), and previous works defined citrus pulp as an ucts have been recently reported as natural cholinesterases inhib- effective HOCl scavenger (Ramful, Tarnus, Aruoma, Bourdon, & itors, such as tomato seeds (IC20 = 2.4 mg/mL) (Ferreres et al., Bahorun, 2011). The addition of papaya to lemon juice (LP) resulted 2010). Other purified extracts reported anticholinergic effects such on the highest activity when compared to controls (AP and L), as Croton gratissimus Burch. (IC50 = 0.21 mg/ml for AChE) and while the addition of noni to lemon juice increased the antioxidant Croton zambesicus Muell.Arg. (IC50 = 0.28 mg/ml for AChE) leaves capacity with respect to noni control (LN vs AN), but not with (Ndhlala, Aderogba, Ncube, & Van Staden, 2013), or Lavandula respect to lemon juice control (LN vs L) (Table 2). As far as we viridis L’Her essential oil (IC50 = 0.42 mg/ml and 0.75 mg/ml for know, HOCl scavenging capacity of noni and papaya fruits has AChE and BuChE, respectively). Moreover, some flavonoids deriva- never been reported. tives have demonstrated potential for acetylcholinesterase inhibi- The antioxidant capacity results of the beverages was in the tion (Li, Wang, Hu, & Kong, 2013), but the LP and LN are not range of previous works in blends of lemon juice with berries con- purified extracts: they are natural juices enriched with powdered centrates (Gironés-Vilaplana, Valentão, Andrade, et al., 2012; fruits that can be taken regularly. Due to this fact, these results Gironés-Vilaplana, Valentão, Moreno, et al., 2012). Moreover, the are of interest for developing natural foods using lemon juice

Fig. 2. Acetylcholinesterase and butyrylcholinesterase inhibitory activity of the lemon juice (L), papaya control (AP), noni control (AN), lemon juice plus 5% papaya fruit (LP) and lemon juice plus 5% noni fruit (LN). A. Gironés-Vilaplana et al. / Food Chemistry 170 (2015) 16–21 21 enriched with papaya and noni, for dietary interventions and nutri- Dussossoy, E., Brat, P., Bony, E., Boudard, F., Poucheret, P., Mertz, C., et al. (2011). tion programs for aging adults. Further research in formulation, Characterization, anti-oxidative and anti-inflammatory effects of Costa Rican noni juice (Morinda citrifolia L.). Journal of Ethnopharmacology, 133, 108–115. bioaccessibility and efficacy is guaranteed. Essa, M. M., Vijayan, R. K., Castellano-Gonzalez, G., Memon, M. A., Braidy, N., & Guillemin, G. J. (2012). Neuroprotective effect of natural products against Alzheimer’s disease. Neurochemical Research, 37, 1829–1842. 4. Conclusions Ferreres, F., Fernandes, F., Oliveira, J. M. A., Valentão, P., Pereira, J. A., & Andrade, P. B. (2009). Metabolic profiling and biological capacity of Pieris brassicae fed with In this work new blends of lemon juice and different exotic kale (Brassica oleracea L. var. acephala). Food and Chemical Toxicology, 47, fruits (papaya and noni) with health-promoting effects were devel- 1209–1220. Ferreres, F., Taveira, M., Pereira, D. M., Valentão, P., & Andrade, P. B. (2010). Tomato oped. The new beverages presented phytochemical profiles rich in (Lycopersicon esculentum) seeds: New flavonols and cytotoxic effect. Journal of bioactive phenolics (flavonols, xanthone, flavones, flavanones and Agricultural and Food Chemistry, 58, 2854–2861. anthraquinone derivatives). New data on scavenging abilities of Gironés-Vilaplana, A., Valentão, P., Andrade, P. B., Ferreres, F., Moreno, D. A., & García-Viguera, C. (2012). Phytochemical profile of a blend of black chokeberry new beverages made of lemon enriched with noni and papaya and lemon juice with cholinesterase inhibitory effect and antioxidant potential. described additional mode of actions in terms of antioxidant Food Chemistry, 134, 2090–2096. capacity and suggest that phenolic compounds are not the only Gironés-Vilaplana, A., Valentão, P., Moreno, D. A., Ferreres, F., García-Viguera, C., & Andrade, P. B. (2012). New beverages of lemon juice enriched with the exotic parameters indicative of the antioxidant potential, as other compo- berries maqui, açaí, and blackthorn: Bioactive components and in vitro nents which possibly exist in papaya and noni, and the interactions biological properties. Journal of Agricultural and Food Chemistry, 60, 6571–6580. between them in the food matrix, that may also play important González, E. A., & Nazareno, M. A. (2011). Antiradical action of flavonoid-ascorbate mixtures. LWT – Food Science and Technology, 44, 558–564. roles on the scavenging of reactive species. The lemon-papaya bev- González-Molina, E., Domínguez-Perles, R., Moreno, D. A., & García-Viguera, C. erage (LP) is the most interesting blend in terms of antioxidant (2010). Natural bioactive compounds of Citrus limon for food and health. Journal capacity, and also in terms of inhibition of cholinesterases (AChE of Pharmaceutical and Biomedical Analysis, 51, 327–345. Ikeda, R., Wada, M., Nishigaki, T., & Nakashima, K. (2009). Quantification of and BuChE), mainly due to the combination with lemon juice. coumarin derivatives in noni (Morinda citrifolia) and their contribution of The development of rich in natural foods with cholinesterases quenching effect on reactive oxygen species. Food Chemistry, 113, 1169–1172. inhibitors, with potential application in preserving cognitive func- Juárez-Rojop, I. E., Díaz-Zagoya, J. C., Ble-Castillo, J. L., Miranda-Osorio, P. H., Castell- tion and managing age-related conditions (i.e. Alzheimer’s and Rodríguez, A. E., Tovilla-Zárate, C. A., et al. (2012). Hypoglycemic effect of Carica papaya leaves in streptozotocin-induced diabetic rats. BMC Complementary and Parkinson’s diseases, senile dementia, among others) is possible , 12. with healthy fruit-based beverages to facilitate daily intake, and Li, R. S., Wang, X. B., Hu, X. J., & Kong, L. Y. (2013). Design, synthesis and evaluation in vivo research for safety and efficacy is guaranteed to support fur- of flavonoid derivatives as potential multifunctional acetylcholinesterase inhibitors against Alzheimer’s disease. Bioorganic and Medicinal Chemistry ther actions. Letters. Lim, Y. Y., Lim, T. T., & Tee, J. J. (2007). Antioxidant properties of several tropical Acknowledgements fruits: A comparative study. Food Chemistry, 103, 1003–1008. Lin, Y. L., Chou, C. H., Yang, D. J., Chen, J. W., Tzang, B. S., & Chen, Y. C. (2012). Hypolipidemic and antioxidative effects of noni (Morinda citrifolia L.) juice on Authors would like to express their gratitude to Spanish Minis- high-fat/cholesterol-dietary hamsters. Plant Foods for Human Nutrition, 67, try of Economy and Competitivity for the funding through the 294–302. Mukherjee, P. K., Kumar, V., Mal, M., & Houghton, P. J. (2007). Acetylcholinesterase CICYT project AGL2011-23690, and the CONSOLIDER-INGENIO inhibitors from plants. Phytomedicine, 14, 289–300. 2010 Research Project FUN-C-FOOD (CSD2007-00063) and to Ndhlala, A. R., Aderogba, M. A., Ncube, B., & Van Staden, J. (2013). Anti-oxidative and Fundação para a Ciência e a Tecnologia (FCT) for grant no. PEst- cholinesterase inhibitory effects of leaf extracts and their isolated compounds from two closely related croton species. Molecules, 18, 1916–1932. C/EQB/LA0006/2011. Part of this work was carried out as an inter- Nguyen, T. T. T., Shaw, P. N., Parat, M. O., & Hewavitharana, A. K. (2013). Anticancer national research collaboration within the CYTED Programme (Ref. activity of Carica papaya: A review. Molecular Nutrition and Food Research, 57, 112RT0460) CORNUCOPIA Thematic Network (URL: www.redcor- 153–164. nucopia.org). AGV would also like to thank CSIC for a JAE Pachauri, S. D., Tota, S., Khandelwal, K., Verma, P. R. P., Nath, C., Hanif, K., et al. (2012). Protective effect of fruits of Morinda citrifolia L. on scopolamine induced Predoctoral Grant, and a special thanks to the Laboratory of Phar- memory impairment in mice: A behavioral, biochemical and cerebral blood macognosy of the Faculty of Pharmacy of Porto University for the flow study. Journal of Ethnopharmacology, 139, 34–41. help in the bioactivity tests. Ramful, D., Tarnus, E., Aruoma, O. I., Bourdon, E., & Bahorun, T. (2011). Polyphenol composition, vitamin C content and antioxidant capacity of Mauritian citrus fruit pulps. Food Research International, 44, 2088–2099. 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