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In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole cashew apple...
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In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds ofwhole cashew apple juice and cashew apple fiber (Anacardium occidentale L.)following simulated gastro-intestinal digestion
Ana Cristina Silva de Lima, Denise Josino Soares, Larissa Morais Ribeiro daSilva, Raimundo Wilane de Figueiredo, Paulo Henrique Machado de Sousa,Eveline de Abreu Menezes
PII: S0308-8146(14)00526-3DOI: http://dx.doi.org/10.1016/j.foodchem.2014.03.123Reference: FOCH 15653
To appear in: Food Chemistry
Received Date: 7 October 2013Revised Date: 20 March 2014Accepted Date: 26 March 2014
Please cite this article as: de Lima, A.C.S., Soares, D.J., da Silva, L.M.R., de Figueiredo, R.W., de Sousa, P.H.M.,de Abreu Menezes, E., In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole cashewapple juice and cashew apple fiber (Anacardium occidentale L.) following simulated gastro-intestinal digestion,Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.03.123
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In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole 1
cashew apple juice and cashew apple fiber (Anacardium occidentale L.) following 2
simulated gastro-intestinal digestion 3
4
Running title: Bioaccessibility of cashew apple following simulated gastro-intestinal 5
digestion 6
7
Ana Cristina Silva de Limaa*, Denise Josino Soaresa, Larissa Morais Ribeiro da Silvaa, 8
Raimundo Wilane de Figueiredoa, Paulo Henrique Machado de Sousab, Eveline de Abreu 9
Menezesc 10
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a Departamento de Tecnologia de Alimentos/Universidade Federal do Ceará, Av. Mister Hull, 12
2977, Campus Universitário do Pici, Fortaleza, Ceara, Brazil 60356-000. E-mail: 13
[email protected]; [email protected]; [email protected]; 14
b Instituto de Cultura e Arte/Universidade Federal do Ceará, Av. Mister Hull, 2977, Campus 16
Universitário do Pici, Fortaleza, Ceara, Brazil. 60356-000. E-mail: 17
c Universidade Estadual do Paiuí, Campus Professor Antonio Geovanne de Sousa Piripiri. 19
Avenida Marechal Castelo Branco, 180 Petecas, Piripiri, Piaui, Brazil, 64260-000. E-mail: 20
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* Corresponding author: E-mail: [email protected]; Fax: +55 85 33669752. 23
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Abstract. Considering the lack of research studies about nutrients’ bioaccessibility in cashew 25
apple, in this study the whole cashew apple juice and the cashew apple fiber were submitted 26
to simulated in vitro gastrointestinal digestion. The samples were analyzed before and after 27
digestion and had their copper, iron, zinc, ascorbic acid, total extractable phenols and total 28
antioxidant activity assessed. As a result, for the whole cashew apple juice, the content of 29
copper and iron minerals bioaccessible fraction was higher than 10% and for zinc this level 30
was lower than 5%. Regarding the cashew apple fiber, the bioaccessible fraction for these 31
minerals was lower than 5%. The ascorbic acid, total extractable polyphenols and total 32
antioxidant activity bioaccessible fraction for whole cashew apple juice showed 33
bioaccessibility percentages higher than 25%, while for the cashew apple fiber, bioaccessibles 34
levels were found to be around 15%. 35
Key-Words: ICP-OES, inorganic compounds, bioactive compounds, antioxidant activity. 36
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In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole 40
cashew apple juice and cashew apple fiber (Anacardium occidentale L.) following 41
simulated gastro-intestinal digestion 42
43
Running title: Bioaccessibility of cashew apple juice and cashew apple fiber compounds. 44
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Abstract. Considering the lack of research studies about nutrients’ bioaccessibility in cashew 46
apple, in this study the whole cashew apple juice and the cashew apple fiber were submitted 47
to simulated in vitro gastrointestinal digestion. The samples were analyzed before and after 48
digestion and had their copper, iron, zinc, ascorbic acid, total extractable phenols and total 49
antioxidant activity assessed. As a result, for the whole cashew apple juice, the content of 50
copper and iron minerals bioaccessible fraction were 15% and 11.5% and for zinc this level 51
was 3.7%. Regarding the cashew apple fiber, the bioaccessible fraction for these minerals was 52
lower than 5%. The ascorbic acid, total extractable polyphenols and total antioxidant activity 53
bioaccessible fraction for whole cashew apple juice showed bioaccessibility percentages of 54
26.2%, 39% and 27%, respectively, while for the cashew apple fiber, low bioaccessibles 55
levels were found. The bioacessible percentage of zinc, ascorbic acid and total extractable 56
polyphenols were higher in cashew apple juice than cashew apple fiber, 57
Key-Words: ICP OES, minerals, bioactive compounds, antioxidant activity. 58
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1. Introduction 60
The cashew tree belongs to the Anacardiaceae family, concentrated in the tropical 61
region of the globe and is widespread in several countries such as Brazil, India, Mozambique, 62
Tanzania, Kenya, Vietnam, Indonesia and Thailand (Mazetto, Lomonaco, & Mele, 2009). The 63
pseudo fruit of the cashew tree is known as cashew apple and has a similar structure to that of 64
4
a fibrous and juicy fruit. The cashew apple pulp is rich in ascorbic acid (Queiroz, Lopes, 65
Fialho, & Valente-Mesquita, 2011), phenolic compounds, minerals (Sivagurunathan, 66
Sivasankari, & Muthukkaruppan, 2010) and carotenoids, giving this fruit the title of 67
functional food. 68
Despite its high level of astringency, cashew apple has a good potential for 69
industrialization, due to its fleshy pulp, soft skin, high sugar and exotic flavor 70
(Sivagurunathan et al., 2010), and it is widely consumed in the form of juice, nectar, jam, 71
among others. During the processing of fruit products byproducts such as skins, seeds and 72
fibers are produced. The utilization of these byproducts can contribute to the improvement of 73
the environment, in view of the large volumes produced and disposed of in inappropriate 74
places, causing serious environmental problems (Sousa, Vieira, Silva, & Lima, 2011). The use 75
of cashew apple fiber for human consumption opens up new perspectives, as it is a natural 76
source of phenolic compounds and antioxidant activity (Broinizi et al., 2007), and also has 77
appreciable amounts of vitamin C (Uchoa, Costa, Maia, Silva, Carvalho, & Meira, 2008). 78
Several studies have focused on the functional compounds present in cashew apple 79
(Brito, Araújo, Lin, & Harnly, 2007; Queiroz et al., 2011), however, in terms of nutrition it is 80
not enough merely to determine the total content of nutrients, it is necessary to know the 81
bioaccessibility, in other words, the amount of compound released from the matrix during 82
gastrointestinal digestion that becomes available for absorption in the intestine. 83
Studies about the bioaccessibility of nutrients in foods can be performed using in vivo 84
and/or in vitro methods. The combination of these methods can provide information that can 85
help in the interpretation of results. The in vitro method is applied to a system of simulated 86
gastrointestinal digestion using pepsin in the gastric phase and a mixture of pancreatin and 87
bile salts during the intestinal tract. The element diffused through a semipermeable membrane 88
5
in the intestinal phase is used as a measure of the element bioaccessibility (Kulkarni, Acharya, 89
Rajurkar, & Reddy, 2007). 90
In this context, the aim of this study was to determine the bioaccessibility of the 91
minerals copper, iron and zinc, of ascorbic acid, total phenolics and total antioxidant activity 92
of cashew apple juice and cashew apple fiber. 93
94
2. Material and Methods 95
2.1. Chemicals 96
ABTS•+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), pancreatin, pepsin, 97
bile extract, Folin–Ciocalteau reagent, ascorbic acid and galic acid were purchased from 98
Sigma Aldrich (Saint Louis, USA). All the other reagents and chemicals of analytical grade 99
were purchased from local sources. Certified reference material SRM 1547 - Peach Leaves, 100
the National Institute of Standard Technology (NIST, Gaithersburg, MD, USA) was used to 101
assess the accuracy of methods for determination of analytes; stock patterns of Cu, Fe and Zn 102
1000 mg L-1 solutions. Titrisol (Merck, Darmstadt, Germany) were used to prepare reference 103
solutions. 104
2.2. Cashew apple juice and cashew apple fiber 105
The experiment was performed with cashew apple juice and cashew apple fiber. Bottles 106
of cashew apple juice (500 mL) from three different batches belonging to a commercial brand, 107
were donated by the production company, located in Ceara/Brazil. For the extraction of 108
cashew apple fiber, whole fruits were used, with red and yellow color, hand-picked for their 109
quality attributes and washed by immersion in chlorinated water (200 ppm of active chlorine). 110
The cashew apples were peeled and the juice was separated from the fiber with the aid of a 111
domestic centrifuge. At the end of the process, the fibers which had been obtained were 112
packed in polyethylene bags, properly sealed in a vacuum and stored in a freezer (-18 ± 1°C) 113
until analysis. 114
2.3. Mineral analysis 115
6
The digestion was performed with the aid of a microwave oven cavity (Model 116
Multiwave 3000) with a temperature sensor and internal and external pressure. Quartz jars 117
were used for digestion of the samples, adapting procedures described by Menezes (2010). 118
For the microwave digestion of the cashew apple samples, 0.5 mL of the juice was used, 119
which had been digested with the diluted oxidant mixture (1 mL of H2O2 and 2 mL of HNO3). 120
Table 1 describes the heating program used during microwave digestion of cashew apple 121
juice. 122
The quantification of copper, iron and zinc was performed by using an external standard 123
curve prepared with stock standard solutions of Cu, Fe e Zn 1000 mg L-1 Titrisol (Merck, 124
Darmstadt, Germany) as a reference. Certified reference material SRM 1547 - Peach Leaves, 125
the National Institute of Standard Technology (NIST, Gaithersburg, MD, USA) was used to 126
assess the accuracy of methods for determination of analytes. 127
After the complete oxidation of the organic matter, the samples were diluted to 20 mL 128
with deionized water and the copper, iron and zinc were measured by Inductively Coupled 129
Plasma Optical Emission Spectrometry (ICP OES) (Table 2). 130
For the microwave digestion of cashew apple fiber crushing of the samples was performed 131
followed by drying in an oven at 60°C/24 hours and then maceration until a powder was 132
obtained. Two hundred milligrams of the sample were digested with the diluted oxidant 133
mixture (1 mL of H2O2, 1 mL of HNO3 and 1 mL of Milli Q water). After the complete 134
oxidation of the organic matter, the samples were diluted to 10 mL with deionized water and 135
the copper, iron and zinc were measured by ICP OES in the same way as described for the 136
cashew apple juice using the heating program for microwave digestion as described in Table 137
1. 138
2.4. Simulated in vitro gastrointestinal digestion 139
The digestions were performed with simulated gastric fluid and simulated intestinal 140
fluid, both prepared according to procedures implemented by Moura and Canniatti-Brazaca 141
(2006). The simulated gastrointestinal digestion was performed with pepsin solubilized with 142
0.1 mol L-1 HCl during the gastric phase and pancreatin-bile salts solubilized with 0.1 mol L-1 143
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NaHCO3 in the intestinal phase. Twenty grams of each sample was added to 100 mL of 0.01 144
mol L-1 HCl and adjusted to pH 2 with 2 mol L-1 HCl solution. After the pH adjustment, 3.2 145
mL of pepsin was added, and the sample was stirred in a thermostat at 37°C/2 hours to 146
simulate the digestion of the food in the stomach. After that, titration with 0.5 mol L-1 NaOH 147
was performed until pH 7.5 to simulate the pH found in the intestine of an individual. Dialysis 148
was performed for two hours in dialysis membranes (33 x 21 mm, molecular weight: 12.000-149
16.000, porosity: 25 Angstrons – INLAB, Brazil) containing 0.1 mol L-1 NaHCO3 equivalent 150
to titratable acidity. After the pH adjustment, the dialysis membranes were added and stirred 151
in a water bath thermostatted at 37°C/30 minutes, then 5.0 mL of pancreatin solution and bile 152
salts were added and stirred in a bath at 37°C/2 hours. This step simulates the digestion of 153
food in the intestine. At the end of this step the contents of the membrane (dialysate) were 154
removed and samples were stored at -20°C until the time of analysis. 155
2.5. Determination of bioacessible levels of cooper, iron and zinc 156
The dialyzed samples obtained from the in vitro simulated gastrointestinal digestion 157
were analyzed by ICP OES according to the conditions mentioned in Table 2. The 158
bioaccessible percentage was calculated according to the equation described by Menezes 159
(2010) (Equation 1). 160
161
% Bioaccessible = (A x 25 mL/B x C) x 100 Equation (1) 162
163
A - element content of the dialysable mineral fraction (mg), B - value of the total mineral 164
(iron, copper or zinc) content of the sample (mg); C - initial weight of sample (g). 165
166
2.6. Ascorbic acid level determination 167
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The level of ascorbic acid was determined according to Scherer, Rybka and Godoy 168
(2008). Analyses were conducted in HPLC Shimadzu, controlled by LC Solution Software, 169
using a manual injector with a fixed volume of 20 µL, pump model LC-20DA, performed at 170
25°C adjusted by CTO-20A oven and UV-VIS detector model SPD-20A. The Nova Pack C18 171
(CLC-ODS, 3µm, 4.6 mm x 25 cm) column was used. The injections were performed in 172
triplicate. The mobile phase used was an aqueous solution of 0.01 M KH2PO4, with pH 173
adjusted to 2.6 with phosphoric acid at a flow rate of 0.5 mL min-1. The quantification was 174
performed by using an external standard curve with seven points prepared with ascorbic acid 175
(Sigma Aldrich, Saint Louis, USA) as a reference. All samples and the mobile phase were 176
filtered in a 0.45 µm membrane (Millipore). For the determination of ascorbic acid before the 177
simulated gastrointestinal digestion in vitro, cashew apple juice and cashew apple fiber were 178
diluted with the mobile phase (1/9, v/v, aqueous solution of 0.01 M KH2PO4), filtered and 179
injected in the chromatograph with a run time of 10 minutes. For the determination of 180
ascorbic acid after in vitro simulated gastrointestinal digestion of cashew apple juice and 181
cashew apple fiber the dialysate was removed, filtered and injected into the chromatograph 182
with a run time of 10 minutes under the same conditions described above. The identification 183
of ascorbic acid in the samples was performed by comparing the retention times obtained for 184
the standard (L-ascorbic acid), and co-injection of samples with the standard solution. The 185
bioaccessible percentage was calculated according to Briones-Labarca, Venegas-Cubillos, 186
Ortiz-Portilla, Chacana-Ojeda, and Maureira (2011) (Equation 2). 187
188
% Bioaccessible = 100 x (D/E) Equation (2) 189
190
D - ascorbic acid dialyzable content (mg 100 g-1), E - ascorbic acid content of the sample (mg 191
100 g-1). 192
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2.7. Total extractable polyphenol level determination 193
The total extractable polyphenols were determined by the Folin-Ciocalteu method, 194
using a standard curve prepared with galic acid (Sigma Aldrich, Saint Louis, USA) as a 195
reference, according to the methodology described by Larrauri, Rupérez, and Saura-Calixto 196
(1997). The determination of total extractable polyphenols before in vitro simulated 197
gastrointestinal digestion of the cashew apple juice and cashew apple fiber was performed 198
according to the methodology described by Larrauri et al. (1997), by reading the extracts in a 199
spectrophotometer (Shimadzu, model UV-1800) at 700 nm. For the determination of total 200
extractable polyphenols after simulated gastrointestinal digestion in vitro the dialysate was 201
analyzed using the same methodology described for the determination of total extractable 202
polyphenols before gastrointestinal digestion. The results were expressed in mg of galic acid 203
equivalent (GAE) 100g-1. The bioaccessible percentage was calculated according to Briones-204
Labarca et al. (2011) (Equation 3). 205
206
% Bioaccessible = 100 x (F/G) Equation (3) 207
208
F – Total extractable polyphenol compounds dialyzable (mg GAE 100 g-1), G - total 209
extractable polyphenol content of the sample (mg GAE 100 g-1). 210
211
2.8. Antioxidant activity 212
The antioxidant activity was determined by the ABTS•+ method, as described by 213
Rufino, Alves, Brito, Pérez-Jiménez, Saura-Calixto, and Mancini-Filho (2010). The extract 214
used for this analysis was the same as that used for the determination of total extractable 215
polyphenols. The readings for the determination of antioxidant activity before and after in 216
vitro digestion were carried out in a spectrophotometer (Shimadzu model UV-1800) to 734 217
10
nm, and the quantification of the antioxidant activity before digestion was performed on the 218
extract prepared with the cashew apple juice and the cashew apple fiber and the quantification 219
after digestion was performed in the dialysate. The quantification was performed by using an 220
external standard curve prepared with Trolox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-221
carboxylic acid) (Sigma Aldrich, Saint Louis, USA) as a reference. The results were 222
expressed as equivalent antioxidant Trolox® (TEAC) in µM g-1. The bioaccessible percentage 223
was calculated according to Briones-Labarca et al. (2011) (Equation 4). 224
225
% Bioaccessible= 100 x (H/I) Equation (4) 226
227
H - dialysable antioxidant activity (µM g-1), I - antioxidant activity of the sample (µM g-1.). 228
229
2.9. Statistical analysis 230
The experiment was conducted according to a completely randomized design with three 231
replications of the experiments. The results were statistically evaluated by variance analysis. 232
As evidenced the significant by the F test, the treatments were compared by Tukey test at 5% 233
probability. 234
235
3. Results and Discussion 236
The content of the minerals, ascorbic acid, total extractable polyphenols and antioxidant 237
activity of cashew apple juice and cashew apple fiber decreased during gastrointestinal 238
digestion, occurring a significant difference (p<0.05) between the native and after digestion 239
level (Tables 3 and 4). 240
3.1. Minerals copper, iron and zinc 241
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Copper is an essential element for plants and animals, its importance lying in the fact 242
that it is present in more than 13 enzymes that are involved in energy production, in the 243
prevention of anemia and bone disease, in reducing cell damage, and which are also required 244
for fetal and infant development. In addition, copper is required for other functions, such as 245
the maintenance of tissue and skin and hair pigmentation (Altundag & Tuzen, 2011). Due to 246
the functions performed by this mineral, its intake is essential, and the recommended daily 247
intake is 2 mg (based on a 2000 calorie intake) (FDA, 2013). 248
The average values for copper in cashew apple juice and cashew apple fiber were 2.10 249
and 12.20 mg L-1, respectively. There was a reduction in copper content after digestion, and 250
the amounts of 0.25 and 0.40 mg L-1 for cashew apple juice and cashew apple fiber, 251
respectively, were observed. Despite the fact that cashew apple juice has a lower copper 252
content than cashew apple fiber, the bioaccessibility of the juice was almost four times higher 253
than that observed in the cashew apple fiber (Table 3). This fact can be explained by the 254
possible presence of phytic acid, which is found naturally in peels, seeds and insoluble fiber, 255
which is capable of chelating minerals, reducing their bioaccessibility (Walter, Marchezan 256
and Avila, 2008) especially when subjected to heating processes (Helbig & Gigante, 2008). 257
Iron’s main function in the body is its presence in the formation of red blood cells, and 258
its deficiency causes anemia, reducing the number of red blood cells and, thereby, decreasing 259
oxygenation (Lehninger, Nelson, & Cox, 2011). The recommended daily intake of iron is 18 260
mg (based on a 2000 calorie intake) (FDA, 2013). The values for iron obtained before and 261
after in vitro simulated gastrointestinal digestion were 1.82 and 0.17 mg L-1 for the cashew 262
apple juice, and 21.60 and 0.20 mg L-1 for cashew apple fiber, respectively (Table 3). Soares, 263
Shishido, Moraes, and Moreira (2004) observed content of 1.27 mg L-1 in cashew apple juice. 264
The bioaccessibility of iron after digestion of cashew apple juice was 11.50% and of 265
cashew apple fiber was 1.2% (Table 3). Khouzam, Pohl, and Lobinskib (2011) in a study on 266
12
the evaluation of the bioaccessibility of essential elements in fruits and vegetables, reported 267
bioaccessible percentages of iron ranging from 6.7 to 12.7%, values close to those found for 268
the cashew apple juice in the present study, and they suggest that the low-iron bioaccessibility 269
in fruits and vegetables is due to the presence of phytate, oxalic acid and carbonate salts 270
which form insoluble polyphenols and impair iron absorption. The reduction of iron 271
bioaccessibility caused by the presence of phytates has also been reported by Cámara, Amaro, 272
Barbera, and Clemente (2005) in lentils. 273
Zinc is required for the operation of over 300 different enzymes and plays a vital role in 274
a number of biological processes (Aberoumand & Deokule 2009). The deficiency of this 275
mineral in humans causes growth retardation, abnormal bone formation and dermatitis 276
(Konoha, Sadakane, & Kawahara, 2006). The recommended daily intake of zinc based on a 277
2000 calorie intake is 15 mg (FDA, 2013). The obtained values for zinc in cashew apple juice 278
and cashew apple fiber were 4.70 and 7.14 U mg-1, and after in vitro simulated 279
gastrointestinal digestion were 0.14 and 0.12 mg L-1, respectively (Table 3). Soares et al. 280
(2004), in a study on the amount of total mineral in fruit juices, reported an average of 0.12 281
mg L-1 of zinc in the concentrate cashew apple juice. 282
In the present study, for both cashew apple juice and cashew apple fiber the 283
bioaccessible fraction of zinc was lower than 5% (Table 3). The bioaccessibility of this 284
mineral is small, as has been reported by Cámara et al. (2005) in rice with meat (7.5%), 285
spinach omelet (5.8%) and pasta with tuna (7.6%). This is attributed to the presence of other 286
minerals, since, according to Andrade, Alves, and Takase (2005), the presence of other 287
elements like Fe, Ca and Cd influence the absorption of zinc by the body. Another factor that 288
can reduce the bioaccessibility of zinc is the presence of phytate, which also may have caused 289
a reduction in the levels of copper and iron in this study. 290
3.2. Ascorbic acid 291
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Ascorbic acid is an important nutrient for the human physiology, and it has a role in the 292
production and maintenance of collagen, wound healing, the reduction in susceptibility to 293
infections, in the formation of bones and teeth, iron absorption and prevention of scurvy 294
(Maia, Sousa, Santos, Silva, Fernandes, & Prado, 2007). Due to these characteristics, the 295
intake of this compound is important and the study of sources of ascorbic acid is necessary. 296
The ascorbic acid levels in cashew apple juice were 49.30 and 12.90 mg 100 g-1 native 297
and after in vitro simulated gastrointestinal digestion, respectively (Table 4). The decrease in 298
the level of ascorbic acid which occurred during digestion may be due to exposure of the 299
cashew apple juice to digestion temperature, since this compound is thermosensitive. The 300
results obtained in cashew apple juice are in agreement with those observed by Scherer et al. 301
(2008) who measured the ascorbic acid in cashew apple juice using the HPLC technique, and 302
found average values of 47.42 mg 100 g-1. Pinheiro, Fernandes, Fai, Prado, Sousa, and Maia 303
(2006) in their study of cashew apple juice observed an average of ascorbic acid of 109.6 mg 304
100 g-1. The differences in ascorbic acid content observed can be due to the soil conditions 305
and climate where the cashew trees were grown, and the maturation of cashews. 306
The presence of ascorbic acid in cashew apple fiber was not observed (Table 4), which 307
differs from the observations by Pinho, Afonso, Carioca, Costa, and Rybka (2011) who 308
reported an ascorbic acid level of 2.7 mg 100 g-1 in cashew apple fiber. The non-detection of 309
ascorbic acid in the present study may be due to the drying step in an oven (60°C/24 hours), 310
since this compound is sensitive to high temperatures. 311
The bioaccessible percentage after in vitro simulated gastrointestinal digestion of 312
ascorbic acid of the cashew apple juice was 26.2% (Table 4). There are no reports in the 313
literature about the bioaccessibility of ascorbic acid in cashew apple juice. Perez-Vicente, Gil-314
Izquierdo, and Garcia-Viguera (2002) in a study about the bioaccessibility of ascorbic acid in 315
pomegranate juice, reported a significant reduction in the concentration of this compound of 316
14
about 95% after in vitro intestinal digestion. The reduction of the bioaccessibility of ascorbic 317
acid is due to the low stability of this compound (Perez-Vicente et al., 2002), to the change in 318
pH and to the presence of oxygen during the process of gastrointestinal digestion (Cilla, 319
Perales, Lagarda, Barberá, Clemente, & Farré, 2011). 320
3.3. Total extractable polyphenols 321
Phenolic compounds are metabolites that have the ability to neutralize reactive species, 322
helping to protect the body against oxidative stress and have antioxidant activity (Wojdylo, 323
Oszmiansk, & Laskowski, 2009). 324
The average content of total extractable polyphenols observed in the present study was 325
338.60 and 566.10 mg GAE 100 g -1 for cashew apple juice and cashew apple fiber and 326
130.60 and 105.03 mg GAE 100 g -1 for cashew apple juice and cashew apple fiber after 327
digestion, respectively (Table 4). Lopes, Miranda, Moura, and Filho (2012) studied the 328
content of total extractable polyphenols in different clones of cashew apple and observed 329
levels of 375.79 mg GAE 100 g-1 for CCP 09 clone and 124.2 mg GAE 100 g-1 to CCP 76 330
clone. The difference in the levels of phenolic compounds observed may be due to the clones 331
and the extraction method used, since, according to Goli, Barzegar, and Sahari (2005), the 332
concentration of phenolic compounds in fruit extracts is dependent on the solvent and on the 333
extraction method employed. 334
Bioaccessible levels of total extractable polyphenols were 39.0 and 18.6% for cashew 335
apple juice and cashew apple fiber, respectively (Table 4). There are reports in the literature 336
demonstrating the potential of the phenolic compounds and their biological effects (Othman, 337
Roblain, Chammen, Thonart, & Hamdi, 2009, He et al., 2011), however, there are no studies 338
on the in vitro bioaccessibility of phenolic compounds of cashew apple and its byproducts. 339
Bouayed, Hoffmann and Bohn (2011) in their study evaluating the bioaccessibility of 340
phenolic compounds in apple found a bioaccessible percentage of about 55%. These authors 341
15
suggested that this bioaccessibility is due to the fact that some phenolic compounds are linked 342
to macromolecular compounds which are not dialyzable, or which can form complex 343
minerals, further decreasing their solubility. This affirmation can also be suggested in this 344
study, especially in relation to the bioaccessible percentage of phenolic compounds found in 345
cashew apple byproducts, which found that the bioaccessible percentages for the minerals 346
iron, copper and zinc were also low. 347
3.4. Antioxidant activity 348
The antioxidant compounds work by blocking the action of free radicals and preventing 349
the development of diseases (Ferreira, Farias, Oliveira, & Carvalho, 2008). The results 350
obtained in the present study for cashew apple juice and cashew apple fiber were 18.10 and 351
51.10 µM Trolox g-1, respectively (Table 4). The higher antioxidant activity observed in 352
cashew apple fiber cashews may have occurred due to the higher content of phenolic 353
compounds present in the fiber. There was a reduction in the antioxidant activity of cashew 354
apple juice and of cashew apple fiber after digestion, and levels of 4.80 and 5.20 µM Trolox 355
g-1 were observed for cashew apple juice and cashew apple fiber, respectively (Table 4). 356
The bioaccessible percentage of antioxidant activity after in vitro simulated 357
gastrointestinal digestion for cashew apple juice and cashew apple fiber were 27.0% and 358
10.2%, respectively. It has been suggested that the better bioaccessibility of the juice in 359
comparison to the fiber is due to the contribution of ascorbic acid, present in higher contents 360
in cashew apple juice, exerting greater antioxidant function than in the fiber. Different studies 361
involving in vitro simulated digestion in plants have pointed to the presence of bioaccessible 362
antioxidant compounds (Bouayed, Hoffmann, & Bohn, 2011; Gawlik-Dziki, Jeżyna, Świeca, 363
Dziki, Baraniak, & Czyż, 2012). However, the data on the antioxidant activity of the 364
bioaccessible fraction of cashew apple, obtained by in vitro digestion, has not been 365
investigated. 366
16
3.5. Correlation analysis 367
The Pearson correlation analysis between bioactive compounds and antioxidant activity 368
showed a high correlation for both ascorbic acid (r = 0.9849, p <0.0001) and total extractable 369
polyphenol (r = 0.9941, p <0.0001). Due to these results, it is possible to infer that these 370
compounds have high importance in the antioxidant activity of cashew apple juice and they 371
contribute significantly to the antioxidant activity of this product. 372
373
4. Conclusion 374
The application of in vitro simulated gastrointestinal digestion has demonstrated that in 375
some cases only a minor fraction of the total quantity of nutrients in foods is potentially 376
bioaccessible. The results obtained with regard to the minerals, copper, iron and zinc, ascorbic 377
acid, total extractable polyphenols and antioxidant activity, show that the percentage of 378
absorption of these compounds varies widely depending on the components of the food 379
matrix elements. 380
The bioacessible percentage of zinc, ascorbic acid and total extractable polyphenols 381
were higher in cashew apple juice than cashew apple fiber, which possible occurred due to the 382
low level of tannins and phytates found in fruit juices, being the consumption of cashew apple 383
juice highly recommended. 384
The results of the present study indicate that there are components of bioaccessibility 385
facilitators, for example, ascorbic acid is easily found in fruits, and it increases the absorption 386
of non-heme iron absorption as depressants components such as tannins, phytates and calcium 387
oxalate. Moreover, phenolic compounds and ascorbic acid contribute in a very positive way 388
for the bioaccessible percentage of the total antioxidant activity, which in turn, may 389
conceivably contribute to protection against several diseases in relation to its antioxidant 390
power. 391
17
392
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509
22
Table 1 - Heating program used during microwave digestion of the cashew apple juice and 510
cashew apple fiber. 511
Steps Cashew apple juice Cashew apple fiber
Potency (W) Time (min.) Potency (W) Time (min.)
1 100 5 100 5
2 800 15 600 5
3 0 15 1000 10
4 - - 0 15
512
513
23
Table 2 - Parameters used in the analysis by ICP OES. 514
Instrumental parameters ICP OES (radial view)
Power radio frequency (kW) 1.3
Flow nebulizer (L min–1) 0.6
Plasma gas flow rate (L min–1) 15
Auxiliary gas flow (L min–1) 1.50
Nebulizer V-Groove
Nebulization chamber Sturman Master
Point of observation (mm) 15
Wavelength (nm) Cu (I) (λ = 324.752)
Fe (II) (λ =259.939)
Zn (II) (λ = 206.200)
Detection limit (mg L-1) Cu: 0.05
Fe: 0.03
Zn: 0.02
515
24
Table 3 - Mean values for the copper, iron and zinc in cashew apple juice and cashew apple 516
fiber before and after in vitro simulated gastrointestinal digestion. 517
Sample Mineral Native (mg L-1) Bioaccessible
(mg L-1)
Bioaccessibility after
digestion (%)
Cashew apple
juice
Cu 2.10 ± 0.14a 0.25 ± 0.00b 15.0
Fe 1.82 ± 0.12a 0.17 ± 0.00b 11.5
Zn 4.70 ± 0.31a 0.14 ± 0.00b 3.7
Cashew apple
fiber
Cu 12.20 ± 0.31a 0.40 ± 0.01b 4.0
Fe 21.60 ± 0.67a 0.20 ± 0.00b 1.2
Zn 7.14 ± 0.40a 0.12 ± 0.00b 2.2
* Mean ± standard deviation (n = 9 for cashew apple juice and n = 3 for cashew apple fiber). 518
** Means with the same letter in the same row are not statistically different by the Tukey test 519
(p≤0.05). 520
521
25
Table 4 - Mean values of ascorbic acid, total extractable polyphenols and antioxidant activity 522
of cashew apple juice before and after in vitro simulated gastrointestinal digestion. 523
Sample Mineral Native (mg L-1) Bioaccessible
(mg L-1)
Bioaccessibility
after digestion
(%)
Cashew
apple
juice
Ascobic acid 49.30 ± 2.05a 12.90 ± 1.84b 26.2
Total extractable
polyphenols
338.60 ± 10.68a 130.60 ± 3.02b 39.0
Antioxidant activity 18.10 ± 1.92a 4.80 ± 0.09b 27.0
Cashew
apple
fiber
Ascobic acid N.D. N.D. -
Total extractable
polyphenols
566.10 ± 11.37a 105.03 ± 2.23b 18.6
Antioxidant activity 51.10 ± 1.49a 5.20 ± 0.10b 10.2
* Mean ± standard deviation (n = 9 for cashew apple juice and n = 3 for cashew apple fiber). 524
** Means with the same letter in the same row are not statistically different by the Tukey test 525
(p≤0.05). 526
*** N.D.: Not Detected. 527
**** Ascorbic acid: mg 100 g-1; Total extractable polyphenols: mg GAE 100 g-1; Antioxidant 528
activity: µM Trolox g-1; Bioaccessibility after digestion: %. 529
530
531
532
533
534
535