Accepted Manuscript

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

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting proof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

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

11

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

anacristinalima.hp@gmail.com; denisejosino@hotmail.com; larissamrs@yahoo.com.br; 14

figueira@ufc.br 15

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

phenriquemachado@gmail.com 18

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

evelineabreu@yahoo.com.br 21

22

* Corresponding author: E-mail: anacristinalima.hp@gmail.com; Fax: +55 85 33669752. 23

24

2

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

37

38

39

3

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

45

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

59

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

7

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

8

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

9

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

11

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

13

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

5. References 393

Aberoumand, A., & Deokule, S. S. (2009). Determination of elements profile of some wild 394

edible plants. Food Analytical Methods, 2, 116–119. 395

Altundag, H., & Tuzen, M. (2011). Comparison of dry, wet and microwave digestion methods 396

for the multi element determination in some dried fruit samples by ICP-OES. Food and 397

Chemical Toxicology, 49, 2800-2807. 398

Andrade, E. C. B., Alves, S. P., & Takase, I. (2005). Avaliação do uso de ervas medicinais 399

como suplemento nutricional de ferro, cobre e zinco. Ciência e Tecnologia de Alimentos, 25, 400

p. 591-596. 401

Bouayed, J., Hoffmann, L., & Bohn, T. (2011). Total phenolics, flavonoids, anthocyanins and 402

antioxidant activity following simulated gastrointestinal digestion and dialysis of apple 403

varieties: Bioaccessibility and potential uptake. Food Chemistry, 128, 14-21. 404

Briones-Labarca, V., Venegas-Cubillos, G., Ortiz-Portilla, S., Chacana-Ojeda, M., & 405

Maureira, H. (2011). Effects of high hydrostatic pressure (HHP) on bioaccessibility, as well 406

as antioxidant activity, mineral and starch contents in Granny Smith apple. Food Chemistry, 407

128, 520-529. 408

Brito, E. S. de, Araújo, M. C. P., Lin, L. Z., & Harnly, J. (2007). Determination of the 409

flavonoid components of cashew apple (Anacardium occidentale) by LC-DAD-ESI/MS. Food 410

Chemistry, 105, 1112-1118. 411

Bronizi, P. R. B., Andrade-Wartha, E. R. S. de, Silva, A. M. de O., Novoa, A. J. V., Torres, R. 412

P., Azeredo, H. M. C., Alves, R. E., & Mancini-Filho, J. (2007). Avaliação da atividade 413

antioxidante dos compostos fenólicos naturalmente presentes em subprodutos do pseudofruto 414

de caju (Anacardium occidentale L.). Ciência e Tecnologia de Alimentos, 27, 902-908. 415

18

Cámara, F., Amaro, M. A., Barbera, R., & Clemente, G. (2005). Bioaccessibility of minerals 416

in school meals: Comparision between dialysis and solubility methods. Food Chemistry, 92, 417

481-489. 418

Cilla, A., Perales, S., Lagarda, M. J., Barberá, R., Clemente, G., & Farré, R. (2011). Influence 419

of storage and in vitro gastrointestinal digestion on total antioxidant capacity of fruit 420

beverages. Journal of Food Composition and Analysis, 24, 87-94. 421

Ferreira, P. M. P., Farias, D. F., Oliveira, J. T. de A., & Carvalho, A. de F. U. (2008). 422

Moringa oleifera: bioactive compounds and nutritional potential. Revista de Nutrição, 21, 423

431-437. 424

Food and Drug Administration (FDA). (2013). Appendix F: calculate the percent daily value 425

for the appropriate nutrients. Available at: < 426

http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/L427

abelingNutrition/ucm064928.htm>. Accessed 09.07.2013. 428

Gawlik-Dziki, U., Jeżyna, M., Świeca, M., Dziki, D., Baraniak, B., & Czyż, J. (2012). Effect 429

of bioaccessibility of phenolic compounds on in vitro anticancer activity of broccoli sprouts. 430

Food Research International, 49, 469-476. 431

Goli, A. H., Barzegar, M., & Sahari, M. A. (2005). Antioxidant activity and total phenolic 432

compounds of pistachio (Pistachia vera) hull extracts. Food Chemistry, 92, 521-525. 433

Helbig, E., & Gigante, D. P. (2008). Análise dos teores de ácidos cianídrico e fítico em 434

suplemento alimentar: multimistura. Revista de Nutrição, 21, 323-328. 435

He, L., Xu, H., Liu, X., He, W., Yuan, F., Hou, Z., & Gao, Y. (2011). Identification of 436

phenolic compounds from pomegranate (Punica granatum L.) seed residues and investigation 437

into their antioxidant capacities by HPLC–ABTS+ assay. Food Research International, 44, 438

1161-1167. 439

19

Khouzam, R. B., Pohl, P., & Lobinskib, R. (2011). Bioaccessibility of essential elements from 440

white cheese, bread, fruit and vegetables. Talanta, 86, 425-428. 441

Konoha, K., Sadakane, Y., & Kawahara, M. (2006). Zinc neurotoxicity and its role in 442

neurodegenerative diseases. Journal of Health Science, 52, 1-8. 443

Kulkarni, S. D., Acharya, R., Rajurkar, N. S., & Reddy, A. V. R. (2007). Evaluation of 444

bioaccessibility of some essential elements from wheatgrass (Triticum aestivum L.) by in vitro 445

digestion method. Food Chemistry, 103, 681-688. 446

Larrauri, J. A., Rupérez, P., & Saura-Calixto, F. (1997). Effect of drying temperature on the 447

stabilitity of polyphenols and antioxidant activity of red grape pomace peels. Journal of 448

Agricultural and Food Chemistry, 45, 1390-1393. 449

Lehninger, A. L., Nelson, D. L., & Cox, M. M. (2011). Princípios de bioquímica de 450

Lehninger. (3rd ed.). São Paulo: Artmed, 1304p. 451

Lopes, M. M. A., Miranda, M. R. A., Moura, C. F. H., & Filho, J. E. (2012). Bioactive 452

compounds and total antioxidant capacity of cashew apples (Anacardium occidentale L.) 453

during the ripening of early dwarf cashew clones. Ciência e Agrotecnologia, 36, 325-332. 454

Maia, G. A., Sousa, P. H. M., Santos, G. M., Silva, D. S., Fernandes, A. G., & Prado, G. M. 455

(2007). Efeito do processamento sobre componentes do suco de acerola. Ciência e Tecnologia 456

de Alimentos, 27, 130-134. 457

Mazetto, S. E., Lomonaco, D., & Mele, G. (2009). Óleo da castanha de caju: oportunidades e 458

desafios no contexto do desenvolvimento e sustentabilidade industrial. Química Nova, 32, 3, 459

732-741. 460

Menezes, E. A. (2010). Determinação da disponibilidade de Ca, Cu, Fe, Mg e Zn em 461

amostras de carnes bovinas, suínas e de frango in natura e processadas termicamente. Tese 462

(Doutorado) - Universidade Federal de São Carlos. 463

20

Moura, N. C., & Canniatti-Brazaca, S. G. (2006). Avaliação da disponibilidade de ferro de 464

feijão comum (Phaseolus vulgaris L.) em comparação com carne bovina. Ciência e 465

Tecnologia de Alimentos, 26, 270-276. 466

Othman, N. B., Roblain, D., Chammen, N., Thonart, P., & Hamdi, M. (2009). Antioxidant 467

phenolic compounds loss during the fermentation of Chétoui olives. Food Chemistry, 116, 468

662-669. 469

Perez-Vicente, A., Gil-Izquierdo, A., & Garcia-Viguera, C. (2002). In vitro gastrointestinal 470

digestion study of pomegranate juice phenolic compounds, anthocyanins, and vitamin C. 471

Journal Agricultural of Food Chemistry, 50, 2308-2312. 472

Pinheiro, A., Fernandes, A. G., Fai, A. E. C., Prado, G. M., Sousa, P. H. M., & Maia, G. A. 473

(2006). Avaliação química, físico-química e microbiológica de sucos de frutas integrais: 474

abacaxi, caju e maracujá. Ciência e Tecnologia de Alimentos, 26, 98-103. 475

Pinho, L. X., Afonso, M. R. A., Carioca, J. O. B., Costa, J. M. C., & Rybka, A. C. P. (2011). 476

Desidratação e aproveitamento de resíduo de pedúnculo de caju como adição de fibra na 477

elaboração de hambúrguer. Alimentos e Nutrição, 22, 571-576. 478

Queiroz, C., Lopes, M. L. M., Fialho, E., & Valente-Mesquita, V. L. (2011). Changes in 479

bioactive compounds and antioxidant capacity of fresh-cut cashew apple. Food Research 480

International, 44, 1459-1462. 481

Rufino, M. S. M., Alves, R. E., Brito, E. S., Jiménez, J. P., Calixto, F. S., & Mancini-Filho, J. 482

(2010). Bioactive compounds and antioxidant capacities of 18 non-traditional tropical fruits 483

from Brazil. Food Chemistry, 121, 996-1002. 484

Santos, G. M., Maia, G. A., Sousa, P. H. M., Figueiredo, R. W., Costa, J. M. C., & Fonseca, 485

A. V. V. F. (2010). Atividade antioxidante e correlações com componentes bioativos de 486

produtos comerciais de cupuaçu. Ciência Rural, 40, 1636-1642. 487

21

Scherer, R., Rybka, A. C. P., & Godoy, H. T. (2008). Determinação simultânea dos ácidos 488

orgânicos tartárico, málico, ascórbico e cítrico em polpas de acerola, açaí e caju e avaliação 489

da estabilidade em sucos de caju. Química Nova, 31, 1137-1140. 490

Sivagurunathan, P., Sivasankari, S., & Muthukkaruppan, S. M. (2010). Characterisation of 491

cashew apple (Anacardium occidentale L.) fruits collected from Ariyalur District. Journal of 492

Biosciences Research, 1, 101-107. 493

Soares, L. M. V., Shishido, K., Moraes, A. M. M., & Moreira, V. A. (2004). Mineral 494

composition of Brazilian concentrate fruit juices. Ciência e Tecnologia de Alimentos, 24, 202-495

206. 496

Sousa, M. S. B., Vieira, L. M., Silva, M. J. M., & Lima, A. (2011). Caracterização nutricional 497

e compostos antioxidantes em sub-produtos de polpas de frutas tropicais. Ciência e 498

Agrotecnologia, 35, 554-559. 499

Uchoa, A. M. A., Costa, J. M. C. da, Maia, G. A., Silva, E. M. C., Carvalho, A. de F. F. U., & 500

Meira, T. R. (2008). Parâmetros físico-químicos, teor de fibra bruta e alimentar de pós 501

alimentícios obtidos de resíduos de frutas tropicais. Segurança Alimentar e Nutricional, 15, 502

58-65. 503

Walter, M., Marchezan, E., & Avila, L. A. (2008). Arroz: composição e características 504

nutricionais. Ciência Rural, 38, 1184-1192. 505

Wojdylo, A., Oszmianski, J., & Laskowski, P. (2009). Polyphenolic compounds and 506

antioxidant activity of new and old apple varieties. Journal Agricultural of Food Chemistry, 507

56, 6520-6530. 508

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