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Bioactive Constituents, Metabolites, and Functions

Bioaccessibility during in vitro digestion and antiproliferative effect of bioactive compounds from Andean berry (Vaccinium meridionale Swartz) juice Carlos Daniel Agudelo, Ivan Luzardo-Ocampo, Rocio CamposVega, Guadalupe Loarca-Piña, and Maria Maldonado J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01604 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 18, 2018

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Journal of Agricultural and Food Chemistry

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Bioaccessibility during in vitro digestion and antiproliferative effect of bioactive compounds from Andean berry (Vaccinium meridionale Swartz) juice

Carlos D. Agudeloa, Ivan Luzardo-Ocampob, Rocio Campos-Vegab, Guadalupe LoarcaPiñab, María E. Maldonado-Celisc*.

a

Instituto de Biología, Facultad de Ciencias Exactas y Naturales, Calle 67 #53-108,

Universidad de Antioquia, Medellín, Colombia. b

Programa de Posgrado del Centro de la República (PROPAC), Research and

Graduate Studies in Food Science, School of Chemistry, Universidad Autónoma de Querétaro, 76010, Santiago de Querétaro, México. c

Escuela de Nutrición y Dietética, Ciudadela de Robledo Cra. 75 # 65-87, Universidad

de Antioquia, Medellín AA 1226, Colombia.

* Corresponding author: Tel: + 57 4 219 9223. Fax: + 57 4 2196400, Ext 6669; E-mail address: [email protected].

1

ACS Paragon Plus Environment


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Abstract

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Berries consumption is associated with colorectal cancer chemoprevention, but

4

digestive conditions can affect this property. The bioaccessibility and apparent

5

permeability coefficients of bioactive compounds from Andean berry juice (ABJ) after

6

in vitro gastrointestinal digestion and colonic fermentation were analyzed. The

7

antiproliferative effect of the fermented non-digestible fraction was evaluated against

8

SW480 colon adenocarcinoma cells. Gallic acid displayed the highest bioaccessibility

9

in the mouth, stomach, small intestine and colon. However, chlorogenic acid exhibited

10

the highest apparent permeability coefficients (up to 1.98 x 10-4 cm/s). The colonic

11

fermented fraction showed an increase of ≥ 50% antiproliferative activity against

12

SW480 cells (19.32% v/v), equivalent to gallic acid: 13.04 µg/g; chlorogenic acid: 7.07

13

µg/g; caffeic acid: 0.40 µg/g; ellagic acid: 7.32 µg/g; rutin: 6.50 µg/g; raffinose: 0.14

14

mg/g; stachyose: 0.70 mg/g and xylose: 9.41 mg/g. Bioactive compounds from ABJ are

15

bioaccessible through the gastrointestinal tract and colon fermentation, resulting in

16

antiproliferative activity.

17 18

Keywords: Vaccinium meridionale Swartz, in vitro gastrointestinal digestion, phenolic

19

compounds, oligosaccharides, bioaccessibility, non-digestible fraction, SW480 human

20

colon cancer cell line.

21 22


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Introduction

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Colorectal cancer is one of the main and most common types of cancer worldwide. Yet,

25

it can be prevented by increasing the consumption of fruits and vegetables, in

26

conjunction with other strategies. This prevention is attributed to their content of

27

phytochemicals such as dietary fiber, including oligosaccharides, phenolic compounds

28

and flavonoids1 substrates which can be fermented by gut microbiota to produce short-

29

chain fatty acids (SCFAs), providing an increased mucosal permeability and protection

30

to the colonic epithelium2.

31

The genus Vaccinium, including blue mulberry, black mulberry, blueberry and other

32

berries, are sources of these compounds identified as chemopreventive agents with

33

antioxidant, anti-inflammatory, and antiproliferative properties involved in the colon

34

carcinogenesis3. Amongst these berries, Vaccinium meridionale Swartz is a native

35

Colombian plant that belongs to the Ericaceae family. When ripe, this fruit is a dark

36

purple globe berry, with two harvest seasons per year (April-May and September-

37

December). The fruit has a sour taste and is frequently processed by hand for wine, jams

38

and desserts4. As a functional food, with an important content of phenolic acids and

39

flavonoids, as well their antioxidant capacity, it is called a “potential new berry” and has

40

been included in the list of species for marketing in the USA since 2006. In Colombia,

41

the domestic consumption reaches up to 20 Ton per year with a value reaching up to

42

US$135.0004.

43

Recently, our research group reported that the non-digested ABJ inhibited the growth of

44

SW480 human colon adenocarcinoma cells5. In fact, similar results have been observed

45

in numerous in vitro and in vivo studies where berry extracts were evaluated, including

46

Vaccinium meridionale Swartz6. Furthermore, the compounds contained in the fruits

47

modulated biomarkers associated with chemopreventive effects. Nevertheless, the direct



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use of extracts to determine the chemopreventive capacity against in vitro colon cancer

49

does not represent the physiological effects after consumption of the berry, in which the

50

fruit is subjected to gastrointestinal digestion before reaching the colon. Also, the

51

phytochemicals present in the fruit that possess antioxidant, cytotoxic and

52

antiproliferative activities might be transformed into other compounds with different

53

biological activity, affecting the potential anticancer properties of these components.

54

In this manner, the in vitro gastrointestinal model mimics the conditions of the

55

gastrointestinal tract, allowing to monitor the bioactive compounds bioaccessibility.

56

This method has been used to understand the stability of anthocyanins from berries and

57

the effect of the digestion on the antiproliferative capacity against different colon

58

adenocarcinoma cells3. Additionally, the authors that conducted this research found that

59

both phenolic acids and anthocyanins are sensitive to the alkaline conditions of the

60

digestion process. Subsequently, these molecules can be transformed into unknown or

61

undetectable metabolites by the usual identification and quantification methods.

62

For this reason, in order to evaluate the physiological conditions which could affect the

63

ABJ functionality, the bioaccessibility, and the apparent intestinal permeability of

64

vitamin C, phenolic compounds, and oligosaccharides were evaluated, as well its

65

antiproliferative activity against SW480 human colon adenocarcinoma cells.

66 67

Materials and Methods

68

Chemicals and reagents

69

All the reagents used were HPLC or analytical grade. The acetic acid, acetonitrile,

70

pentobarbital sodium salt, HCl, NaCl, KCl, MgSO4, KH2PO4, NaHCO3, glucose, CaCl2,

71

pepsin, pancreatin, gall ox, raffinose and HPLC-grade standards were purchased from

72

Sigma-Aldrich Chemical Company (St. Louis, MO, USA) and J. T. Baker (Mexico


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City, Mexico). The animals (14 Wistar male rats, 6-8 weeks’ age) were provided by

74

“Instituto Nacional de Neurobiología” (UNAM, Campus Juriquilla, Mexico). All of the

75

procedures that involved human/animals were previously approved by the Universidad

76

Autónoma de Querétaro Human and Animal Internal Committee and complied with the

77

National Institute of Health Guide for Care and Use of Laboratory Animals.

78 79

Plant material

80

Fresh ripe berries of Andean berry (Vaccinium meridionale) were harvested at the

81

vicinity of El Retiro (Antioquia, Colombia) (16 ºC, 2175 meters above the sea level and

82

16 ºC) on May 2015. The berries were selected, washed disinfected (sodium

83

hypochlorite, 100 ppm) and dried. Afterwards, the berries were blended (2 mi, 2500

84

rpm) and frozen-dried in a vacuum chamber under pressure and low temperature (4.27 ±

85

0.5 mm Hg, -50 ºC).

86

protected from light (PET aluminum package) and stored at room temperature.

After this procedure, the resulting lyophilized powder was

87



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Preparation of Andean Berry Juice

89

The Andean berry juice (ABJ) was prepared as previously described7. Briefly, the

90

lyophilized Andean berry powder was dissolved in a water/sucrose solution (9% w/v) to

91

obtain a juice of 11.1 ºBrix, acidity of 4.33 mg citric acid/mL and pH 3.06. On the

92

procedure that followed, ABJ was sonicated (42 kHz, 135 W; Branson B3510,

93

Ultrasonic Corporation, USA) for 15 min at room temperature (25 ± 1 ºC). Prior to their

94

use, the sonicated solution was protected from light and stored at -70 ºC.

95 96

Free phenolic compounds extraction and quantification

97

For the ABJ, the free phenolic compounds were extracted as described by Cardador-

98

Martínez et al. 8. Briefly, the lyophilized ABJ powder was ground using a coffee grinder

99

(KRUPS GX4100) and through size 40 mesh. One gram of the resulting ABJ powder

100

was placed in a 50 mL flask and mixed with methanol (10 mL). The flask was protected

101

from light and stirred (450 rpm, IKA WERKER 015 magnetic stirrer, IKA Works,

102

USA) for 24 h at room temperature (25 ± 1 ºC). The mixture was then centrifuged (2166

103

g, 10 min; Hermle Z323K, Wehingen, Germany). The supernatant was collected and

104

filtered through a 0.45 µm filter (Econofilter PTFE, Agilent Technologies, Santa Clara,

105

CA, USA).

106 107

A high-performance liquid chromatography-diode array detection (HPLC-DAD)

108

analysis was conducted on an Agilent 1100 Series HPLC System (Agilent

109

Technologies, Palo Alto, CA, USA) using a Zorbax Eclipse XDB-C18 column (Agilent

110

Technologies, 4.6 x 250 mm, 5µm) as reported9. The quantification was carried out

111

using the external standard method with the commercial standards of (+)-catechin, rutin,

112

kaempferol, and ascorbic, gallic, chlorogenic, caffeic, p-Coumaric, sinapic, ferulic and


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ellagic acid. Additional information about the analytical method used is provided in the

114

supplementary Table S1.

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Oligosaccharides extraction and quantification

117

Oligosaccharides were extracted from ABJ

118

procedure10. Briefly, 1.0 g of ABJ lyophilized powder was homogenized with 100 mL

119

of distilled water and incubating at 80 ºC for 60 min using an oscillating water bath. The

120

sample was then centrifuged (Hermle Z326K, Wehingen, Germany) for 10 min at 984 x

121

g. The supernatant was collected, filtered through a 0.45 µm filter (Econofilter PTFE,

122

Agilent Technologies, Santa Clara, CA, USA).

123

A high-performance liquid chromatography-refractive index detector (HPLC-RID)

124

analysis was done with an Agilent 1100 Series HPLC system (Agilent Technologies,

125

Palo Alto, CA, USA) using a Zorbax Eclipse XDB-C18 column (Agilent Technologies,

126

4.6 250 mm) as previously described11. Complementary information about the

127

analytical method used is provided in the supplementary Table S1.

accordingly to a previously reported

128 129

In vitro gastrointestinal digestion

130

For the simulation of the physiological conditions, the procedure described by Campos-

131

Vega et al.12 was followed. All the human subjects (four) provided a written consent

132

before participating in the study. The participants washed their mouths with ABJ (25

133

mL) 15 times for 15 s. For the stomach stage, the product was then expectorated into a

134

beaker containing 5 mL of distilled water, followed by another rinsing with 5 mL of

135

distilled water for 60 s. This procedure was repeated four times for the ABJ and salivary

136

samples used as blanks.



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Afterwards, the suspension of each sample was mixed in a single beaker and the pH was

138

adjusted to 2.0 using an HCl solution (150 mM). Pepsin (0.055 g, Sigma-Aldrich) was

139

dissolved in 0.94 mL of 20 mM HCl solution and added to each aliquot (∼10 mL),

140

following an incubation period of 2 h at 37 °C. The intestinal extract was prepared 30

141

min before use, mixing 3 mg of Gall Ox and 2.6 mg of pancreatin (Sigma) in a 5 mL

142

Krebs-Ringer buffer [118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4,

143

25 mM NaHCO3, 11 mM glucose and 2.5 mM CaCl2, pH 6.8). This solution (5 mL) was

144

added to each sample and gasified for 30 min before using a gas mixture (10:10:80

145

H2:CO2:N2) in order to guarantee the anaerobic conditions. The suspension (15 mL) was

146

transferred to a conical tube containing an everted gut sac, prepared from male Wistar

147

rats (body wt. 250–300 g, n=14). Before the surgical procedure, the rats were fasted

148

overnight (16–20 h) with water ad libitum. The rats were anesthetized with

149

pentobarbital sodium (60 mg/kg, i.p.). The intestine of the rats was exposed by a

150

midline abdominal incision and a 20–25 cm segment of the proximal rat jejunum was

151

excised and placed in the gasified buffer solution of Krebs–Ringer at 37°C. The

152

intestine was previously washed with the same buffer and everted over a glass rod,

153

divided into segments (6 cm) and filled on the serosal side with ~1 mL of Krebs-Ringer

154

buffer to prevent tissue viability loss. This everted gut sac was added to a 15 mL

155

suspension of the gastric phase and incubated in an oscillating water bath (37 ºC, 80

156

cycles/min, for 15, 30, 60 and 120 min). After the incubation period, the sacs were

157

removed; the sample from the mucosal side (outside of the everted gut sac) was

158

considered as the non-digestible fraction of the Andean berry juice (NDFAB). The 120-

159

min NDFAB solution was subsequently subjected to in vitro colonic fermentation to

160

obtain the fermented non-digestible fraction of ABJ (FNDFABJ). The experiments were

161

performed in triplicate and the complete digestion procedure was carried out twice. The


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samples were protected from light and stored in 15 mL conical tubes at -72 ºC. Prior to

163

the HPLC analysis, the samples were defrosted and centrifuged at 5400 rpm for 10 min

164

(HERMLE Z 323 K centrifuge, Wehingen, Germany) at 4 ºC. The obtained supernatant

165

was collected and filtered (0.45 µm). Figure 1 illustrates a scheme for this procedure,

166

including the analyzed bioactive compounds.

167 168

In vitro colonic fermentation

169

For the in vitro colonic fermentation to obtain the fermented non-digestible fraction of

170

Andean berry (Vaccinium meridionale Swartz) juice (FNDFABJ), the procedure

171

reported in a previous study by Campos-Vega et al.13was followed. The fresh fecal

172

samples were collected from one selected healthy donor who was instructed to follow a

173

regular, strict and monitored diet and manifested not having a previous intestinal disease

174

nor being under antibiotic treatment within the last three months.. The pH was measured

175

at 0, 6, 12 and 24 h-fermentation times, vortexing for at least 1 min before the

176

measurement. At the end of each fermentation time, the fermented samples (FNDFABJ)

177

were protected from light and stored at -72 ºC before their centrifuging and filtering for

178

the HPLC quantification. Raffinose (100 mg, R0514, Sigma Aldrich, St. Louis, MO,

179

USA) was used as a fermentable sugar reference. Three independent assays were done

180

at 37°C simulating human conditions. A blank was also prepared using the 120 min-

181

digested salivary solution instead NDFABJ. This procedure was conducted three times

182 183

Calculations of the apparent permeability coefficients (Papp)

184

The apparent permeability coefficients (Papp, x 10-4 cm/s) were estimated as follows14:

185

Papp= (dQ/dt)(1/AC0) where Papp (cm/s) is the apparent permeability coefficient, dQ/dt

186

(mg/s) is the amount of compound (phenolic acids or flavonoids) transported across the

187

membrane per time (15-120 min), A (cm2) is the total intestinal surface area for the



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permeation of compounds and C0 (mg/mL) is the initial concentration of the phenolic

189

compound outside the everted gut sacs.

190 191

Water flux measurement.

192

The water flux, accounting for both water absorption and efflux in

193

segment was determined by applying the following equation15: WF = (W3-W2)/W1,

194

where WF (g water/g fresh intestine) is the water flux and, for the small intestine, W1

195

equals to the initial weight, W2 is equivalent to the weight before the incubation period

196

and W3 is the weight after incubation at the same time periods.

the intestinal

197 198

Bioaccessibility and absorption measurement

199

The bioaccessibility of free phenolic compounds and oligosaccharides along the

200

gastrointestinal digestion and colonic fermentation was computed the following

201

equation: B=[(C0-Cf)/Cf]*100 where B represents the bioaccessibility percentage (%) of

202

both free phenolic compounds and oligosaccharides, C0 is the initial concentration of

203

the compound at a specified incubation time (15-120 min) and Cf the final concentration

204

of the compound at the same time of incubation.

205

The absorption percentages were calculated as the percentage relationship between the

206

amount of free phenolic compound in the digestible fraction and the total amount of

207

compound on both digestible and non-digestible fraction.

208 209

Cell viability assay and determination of IC50

210

The SW480 human colon adenocarcinoma cell line was used. The cells were cultured as

211

previously described16. The crystal violet staining was used to quantify the amount of

212

viable SW480 cells exposed to the FNDFABJ. Briefly, the SW480 cells were seeded in


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96-well culture plates at a concentration of 2 x 103 cells per well and cultured at 37°C in

214

5% CO2. After 24 h, the different concentrations of the FNDFABJ (3, 6, 9, 12 and 18%

215

v/v) were evaluated in the cells for 24 h. After the incubation, the adherent cells were

216

washed with PBS twice and the viable cells were determined at 570 nm in a microplate

217

reader (Thermoscientific Varioskan 51119300) after staining for 10 min at 37°C with

218

50 µL of crystal violet solution per well (0.5% w/v crystal violet, 4% w/v formaldehyde,

219

30% v/v ethanol, and 0.17% w/v NaCl). All the experiments were performed in

220

triplicate. The half maximal lethal concentration 50% of cells (IC50) was calculated

221

using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA). The effect of

222

FNDFABJ was normalized to the blank control (FNDFABJ without cells) and to the

223

non-treated control (cells without treatment). The inhibition (%) was calculated by

224

using the following equation: Inhibition (%) = [1 - (ODt / ODc)] × 100, where ODt is

225

the optical density of the treated cells and ODc from the control cells (without

226

treatment).

227 228

Statistical analysis

229

The results were expressed as means ± standard deviation. The data were analyzed with

230

JMP v. 8.0 software and a statistical analysis was done following one-way ANOVA and

231

Tukey-Kramer’s Test to identify significant differences amongst the groups. Significant

232

differences (p < 0.05) with the control at the cell viability test was done using the

233

Dunnett’s Test. The level of significance was established at p = 0.05.

234 235

Results

236

Bioaccessibility of free phenolic compounds during the in vitro gastrointestinal

237

digestion and simulated colonic fermentation



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As shown in Table 1, free phenolic compounds exhibited bioaccessibility ranging from

239

2.06 to 2813.33 µg/g FW, with gallic acid displaying the highest bioaccessibility at

240

mouth and stomach (over 84-fold) followed by chlorogenic acid that exhibited the

241

highest bioaccessibility values at 15 and 60 minutes in the small intestine (13.65-16.10

242

fold). When compared against the mouth stage, both caffeic and ellagic acid increased

243

their bioaccessibility at the stomach step (up to 2.81-fold), whereas the gallic and

244

chlorogenic acid decreased (-0.73 and -0.75, respectively).

245

Kaempferol, a flavonoid which was undetected in the undigested samples, was

246

bioaccessible at the mouth stage, but was not detected at the following periods (Table

247

1); while the ascorbic acid followed a similar trend during gastrointestinal digestion.

248

Quercetin reached the lowest bioaccessibility values at the stomach step (∼ 4 µg/g), and

249

rutin remained with no significant differences (p > 0.05) at the last stages of the small

250

intestine incubation (NDFABJ). Regarding the absorption of some free phenolic

251

compounds, gallic, chlorogenic and ellagic acid presented the highest overall values at

252

the end of the evaluation (120 min) (Fig. 1). These results are reflected in the

253

representative chromatograms of ABJ (Fig.2).

254 255

Transport of bioactive compounds using the everted intestinal gut sacs model

256

Table 2 shows the apparent permeability coefficients (Papp) and efflux ratios of certain

257

free phenolic compounds. From apical to basolateral side (Papp

258

exhibited their higher Papp at 15 mins, where chlorogenic acid presented the highest

259

permeability coefficients (0.38 x 10-4 cm/s), followed by gallic acid, ellagic acid, and

260

caffeic acid. Chlorogenic and caffeic acid Papp A to B values remained with no significant

261

differences (p > 0.05) at 30 and 60 min, a trend that is similar to those of ellagic and

262

gallic acids. Despite the differences found amongst the assessed compounds, it can be


A to B),

phenolic acids

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observed an increase in the efflux ratios between 15-30 min, and a decrease at the end

264

of the intestinal incubation, suggesting a stabilization of each phenolic compound flux

265

from both intestinal sides.

266 267

Bioaccessibility of oligosaccharides during the in vitro gastrointestinal digestion and

268

simulated colonic fermentation

269

The representative chromatograms of the pure oligosaccharides standards and ABJ are

270

shown in Fig.3. Four oligosaccharides were identified in the juice (xylose, mannose,

271

raffinose, and stachyose) and all of them exhibited their highest bioaccessibility at the

272

small intestine stage (Table 3). Xylose and mannose were the major oligosaccharides

273

found. These bioaccessibility values diminished in the large intestine.

274

Effect of the fermented non-digestible fraction of Andean berry juice (FNDFABJ) on

275

the colonic pH

276

Table 4 shows the effect of the fermented non-digestible fraction of Andean Berry Juice

277

(FNDFABJ) on the colonic pH. During the first 6 hours of fermentation, the pH values

278

significantly decreased (p < 0.05), especially raffinose (positive control) (up to

279

26.30%). From 6 to 12 hours there is a significant (p < 0.05) increase of the pH for all

280

samples except for raffinose, which exhibits a pH even lower. Within the last 12 hours,

281

only the FNDFABJ exhibited a significant (p < 0.05) increase in pH.

282 283

Effect of FNDFABJ on SW480 cell growth

284

The SW480 cell proliferation was inhibited in a concentration-dependent manner

285

(Fig.5). After 24 h, there is a significant inhibition of SW480 cell growth (p mannose whereas Bifidobacterium pseudolongum have a sequential sugar uptake

373

(galactose > glucose > xylose). As of this date and to our knowledge, there are no

374

reports of the in vitro gastrointestinal profile of oligosaccharides from ABJ.

375

Fecal samples provided a naturally-origin microbiota source for the in vitro colonic

376

fermentation. Despite concerns about differences amongst bacterial composition, our

377

research group has reported a neglectable effect of the fecal source in the pH behavior

378

and similarities amongst gallic, chlorogenic and (+)-catechin bioaccessibilities between

379

normal and overweight donors28. Furthermore, when the pH values were compared with

380

commercial bacterial sources (Lactipan, Danisco, Lactipan/Danisco Mixture), there

381

were similar pH variations during the in vitro colonic fermentation. For this reason, the

382

human-origin fecal samples are a suitable source of gut microbiota for the in vitro

383

colonic fermentation experiments29.

384

The fermentation of the non-digestible fraction of ABJ induced a decrease in the colonic

385

pH during the first 6 hours of fermentation, followed by an increase of the pH at the 24

386

hours. As a completion date of the present research, there are no reports of the in vitro

387

gastrointestinal digestion of berries. Nonetheless, for other food matrices, there are



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publications of a similar trend, reflecting the fermentation process and the production of

389

metabolites which regulate the whole gut ecosystem30. Low pH environments (< 5.7)

390

have resulted in a low acetate-propionate ratio and low ammonia concentrations. As

391

long the pH increases, fiber digestion is improved but it has been observed that pH

392

values over 6.0 had negligible results in the fermentation profile of a diet made from

393

several fibrous food sources (alfalfa hay, ground corn grain, ground barley grain,

394

soybean meal, molasses and lupin seeds, among others). Raffinose is a recognized

395

prebiotic, and the third most fermentable after inulin and lactulose by lactobacilli,

396

increasing the counts of viable cells by 3-4 fold31.

397

The consumption of fiber-rich foods such as fruits can prevent the initiation of

398

colorectal carcinogenesis or retard such process towards the promoting stages attributed

399

to the products of fermented dietary fiber in colon such as SCFAs, capable for binding

400

or excreting carcinogens at the intestinal lumen2. In addition, the anticancer potential of

401

fruits has been associated with the content of phenolic acids and flavonoids based on

402

their antioxidant capacity and the modulation of several metabolic pathways such as cell

403

cycle arrest, apoptosis and inflammation (NF-κB)1,2.

404

After the intake of berry fruits, a substantial portion of the digested phenolic acids and

405

flavonoids pass into the colon, altering their structures and potential function.

406

Therefore, it can be inferred that the colonic epithelium is directly contacting the

407

digested compounds and their degradation products. Most studies have used in vitro

408

colonocyte models with phenolic compounds rich extracts from berries1,2 and have not

409

considered the effects of their in vivo metabolites. Thus, the objective of this study was

410

to evaluate the antiproliferative effect of the fermented non-digestible fraction of ABJ

411

(FNDFABJ). In the present study, we showed that FNDFABJ inhibited the growth of

412

SW480 human colon adenocarcinoma cells, as it was previously observed using non-


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digested ABJ5. Moreover, there were no significant differences in the IC50 value at 24h

414

between the FNDFABJ (IC50 = 19.3% v/v) and the non-digested whole juice (IC50 =

415

19.8% v/v).

416

It has been shown that over a biologically relevant dose range (0-50 µg/mL gallic acid

417

equivalents), the digested and fermented extracts of raspberries, strawberries and

418

blackcurrants demonstrated a significant anti-proliferative, anti-mutagenic and anti-

419

invasive activity on HT29 human colon cancer cells as the MTT, the mutation

420

frequency, and matrigel invasion/migration assays showed3. On the other hand, a colon

421

raspberry extract containing phytochemicals after a digestion procedure, not only

422

decreased significantly the population of SW480 cells in the G1 phase but also caused

423

significant protective effects against DNA damage induced by H2O2 in HT29 cells32. All

424

of the above data, together with our findings, indicate that the non-digestible fermented

425

fractions of juice or whole berry fruits that are in direct contact with the colonic

426

epithelium may have an antiproliferative effect in vivo on colon adenocarcinoma cells.

427

Our results suggest that the in vitro gastrointestinal digestion and simulated colonic

428

fermentation of the ABJ influence the bioaccessibility of their phenolic compounds (up

429

to 2813.33 µg/g) and oligosaccharides (up to 32601.86 mg equivalents/ g), depending

430

on the evaluated compounds and the stage of the in vitro gastrointestinal digestion,

431

considering the changes in pH and enzymatic conditions. Most of these compounds,

432

retained in the non-digestible fraction (NDFABJ), are fermented by the human gut

433

microbiota, showing pH changes as long fermentation is carried out. The FNDFABJ

434

exhibited an antiproliferative effect on SW480 in a dose-dependent manner. To our

435

knowledge, this is the first study that evaluates the effect of a digested putative

436

Colombian berry on human colon cancer cells. However, additional in vitro and in vivo



Page 20 of 39

20 437

studies are required to demonstrate the chemical compounds and mechanisms of action

438

involved in the results obtained here.

439 440

Abbreviations used:

441

ABJ: Andean berry juice; DFABJ: Digestible fraction of Andean berry (Vaccinium

442

meridionale Swartz) juice; NDFABJ: Non-digestible fraction of Andean berry juice;

443

FNDFABJ: Fermented non-digestible fraction of Andean berry juice; PET:

444

Polyethylene terephthalate; SCFAs: Short-chain fatty acids.

445 446

Acknowledgements

447

Authors would like to thank to M.Sc. Kenia Vázquez Sánchez and M.Sc. M. L. Cuellar-

448

Nuñez for her support in the preparation of samples and Mr. Mario González by

449

providing the Andean berry (Vaccinium meridionale Swartz) fruit. English edition by:

450

Agustin Ruiz Esparza y Ballesteros, U. of St. Thomas (Houston, TX, USA), C2 Oxford

451

College (Oxfordshire, U.K.).

452 453

Supporting Information description

454

Table S1. Analytic Description of The HPLC-DAD and HPLC-IR Detection of The

455

Assessed Bioactive Compounds

456 457

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Author Carlos Daniel Agudelo was supported by a scholarship from the Francisco José

581

de Caldas Institute for the Development of Science and Technology (COLCIENCIAS).

582

Author Ivan Luzardo-Ocampo was supported by a scholarship from the Consejo

583

Nacional de Ciencia y Tecnología (CONACYT) [grant number: 384201]. This work

584

was supported by a grant from the Comité para el Desarrollo de la Investigación

585

(CODI) from Universidad de Antioquia [grant number 2015-7505].

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Page 26 of 39

26 Figure Captions Figure 1. In vitro gastrointestinal digestion procedure and bioactive compounds analyzed at each stage. Figure 2. Small intestine absorption (everted gut sac model) of free phenolic acids during 15, 30, 60 and 120 min of in vitro gastrointestinal digestion. The results were expressed as media ± SD at the different small intestine incubation times (15, 30, 60 and 120 min). Different letters represent significant differences (p

Bioaccessibility during In Vitro Digestion and ... - ACS Publications

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