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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].
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Abstract
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Berries consumption is associated with colorectal cancer chemoprevention, but
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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,
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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
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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
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does not represent the physiological effects after consumption of the berry, in which the
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fruit is subjected to gastrointestinal digestion before reaching the colon. Also, the
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phytochemicals present in the fruit that possess antioxidant, cytotoxic and
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antiproliferative activities might be transformed into other compounds with different
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biological activity, affecting the potential anticancer properties of these components.
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In this manner, the in vitro gastrointestinal model mimics the conditions of the
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gastrointestinal tract, allowing to monitor the bioactive compounds bioaccessibility.
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This method has been used to understand the stability of anthocyanins from berries and
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the effect of the digestion on the antiproliferative capacity against different colon
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adenocarcinoma cells3. Additionally, the authors that conducted this research found that
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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
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undetectable metabolites by the usual identification and quantification methods.
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For this reason, in order to evaluate the physiological conditions which could affect the
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ABJ functionality, the bioaccessibility, and the apparent intestinal permeability of
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vitamin C, phenolic compounds, and oligosaccharides were evaluated, as well its
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antiproliferative activity against SW480 human colon adenocarcinoma cells.
66 67
Materials and Methods
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Chemicals and reagents
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All the reagents used were HPLC or analytical grade. The acetic acid, acetonitrile,
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pentobarbital sodium salt, HCl, NaCl, KCl, MgSO4, KH2PO4, NaHCO3, glucose, CaCl2,
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pepsin, pancreatin, gall ox, raffinose and HPLC-grade standards were purchased from
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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
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National Institute of Health Guide for Care and Use of Laboratory Animals.
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Plant material
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Fresh ripe berries of Andean berry (Vaccinium meridionale) were harvested at the
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vicinity of El Retiro (Antioquia, Colombia) (16 ºC, 2175 meters above the sea level and
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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
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Preparation of Andean Berry Juice
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The Andean berry juice (ABJ) was prepared as previously described7. Briefly, the
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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-
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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
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from light and stirred (450 rpm, IKA WERKER 015 magnetic stirrer, IKA Works,
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USA) for 24 h at room temperature (25 ± 1 ºC). The mixture was then centrifuged (2166
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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,
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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
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supplementary Table S1.
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Oligosaccharides extraction and quantification
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Oligosaccharides were extracted from ABJ
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procedure10. Briefly, 1.0 g of ABJ lyophilized powder was homogenized with 100 mL
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of distilled water and incubating at 80 ºC for 60 min using an oscillating water bath. The
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sample was then centrifuged (Hermle Z326K, Wehingen, Germany) for 10 min at 984 x
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g. The supernatant was collected, filtered through a 0.45 µm filter (Econofilter PTFE,
122
Agilent Technologies, Santa Clara, CA, USA).
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A high-performance liquid chromatography-refractive index detector (HPLC-RID)
124
analysis was done with an Agilent 1100 Series HPLC system (Agilent Technologies,
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Palo Alto, CA, USA) using a Zorbax Eclipse XDB-C18 column (Agilent Technologies,
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4.6 250 mm) as previously described11. Complementary information about the
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analytical method used is provided in the supplementary Table S1.
accordingly to a previously reported
128 129
In vitro gastrointestinal digestion
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For the simulation of the physiological conditions, the procedure described by Campos-
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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
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mL) 15 times for 15 s. For the stomach stage, the product was then expectorated into a
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beaker containing 5 mL of distilled water, followed by another rinsing with 5 mL of
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distilled water for 60 s. This procedure was repeated four times for the ABJ and salivary
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samples used as blanks.
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Afterwards, the suspension of each sample was mixed in a single beaker and the pH was
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adjusted to 2.0 using an HCl solution (150 mM). Pepsin (0.055 g, Sigma-Aldrich) was
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dissolved in 0.94 mL of 20 mM HCl solution and added to each aliquot (∼10 mL),
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following an incubation period of 2 h at 37 °C. The intestinal extract was prepared 30
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min before use, mixing 3 mg of Gall Ox and 2.6 mg of pancreatin (Sigma) in a 5 mL
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Krebs-Ringer buffer [118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4,
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25 mM NaHCO3, 11 mM glucose and 2.5 mM CaCl2, pH 6.8). This solution (5 mL) was
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added to each sample and gasified for 30 min before using a gas mixture (10:10:80
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H2:CO2:N2) in order to guarantee the anaerobic conditions. The suspension (15 mL) was
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transferred to a conical tube containing an everted gut sac, prepared from male Wistar
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rats (body wt. 250–300 g, n=14). Before the surgical procedure, the rats were fasted
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overnight (16–20 h) with water ad libitum. The rats were anesthetized with
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pentobarbital sodium (60 mg/kg, i.p.). The intestine of the rats was exposed by a
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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
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intestine was previously washed with the same buffer and everted over a glass rod,
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divided into segments (6 cm) and filled on the serosal side with ~1 mL of Krebs-Ringer
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buffer to prevent tissue viability loss. This everted gut sac was added to a 15 mL
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suspension of the gastric phase and incubated in an oscillating water bath (37 ºC, 80
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cycles/min, for 15, 30, 60 and 120 min). After the incubation period, the sacs were
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removed; the sample from the mucosal side (outside of the everted gut sac) was
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considered as the non-digestible fraction of the Andean berry juice (NDFAB). The 120-
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min NDFAB solution was subsequently subjected to in vitro colonic fermentation to
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obtain the fermented non-digestible fraction of ABJ (FNDFABJ). The experiments were
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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
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the HPLC analysis, the samples were defrosted and centrifuged at 5400 rpm for 10 min
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(HERMLE Z 323 K centrifuge, Wehingen, Germany) at 4 ºC. The obtained supernatant
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was collected and filtered (0.45 µm). Figure 1 illustrates a scheme for this procedure,
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including the analyzed bioactive compounds.
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In vitro colonic fermentation
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For the in vitro colonic fermentation to obtain the fermented non-digestible fraction of
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Andean berry (Vaccinium meridionale Swartz) juice (FNDFABJ), the procedure
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reported in a previous study by Campos-Vega et al.13was followed. The fresh fecal
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samples were collected from one selected healthy donor who was instructed to follow a
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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
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at 0, 6, 12 and 24 h-fermentation times, vortexing for at least 1 min before the
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measurement. At the end of each fermentation time, the fermented samples (FNDFABJ)
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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,
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USA) was used as a fermentable sugar reference. Three independent assays were done
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at 37°C simulating human conditions. A blank was also prepared using the 120 min-
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digested salivary solution instead NDFABJ. This procedure was conducted three times
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Calculations of the apparent permeability coefficients (Papp)
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The apparent permeability coefficients (Papp, x 10-4 cm/s) were estimated as follows14:
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Papp= (dQ/dt)(1/AC0) where Papp (cm/s) is the apparent permeability coefficient, dQ/dt
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(mg/s) is the amount of compound (phenolic acids or flavonoids) transported across the
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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
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compound outside the everted gut sacs.
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Water flux measurement.
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The water flux, accounting for both water absorption and efflux in
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segment was determined by applying the following equation15: WF = (W3-W2)/W1,
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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
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and W3 is the weight after incubation at the same time periods.
the intestinal
197 198
Bioaccessibility and absorption measurement
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The bioaccessibility of free phenolic compounds and oligosaccharides along the
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gastrointestinal digestion and colonic fermentation was computed the following
201
equation: B=[(C0-Cf)/Cf]*100 where B represents the bioaccessibility percentage (%) of
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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
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of the compound at the same time of incubation.
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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.
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Cell viability assay and determination of IC50
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The SW480 human colon adenocarcinoma cell line was used. The cells were cultured as
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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
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5% CO2. After 24 h, the different concentrations of the FNDFABJ (3, 6, 9, 12 and 18%
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v/v) were evaluated in the cells for 24 h. After the incubation, the adherent cells were
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washed with PBS twice and the viable cells were determined at 570 nm in a microplate
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reader (Thermoscientific Varioskan 51119300) after staining for 10 min at 37°C with
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50 µL of crystal violet solution per well (0.5% w/v crystal violet, 4% w/v formaldehyde,
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30% v/v ethanol, and 0.17% w/v NaCl). All the experiments were performed in
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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
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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
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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
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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
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observed an increase in the efflux ratios between 15-30 min, and a decrease at the end
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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.
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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
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hours. As a completion date of the present research, there are no reports of the in vitro
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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
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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
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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).
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Author Ivan Luzardo-Ocampo was supported by a scholarship from the Consejo
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Nacional de Ciencia y Tecnología (CONACYT) [grant number: 384201]. This work
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was supported by a grant from the Comité para el Desarrollo de la Investigación
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(CODI) from Universidad de Antioquia [grant number 2015-7505].
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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