Subscriber access provided by University at Buffalo Libraries
Article
The deleterious effect of p-cresol on human colonic epithelial cells is prevented by proanthocyanidin-containing polyphenol extracts from fruits and proanthocyanidin bacterial metabolites Ximena Wong, Catalina Carrasco-Pozo, Elizabeth Escobar, Paola Navarrete, François Blachier, Mireille Andriamihaja, Annaïg Lan, Daniel Tomé, Maria Jose Cires, Edgar Pastene, and Martin Gotteland J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00656 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 4, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 36
Journal of Agricultural and Food Chemistry
The deleterious effect of p-cresol on human colonic epithelial cells is prevented by proanthocyanidin-containing polyphenol extracts from fruits and proanthocyanidin bacterial metabolites
Ximena Wong1, Catalina Carrasco-Pozo1, Elizabeth Escobar1, Paola Navarrete2, Franςois Blachier3, Mireille Andriamihaja3, Annaig Lan3, Daniel Tomé3, Maria Jose Cires1, Edgar Pastene4, Martin Gotteland1, 2 *
1
Department of Nutrition, Faculty of Medicine, University of Chile, Independencia 1027, Independencia, Santiago, Chile. 2
Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile.
3
INRA/AGROPARISTECH, UMR 914 Nutrition Physiology and Ingestive Behavior, Paris, France 4
Laboratory of Pharmacognosy, Faculty of Pharmacy, University of Concepción, Concepción, Chile
*Corresponding author: Phone: 56-2-29786977. E-mail:
[email protected] 1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
2
Page 2 of 36
Abstract
3
The protective effect of proanthocyanidin-containing polyphenol extracts from apple,
4
avocado, cranberry or grape, or proanthocyanidin microbial metabolites was evaluated in
5
colonic epithelial cells exposed to p-cresol, a deleterious compound produced by the
6
colonic microbiota from L-tyrosine. In HT29 Glc-/+ cells, p-cresol significantly increased LDH
7
leakage and decreased ATP contents while in Caco-2 cell monolayers it significantly
8
decreased the transepithelial electrical resistance and increased the paracellular transport
9
of FITC-dextran. The alterations induced by p-cresol in HT29 Glc-/+ cells were prevented by
10
the extracts from cranberry and avocado while they were worsened by those from apple
11
and grape. The proanthocyanidin bacterial metabolites decreased LDH leakage,
12
ameliorating cell viability without improving intracellular ATP. All the polyphenol extracts
13
and proanthocyanidin bacterial metabolites prevented the p-cresol-induced alterations of
14
barrier function. These results suggest that PAC-containing polyphenol extracts and
15
proanthocyanidin metabolites likely contribute to the protection of the colonic mucosa
16
against the deleterious effects of p-cresol.
17
Keywords: p-cresol, mitochondria, proanthocyanidins, colonic cells, microbiota, avocado,
18
apple, cranberry, grapes, gut barrier function
19
2 ACS Paragon Plus Environment
Page 3 of 36
20
Journal of Agricultural and Food Chemistry
Introduction
21
Polyphenols are secondary metabolites including phenolic acids and flavonoid compounds
22
(flavanols, flavonols and anthocyanins) that are implicated in the protection of plants
23
against depredators, infection and diverse type of stress. Due to their chemical structure,
24
polyphenols exhibit a great number of health-promoting properties such as antioxidant,
25
antibacterial, anti-inflammatory, anti-hypertensive, anti-proliferative, regulatory of the
26
mitochondrial function, etc.1 For this reason, polyphenols are considered as beneficial
27
dietary non-nutrients whose intake, under the form of fruits and vegetables, is
28
recommended to decrease the risk of chronic non-communicable diseases in the
29
population. Among dietary polyphenols, proanthocyanidins (PACs), also known as
30
condensed tannins, are oligomeric forms of flavan-3-ols abundant in plant-derived
31
foodstuffs such as red wine, green tea, grape seed and skin, cocoa, avocados, cranberries,
32
peanut cuticle, cinnamons and apples.2, 3 In the U.S. population, they are the second most
33
abundant phenolic compounds in the diet after lignin, being the estimated PAC daily
34
intake of 224 mg, approximately.2, 3 Dietary PACs with a degree of polymerization higher
35
than 3 are in general poorly absorbed in the intestine, thus reaching the colon where they
36
are metabolized by the colonic microbiota.4-7 Studies in humans and animals consuming
37
cocoa, red wine, apple or green tea have shown that the PACs present in these foodstuffs
38
stimulate the growth of bacterial populations from the autochthonous microbiota in the
39
colon, including C. histolyticum, E. rectale Lactobacillus and Bifidobacterium spp.8-13 It is
40
therefore possible that this prebiotic-like effect explains some of their health promoting
41
properties. The bacterial metabolism of dietary PACs generates aromatic acids such as 3-
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 36
42
hydroxyphenyl acetic acid (3-HPAA), 4-hydroxyphenyl valeric acid (4-HPVA), 4-
43
hydroxyphenyl acetic acid (4-HPAA), 3,4-dihydroxyphenyl propionic acid (3,4-DHPPA), 3-
44
(3-hydroxyphenyl) propionic acid (3-HPPA) and 3-phenylpropionic acid (3-PPA) that may
45
be detected in high concentrations in stools or fecal water of healthy subjects.6, 8, 13, 14
46
These compounds may be absorbed in the colon and, eventually, they may be detected in
47
the bloodstream at micromolar concentrations (2-50µM) and exert systemic activities. For
48
example, 3,3-DHPPA has been shown to act as a potent vasodilator in rats while 3,4-
49
DHPPA exhibit cytotoxic and anti-inflammatory properties.15-17
50
On the other hand, the colonic microbiota also expresses proteases and peptidases
51
capable of partially degrading dietary and endogenous proteins that remain in the small
52
intestine and reach the colon. The phenylalanine, tyrosine and tryptophan released
53
through this process are subsequently fermented, resulting in the formation of phenolic
54
metabolites including p-cresol (4-methylphenol).18,
55
produced from L-tyrosine by the anaerobic bacteria, a process which occurs preferentially
56
in the distal colon where protein breakdown is more intense and then, the concentrations
57
of p-cresol more elevated.20 p-cresol is absorbed by the epithelial colonic cells, transferred
58
into the bloodstream, metabolized in the liver and excreted in the urine where it
59
represents more than 90% of the phenolic compounds recovered.21 Interestingly, higher
60
urinary concentrations of p-cresol have been described in patients with colorectal
61
cancer.22 In this respect, we recently showed that p-cresol negatively affects human
62
colonic epithelial cells (HT-29 Glc-/+) in vitro, inhibiting their proliferation and respiration,
63
decreasing their ATP content and increasing DNA damage.23 Additionally, p-cresol is also
19
This compound is specifically
4 ACS Paragon Plus Environment
Page 5 of 36
Journal of Agricultural and Food Chemistry
64
recognized as a uremic toxin and in patients with chronic kidney disease its accumulation
65
or that of its conjugate, p-cresyl-sulphate, contributes to the development of renal and
66
cardiovascular complications.24-26 It is noteworthy that the intracolonic concentrations of
67
p-cresol and, eventually, its subsequent deleterious effects in the epithelium increased in
68
individuals consuming high protein diets27 while they decreased in those consuming diets
69
rich in resistant starch or pre- and probiotics.28, 29 Interestingly, a decrease of p-cresol has
70
also been reported in the colon of rats fed a diet supplemented with epigallocatechin
71
gallate (EGCG), the main phenolic compound present in green tea.30
72
In this context, the aim of the present study was to determine whether polyphenol
73
extracts containing PACs from apple, cranberry, grape and avocado, or PAC microbial
74
metabolites may protect colonic epithelial cells from the deleterious effects of p-cresol in
75
human colonic cell lines in terms of cell viability, mitochondrial function and epithelial
76
integrity.
77
78 79
Materials and methods •
Chemicals
80
CytoTox-ONE Homogeneous Membrane Integrity Assay kits and CellTiter-Glo luminescent
81
cell viability assay kits were from Promega (Madison, USA). Flavonol glycosides quercetin
82
3-O-galactoside (hyperoside), quercetin 3-O-glucoside (isoquercitrin) and quercetin 3-O-
83
rhamnoside (quercitrin) were from Roth (Karlsruhe, Germany). 5-caffeoyl quinic acid and
84
4-caffeoyl quinic acid were obtained from Phytolab (Vestenbergsgreuth, Germany). All
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 36
85
solvents (HPLC-grade acetonitrile, methanol and TFA) were purchased from Merck
86
(Darmstadt,
87
dihydroxyphenyl propionic acid (3,4-DHPPA), 3-phenylpropionic acid (3-PPA), fluorescein
88
isothiocyanate-dextran (FD-4), rhodamine 123 quercetin, rutin, apigenin, kaempferol,
89
myricetin, isorhamnetin, epicatechin, catechin, epicatechin gallate, catechin gallate,
90
phloretin-2´-xyloglucoside, phloretin-2´-glucoside (phloridzin), cyanidin-3-O-glucoside,
91
cyanidin-O-arabinoside, peonidin-O-arabinoside, gallic acid, syringic acid, vanillic acid,
92
chlorogenic acid, caffeic acid, ellagic acid, sinapic acid, ferulic acid, anisic acid, p-coumaric
93
acid, cinnamic acid, protocatechuic acid, p-hydroxybenzoic acid, m-hydroxybenzoic acid,
94
caffeoyl glucoside, feruloyl glucoside, coumaroyl glucoside and the Folin-Ciocalteu reagent
95
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
96
•
Germany).
p-cresol,
4-hydroxyphenylacetic
acid
(4-HPAA),
3,4-
Preparation of the polyphenol extracts from fruits
97
The polyphenol extracts from cranberry, avocado, grape and apple were prepared from
98
concentrated cranberry juice (Agricola Cran Chile Ltda., Lanco, Chile), grape skin and seeds
99
(Cabernet Sauvignon variety, De Neira Vinyard, Guarilihue, Chile), apple peels (Granny
100
Smith variety, Surfrut Ltda, Chile) and avocado peels (Hass variety, purchased in local
101
market), respectively.31 500g of frozen samples (−20 °C) of grape, apple and avocado peels
102
were mixed with hot water (5 min, 90 °C) and filtered on a sintered glass funnel (70-100
103
μm). Marcs were homogenized for one min with an Ultraturrax and extracted again with
104
water at 65 °C. After 60 min of maceration, the pH of the pooled extracts was adjusted to
105
2.5 with formic acid and these were poured on a glass column (150 mm i.d. x 300 mm)
106
packed with Sepabeads SP-850 (Supelco, Bellefonte, USA) pre-conditioned with acidic 6 ACS Paragon Plus Environment
Page 7 of 36
Journal of Agricultural and Food Chemistry
107
water. Sugars and other water soluble compounds were removed by washing with 5L of
108
distilled water and the polyphenol extracts were obtained after elution with absolute
109
ethanol26. Cranberry extract was obtained from concentrated juice (500mL) by five-fold
110
dilution with acidic water (pH 2.5) and adsorption on Amberlite XAD7HP column. The
111
ethanolic fractions were evaporated under vacuum (
