Intestinal anti-inflammatory effects of Passiflora edulis peel in the dextran sodium sulphate model of mouse colitis
Introduction
Inflammatory bowel disease (IBD) mainly comprises two related conditions – Crohn's disease (CD) and ulcerative colitis (UC) – which affect millions of people worldwide (Molodecky et al., 2012). The aetiology of IBD has not been fully elucidated; nevertheless, a combination of genetic and environmental factors is involved in its pathogenesis. They promote an abnormal exacerbated immune response in the intestine that generates an inflammation (Bamias, Nyce, De La Rue, & Cominelli, 2005). One of the key environmental factors involved in the increasing incidence of IBD is the lower intake of fibres (Hansen et al., 2011).
Dietary fibres are plant substances that resist the digestive tract hydrolysis and are fermented by colonic microbiota producing short-chain fatty acids (SCFA – acetic, propionic and butyric acids), which play an important role in the maintenance of colonic homeostasis, especially butyric acid, which provides the main fuel for colonocytes (Galvez, Rodriguez-Cabezas, & Zarzuelo, 2005), contributing, this way, to preserve mucosal integrity. In addition, any imbalance in the microbiota homeostasis can up-regulate the immune response leading to mucosal damage and impairment of the barrier function, and thus increasing translocation of antigens and intestinal inflammation (MacDermott, 1996). This barrier is formed by a monolayer of epithelial cells regulated by an apical intercellular junctional protein complex, essential to maintaining the barrier function (Bruewer, Samarin, & Nusrat, 2006).
Passiflora edulis is largely cultivated in Brazil for juice and pulp production. Since more than half of the fruit is peel, which is a great source of pectin (Pinheiro et al., 2008) and flavonoids (Zeraik, Yariwake, Wauters, Tits, & Angenot, 2012), a great amount of waste is generated. According to our previous works this by-product of food industry could be used to increase fibre intake, and modulate some inflammatory markers in ulcerative colitis (Cazarin et al., 2014a) and microbiota composition (da Silva, Cazarin, Bogusz Junior, Augusto, & Maróstica Junior, 2014). Therefore as our previous data suggest P. edulis peel could be considered a functional food source of fibres and polyphenols.
The aim of this study was to examine the preventative effects of P. edulis peel flour in the dextran sodium sulphate (DSS) model of experimental colitis. The DSS model was chosen because it reflects some clinical and histopathological features seen in human IBD, mostly UC (Dieleman et al, 1998, Gaudio et al, 1999, Kullmann et al, 2001), and exhibits good reproducibility. Special attention was given to the effects on the expression of some of the mediators involved in the inflammatory response, such as pro-inflammatory cytokines (tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-12 and IL-17), chemokines like monocyte chemotactic protein (MCP-1) and adhesion molecules (intercellular adhesion molecule (ICAM)-1), as well as different markers of epithelial integrity in the mucosa, like the mucins MUC-2 and MUC-3, tight junction proteins, occludin and zonula occludens (ZO)-1 and the matrix metalloproteinases (MMP) 2 and 9.
Section snippets
Materials and methods
Analytical standards of vicenin, vitexin, isovitexin, orientin and isoorientin used for quantitation and identification of C-glycosyl flavonoids were purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile, methanol and ethanol were purchased from J.T. Baker (J.T. Baker, USA) and analytical grade acetic acid was obtained from Dinâmica Química Comtemporânea (Diadema, SP, Brazil). Ultrapure water used was obtained from a Milli-Q system from Millipore (Bedford, MA, USA).
Identification of phenolic compounds in P. edulis peel by UPLC–ESI-MS/MS
The P. edulis peel flavonoids were extracted by adding 5 mL of 50:50 (v/v) ethanol:water to 250 mg of sample. The mixture was rotated at room temperature for 60 min. Afterwards, the sample was filtered and the supernatant was stored in amber flasks at 4–8 °C to evaluate the presence of C-glycosyl flavonoids by UPLC–MS/MS and stored in amber flasks at 4–8 °C. This procedure was repeated 10 times until C-glycosyl flavonoid total extraction and the filtrates were combined in the same flask.
The
Quantitative analysis of C-glycosyl flavonoids
Initially matrix effect was assessed by comparing the slope of analytical curves prepared in matrix (P. edulis flour) and in methanol (Economou, Botitsi, Antoniou, & Tsipi, 2009). Since matrix effect was not verified, the quantitation was conducted through external calibration. Each stock standard solution of vicenin, vitexin, isovitexin, orientin and isoorientin was prepared by accurately weighing and dissolving the standards in pure methanol. The analytical method was validated based on the
Animals and diets
This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals as promulgated by the National Institutes of Health. The experimental protocol was approved by the Commission of Ethics in Animal Experimentation (Protocol CEEA 2010-286) of the University of Granada (Spain).
Female Wistar rats and C57BL/6 mice were obtained from Janvier (St Berthevin Cedex, France). They were housed in Makrolon cages, maintained in an air-conditioned atmosphere with a 12-h
Statistical analyses
All results were expressed as means ± standard error of mean (SEM). The statistical analyses were carried out using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA) software. Parametric data were analysed by using ANOVA with Tukey (significance limit was set at P <0.05). Non-parametric data were analysed using Kruskal–Wallis test.
Results and discussion
Functional foods show beneficial effects on the body by decreasing the risk of diseases and promoting regular nutrition (Roberfroid, 2002). The prebiotics are an example of functional food that could improve health by selectively modulating the growth and/or activity of one or a limited number of bacteria in the colon through fermentation and short chain fatty acid formation (Roberfroid, 2007).
We decided to investigate the prebiotic effects of the consumption of P. edulis peel, source of fibres
Conclusion
An analytical method using UPLC–ESI-QToF-MS was developed and validated and demonstrated their fitness for the identification and quantitation of the unbounded C-glycosyl flavonoids vicenin, isovitexin, orientin and isoorientin in the P. edulis peel. Since vitexin level was below the LOQ it was only identified. In addition, the compounds lucenin-2, schaftoside and violanthin were tentatively identified.
The consumption of P. edulis peel showed intestinal anti-inflammatory effects in the DSS
Acknowledgements
This work was supported by FAPESP (grant number 2012/24262-1) and the Spanish Ministry of Economy and Competitiveness (SAF2011-29648 and AGL2015-67995-C3-3-R) and Junta de Andalucia (AGR-6826 and CTS 164) with funds from the European Union; J. Garrido-Mesa is a predoctoral fellow from the Spanish Ministry of Education and Science; F. Algieri is a predoctoral fellow of Junta de Andalucia; M.E. Rodriguez-Cabezas is a postdoctoral fellow of CIBER-EHD. The CIBER-EHD is funded by the Instituto de
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