Intestinal anti-inflammatory effects of total alkaloid extract from Fumaria capreolata in the DNBS model of mice colitis and intestinal epithelial CMT93 cells

doi:10.1016/j.phymed.2016.05.003

Phytomedicine

Volume 23, Issue 9, 15 August 2016, Pages 901-913

https://doi.org/10.1016/j.phymed.2016.05.003 Get rights and content

Abstract

Background

Fumaria capreolata L. (Papaveraceae) is a botanical drug used in North Africa for its gastro-intestinal and anti-inflammatory properties. It is characterized for the presence of several alkaloids that could be responsible for some of its effects, including an immunomodulatory activity.

Purpose

To test in vivo the intestinal anti-inflammatory properties of the total alkaloid fraction extracted from the aerial parts of F. capreolata (AFC), and to evaluate its effects on an intestinal epithelial cell line.

Study design and methods

AFC was chemically characterized by liquid chromatography coupled to diode array detection and high resolution mass spectrometry. Different doses of AFC (25, 50 and 100 mg/kg) were assayed in the DNBS model of experimental colitis in mice, and the colonic damage was evaluated both histologically and biochemically. In addition, in vitro experiments were performed with this alkaloid fraction on the mouse intestinal epithelial cell line CMT93 stimulated with LPS.

Results

The chemical analysis of AFC revealed the presence of 23 alkaloids, being the most abundants stylopine, protopine and coptisine. Oral administration of AFC produced a significant inhibition of the release and the expression of IL-6 and TNF-α in the colonic tissue. It also suppressed in vivo the transcription of other pro-inflammatory mediators such as IL-1β, iNOS, IL-12 and IL-17. Furthermore, AFC showed an immunomodulatory effect in vitro since it was able to inhibit the mRNA expression of IL-6, TNF-α and ICAM-1. Moreover, the beneficial effect of AFC in the colitic mice could also be associated with the normalization of the expression of MUC-2 and ZO-1, which are important for the intestinal epithelial integrity.

Conclusion

The present study suggests that AFC, containing 1.3% of stylopine and 0.9% of protopine, significantly exerted intestinal anti-inflammatory effects in an experimental model of mouse colitis. This fact could be related to a modulation of the intestinal immune response and a restoration of the intestinal epithelial function.

Graphical abstract

Introduction

Inflammatory bowel disease (IBD) comprises different chronic inflammatory disorders of the gastrointestinal tract, mainly Crohn's disease and ulcerative colitis, which are characterized by remitting and relapsing episodes of intestinal inflammation. The most common symptoms are intermittent abdominal pain, rectal bleeding, fever, weight loss, fatigue and diarrhoea, which seriously compromise the quality of life of these patients (Braus and Elliott, 2009). The precise aetiology of IBD has not been completely identified, but the chronic relapsing inflammation is thought to be the consequence of a genetic predisposition that triggers a deregulated and exaggerated immune response against the intestinal microbiota (Ardizzone and Bianchi Porro, 2005, Sanchez-Muñoz et al., 2008). This response results in a dysregulation of the synthesis and release of pro-inflammatory cytokines, such as tumour necrosis factor α (TNFα), interferon-γ, (IFN-γ), interleukin (IL)-1β, IL-6, and IL-12, and anti-inflammatory cytokines, including IL-10 or transforming growth factor (TGF)-β (Neurath, 2014), and an excessive production of reactive oxygen/nitrogen species that are not conveniently scavenged and lead to oxidative/nitrosative stress (Piechota-Polanczyk and Fichna, 2014). The final consequence of this unbalance is tissue damage together with lipid and protein modifications and DNA damage or apoptosis, which also contribute to the pathogenesis of the intestinal inflammation.

The conventional pharmacological treatments for human IBD include aminosalicylates, corticosteroids, immunosuppressants and biological agents, and although they show efficacy, in many patients they are not fully effective and can be associated with major adverse effects that limit their required chronic use (Siegel, 2011). These facts have promoted the development of emerging and alternative therapeutic strategies that may be useful for the management of chronic intestinal inflammation, including traditional plant–based remedies, which show immunomodulatory and antioxidant properties (Hur et al., 2012).

Different species from genus Fumaria (Papaveraceae) have been traditionally used against quite diverse disorders. Thus, in Anatolian folk medicine, these plants have been reported to act as a blood purifier and as an anti-allergic agent, as well as in the treatment of some skin diseases (rashes or conjunctivitis) (Orhan et al., 2012). Furthermore, their beneficial effects as anti-hypertensives, diuretics or in hepatobiliary and gastrointestinal complaints have been also reported (Suau et al., 2002a). Typically, the biological activities associated with these plants have been related to the presence of isoquinoline alkaloids in their composition, such as aporphine, protoberberine, protopine and benzophenanthridine type (Suau et al., 2002a, Suau et al., 2002b, Grycova et al., 2007). Isoquinoline alkaloids are considered as a major group of pharmacologically important compounds, and some of them have demonstrated, among others, biological, antimicrobial, antibacterial, antifungal and antitumor properties (Dembitsky et al., 2014).

In a previous study, it was reported that the total alkaloid fraction from Fumaria capreolata L., in addition to exert antioxidant activity, was devoid of significant toxicity when administered orally to mice at doses up to 2 g/kg (Bribi et al., 2013). Moreover, it has been also recently described the antinociceptive and anti-inflammatory effects of this extract (Bribi et al., 2015). The aim of the present study was to evaluate the effects of a total alkaloid fraction from F. capreolata (AFC) in the dinitrobenzenesulphonic acid (DNBS) model of experimental colitis in mice, correlating its potential anti-inflammatory activity to the expression of some of the mediators involved in the intestinal inflammatory response, such as pro-inflammatory cytokines, like IL-6, IL-1β, TNF-α, IL-12 and IL-17, the chemokine intercellular adhesion molecule (ICAM)-1, the enzymes inducible nitric oxide synthase (iNOS) and the metalloproteinase (MMP)-9, as well as two markers of epithelial integrity in the mucosa, the mucin MUC-2 and the transmembrane protein zonula occludens (ZO)-1. Furthermore, some in vitro studies were performed to evaluate the impact of this alkaloid fraction on the mouse intestinal epithelial cell line CMT93.

Section snippets

Drugs and chemicals

All the drugs and chemicals used were purchased from Sigma–Aldrich Chemical (Madrid, Spain), unless otherwise stated. The test substances were dissolved in distilled water and prepared fresh daily for administration to the animals. In addition, methanol, acetonitrile, ultrapure water of MS quality and formic acid were purchased from Fisher Chemicals (ThermoFisher, Waltham, MA, USA). Protopine hydrochloride was obtained from Sigma-Aldrich Chemical (Madrid, Spain) and a methanolic stock solution

Statistical analysis

All data were expressed as mean ± standard error of the mean (SEM), except score data that were expressed as median (range). The statistical analysis of all the observations was carried out using a one-way analysis of variance (ANOVA) followed by a multiple comparison Dunnett's test, when necessary. Non-parametric data (score) were analyzed using the Kruskal-Wallis test. All statistical analyses were carried out with the GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA)

Phytochemical analysis of the total alkaloids extract from F. capreolata

The yield of the alkaloids extract from the aerial part of F. capreolata was 1.17% (w/w), when calculated based on the dry powdered plant material. The chemical analysis was conducted to characterize the composition of the alkaloid fraction obtained from F. capreolata. Fig. 1A depicts the base peak chromatogram (BPC) in the positive ionization mode. The results of LC–DAD–MS and –MS/MS analysis are given in Table 2, which shows RT, molecular formula, experimental m/z, mass error (ppm), mσ value,

Discussion

At present, IBD therapy is mainly based on the administration of anti-inflammatory or immunosuppressive drugs such as salicylates, glucocorticoids and other immunosuppressants, as well as biologicals. Although they have shown efficacy in most cases, some patients obtain little benefit after their administration, and all these drugs are often associated with important side effects that limit their long-term use (Siegel, 2011). For this reason there is a clinical need to identify new and safe

Conclusion

In conclusion, the present study suggests that the AFC, containing stylopine and protropine, significantly exerted intestinal anti-inflammatory effects in an experimental colitis model in mice, which resembles human IBD. This fact could be associated with a modulation of the intestinal immune response since AFC was able to inhibit the expression and/or release of different pro-inflammatory mediators, as well as to improve epithelial barrier integrity. This observation was supported by the

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome

Acknowledgments

This work was supported by the Junta de Andalucía (the Excellence Projects P10-AGR-6826, P11-CTS-7625 and CTS 164) and by the Spanish Ministry of Economy and Competitiveness (AGL2015-67995-C3-2-R and AGL2015-67995-C3-3-R) with funds from the European Union. J. Garrido-Mesa is a predoctoral fellow from the Spanish Ministry of Education and Science; A. Rodriguez-Nogales and F. Algieri are postdoctoral fellows of University of Granada; M.E. Rodriguez-Cabezas is a postdoctoral fellow of CIBER-EHD;

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¹: Both authors contributed equally to the supervision of the study.

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Original ArticleIntestinal anti-inflammatory effects of total alkaloid extract from Fumaria capreolata in the DNBS model of mice colitis and intestinal epithelial CMT93 cells

Abstract

Background

Purpose

Study design and methods

Results

Conclusion

Graphical abstract

Introduction

Section snippets

Drugs and chemicals

Statistical analysis

Phytochemical analysis of the total alkaloids extract from F. capreolata

Discussion

Conclusion

Conflict of interest

Acknowledgments

J. Ethnopharmacol.

Clin. Immunol.

Gastroenterology

J. Ethnopharmacol.

J. Ethnopharmacol

Phytochemistry

Nutr. Res.

J. Pharmacol. Toxicol. Methods

Exp. Toxicol. Pathol.

Bioorg. Med. Chem. Lett

Phytomedicine

Biochem. Syst. Ecol.

Toxicol. Lett

Di-D-fructose dianhydride-enriched caramels: effect on colon microbiota, inflammation, and tissue damage in trinitrobenzenesulfonic acid-induced colitic rats

J. Agric. Food Chem.

Biologic therapy for inflammatory bowel disease

Drugs

Protopine reduces the inflammatory activity of lipopolysaccharide-stimulated murine macrophages

BMB Rep

Evaluation of erythrocytes toxicity and antioxidant activity of alkaloids of Fumaria capreolata

Int. J. Pharm. Bio. Sci.

Antinociceptive and Anti-Inflammatory Effects of Total Alkaloid Extract from Fumaria capreolata

Evid. Based Complement. Alternat. Med

The intestinal anti-inflammatory effect of quercitrin is associated with an inhibition in iNOS expression

Br. J. Pharmacol.

Intestinal mucosal barrier is injured by BMP2/4 via activation of NF-κB signals after ischemic reperfusion

Mediators Inflamm.

Original Article
Intestinal anti-inflammatory effects of total alkaloid extract from Fumaria capreolata in the DNBS model of mice colitis and intestinal epithelial CMT93 cells