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

ABSTRACT

Background: Fumaria capreolata L. (Papaveraceae) is a botanical drug used in North Africa for its gastrointestinal 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 100mg/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-ct in the colonic tissue. It also suppressed in vivo the transcription of other pro-inflammatory mediators such as (IL)-1[beta], iNOS, 1L-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-[alpha] and 1CAM-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.

Keywords:

DNBS mouse colitis

Intestinal inflammation

Cytokine, Stylopine

protopine

Fumaria capreolata

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-Munoz et al., 2008). This response results in a dysregulation of the synthesis and release of pro-inflammatory cytokines, such as tumour necrosis factor [alpha] (TNF[alpha]), interferon-[gamma], (IFN-[gamma]), interleukin (IL)-1[beta], IL-6, and IL-12, and anti-inflammatory cytokines, including IL-10 or transforming growth factor (TGF)-[beta] (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)-1[beta], TNF-[alpha], IL-12 and IL-17, the chemokine intercellular adhesion molecule (ICAM)-l, 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.

Materials and methods

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 (1 mg/ml) was prepared.

Extraction of alkaloids

Aerial parts of F. capreolata were collected from Bejaia area, in the North East of Algeria in May 2013 when they were at the flowering and fruit setting stage. Dr Benabdesselam authenticated the plant and a voucher specimen was deposited in a reference collection or the Herbarium of the Laboratory of Plant Biotechnology and Ethnobotany (University of Bejaia, Algeria) (Reference No. FC015). The alkaloid extract of F. capreolata (AFC) was obtained following the procedure previously reported (SouSek et al., 1999). Briefly, the aerial parts of the plant were dried in an oven at 40[degrees]C overnight and ground into a fine powder using a grinder. The powder samples (1 kg) were extracted with ethanol in a Soxhlet apparatus for 8h, then evaporated under reduced pressure, acidified with 2.5% HC1 to pH 1-2 and filtered, and stored overnight at room temperature. The aqueous acid solution was adjusted to pH 9.5 with concentrated ammonium hydroxide and extracted with dichloromethane. The extracts were dried over magnesium sulphate and the solvent evaporated to afford a crude extract of total alkaloids. After evaporation, the yield of each fraction was calculated, and the AFC obtained was stored at 4[degrees]C until use.

Characterization of the alkaloid fraction by liquid chromatography-coupled to diode array detection and high-resolution mass spectrometry (LC-DAD-MS)

Analyses were made with an Agilent 1200 series rapid resolution (Agilent, Palo Alto, CA, USA) equipped with a binary pump, an autosampler and a DAD. The mobile phases consisted of water with 0.2% formic acid (mobile phase A) and acetonitrile (mobile phase B). A multistep linear gradient was then applied: 0-5.5 min, 1-7% B; 5.5-11 min, 7-14% B; 11-17.5 min, 14-24% B; 17.5-22.5 min, 24-40% B; 22.5-27.5 min, 40-100% B; 27.5-28.5 min, 100-100% B; 28.5-29.5 min, 100-1% B. The latter value (99% A and 1% B) was held for 5.5 min to equilibrate the column to initial conditions before the next injection. The flow rate was set at 0.5 ml/min throughout the gradient. Separation was carried out with a Zorbax Eclipse XDB-C18 column (4.6 x 50 mm, 1.8 [micro]m of particle size) (Agilent, Palo Alto, CA, USA) at 25[degrees]C. The absorbance was monitored between 190 and 600 nm. The injection volume was 2 pi

The effluent from the analytical column was reduced using a "T" type splitter before being introduced into the mass spectrometer (split ratio 1:3), a micrOTOF[TM] (Bruker Corporation, Bremen, Germany). This was equipped with an electrospray ionization (ESI) interface operating in positive ionization mode using a capillary voltage of -4.5 kV. The other optimum values of the ESI-TOF parameters were drying gas temperature, 190 [degrees]C; drying gas flow, 7Lmin-l, and nebulizing gas pressure, 1.5 bar. The detection was carried out considering a mass range of 50-1000 m/z. During the development of the HPLC method, external instrument calibration was performed using a 74900-00-05 Cole-Parmer syringe pump (Cole-Parmer, Vernon Hills, IL, USA), which was directly connected to the interface, and with a sodium formate cluster solution (5 mM sodium hydroxide and 0.2% formic acid in water: isopropanol, 1:1, v/v) passing through. The calibration solution was injected at the beginning of each run and all the spectra were calibrated prior to the compound characterization. By using this method, an exact calibration curve based on numerous cluster masses each differing by 68 Da (NaCOOH) was obtained. Furthermore, to perform tandem MS experiments, the LC system was coupled to a 6540 Agilent Ultra-High-Definition Accurate-Mass quadrupole-time-of-flight mass spectrometer, which was equipped with an Agilent Dual Jet Stream ESI interface (Agilent technologies, Palo Alto, CA, USA).

The data processing was performed with DataAnalysis 4.0 software (Bruker Corporation, Bremen, Germany). The SmartFormula[TM] editor was used to provide a list of possible elemental formulae based on a CHNO algorithm, the deviation between the measured mass and theoretical mass (error, expressed as ppm) and the comparison of the theoretical with the measured isotope patterns (movalue) for increasing the confidence in the suggested molecular formula. The found compounds--chromatogram algorithm (version 2.1) was used to easily find the most abundant alkaloids, with an area and intensity thresholds of 5%. All alkaloids were characterized by the generation of their candidate formulae with a mass accuracy limit of 5 ppm, and also considering the mo value. In this case the lower the better. The retention time (RT), UV-Vis and MS/MS spectra were also taking into account together information about Papaveraceae alkaloids. For that, following literature and chemical structure databases were consulted: Reaxys (https: //www.elsevier.com/solutions/reaxys), SciFinder (https://scifinder.cas.org/), KNApSAcK Core System (http://kanaya.naist.jp/knapsack_jsp/top.html) and MassBank (http://www.massbank.jp/).

For quantification, protopine hydrochloride stock solution was conveniently diluted with methanol to prepare calibration points (0.02-100 [micro]g/ml). The external standard method was used and validated according to according to the guidelines of the European Medicines Agency (CPMP/ICH/381/95--ICH Q2 (R1)). A linear regression for the calibration curve was estimated using the area under the curve of the extracted ion chromatogram (EIC) of protopine (at m/z 354.1336 [+ or -] 0.1) against concentration and after log-log transformation: y = 0.834x + 3.159. In this sense, we used the EIC mode since it provides high selectivity when there are overlapping peaks, where spectrophotometric detection is limited. The value of the coefficient of determination was 0.997, the regression coefficients were significantly different from zero (P<0.05) and the regression was significant (P<0.01). Finally, the alkaloid extract (5 mg) was dissolved in methanol (1 ml) of MS quality, agitated, sonicated and passed through a 0.20 [micro]m syringe filter of polytetrafluoroethylene (13 mm) (ThermoFisher, Waltham, MA, USA). All analyses were done in triplicate.

In vivo experiments: effects of AFC on DNBS-induced experimental colitis

This study was carried out in accordance with the 'Guide for the Care and Use of Laboratory Animals' as promulgated by the National Institute of Health, and the protocols approved by the Ethic Committee of Laboratory Animals of the University of Granada (Spain) (Ref. No. CEEA-2010-286). Male CD1 mice, weighing 25-30 g, obtained from the Laboratory Animal Service of the University of Granada (Granada, Spain), were housed in makrolon cages, maintained in an air-conditioned atmosphere with a 12 h light-dark cycle, and provided with free access to tap water and food. Mice were assigned to different experimental groups (n = 6): a non-colitic group and five DNBS colitic groups, including an untreated group, three that received AFC treatment (25, 50, or 100 mg/kg), and the remaining administered with dexamethasone (2.4 mg/kg); all treatments were dissolved in 0.2 ml of phosphate buffer saline (PBS). Colitis was induced by intracolonic instillation of DNBS as previously reported (Cannarile et al., 2009). Briefly, mice were anesthetized with ketamine (Ketamidor[R], Richter Pharma AG, Weis, Germany) (100 mg/kg) and Xylazine (Xilagesic[R] 2%, Calier, Barcelona, Spain) (7.5 mg/kg), and DNBS (3 mg/mouse dissolved in 0.1 ml ethanol/water 50% v/v) was instilled in the colon inserting a polyethylene catheter in the rectum, and maintained in a downright position until recovery from anaesthesia. Mice from the non-colitic group (n = 6) received 0.1 ml PBS instead of the DNBS solution. The treatment with the different doses of AFC or dexamethasone started the same day of colitis induction, to avoid their possible preventive effect, and continued for five days until the sacrifice of the mice by cervical dislocation. Animal body weights, occurrence of diarrhoea, and water and food intake were recorded daily throughout the experiments. Once the animals were sacrificed, the colon was removed aseptically and rinsed with ice-cold saline. Afterwards, the colonic segment was weighed and its length measured under a constant load (2 g). Representative whole gut specimens were taken from a region of the inflamed colon corresponding to the adjacent segment to the gross macroscopic damage and were fixed in 4% buffered formaldehyde for the histological studies. Equivalent colonic segments were also obtained from the non-colitic group. The remaining colon samples were subsequently minced for RNA isolation.

Histological studies

Cross-sections were selected and embedded in paraffin, and subsequently 5 [micro]m full-thickness sections were taken at different levels, which were stained with haematoxylin and eosin. The histological damage was evaluated according to the criteria previously described (Arribas et al., 2010) by an independent pathologist unaware of the experimental groups assignment.

In vitro studies: effects of AFC on intestinal epithelial CMT93 cell activity

The intestinal epithelial cell line CMT93 was obtained from the Cell Culture Unit of the University of Granada (Granada, Spain). The cells were cultured in Dulbecco's Modified Eagle Medium, supplemented with 10% FBS and 2mM L-glutamine, in a humidified 5% C02 atmosphere at 37[degrees]C. CMT93 cells were treated with different concentrations of AFC (1, 10, and 100 [micro]g/ml) or dexamethasone (10 [micro]m) for 24 h an then stimulated with lipopolysaccharide (LPS) from Escherichia coli 055: B5 (10 [micro]g/ml). After 24 h, the culture media was collected and the levels of IL-6 and TNF[alpha] were determined using commercial available ELISA kits (R&D Systems, Abingdon, UK) according to the manufacturer's instructions.

A similar protocol was followed in order to evaluate the effect of AFC and dexamethasone on gene expression of different mediators of inflammation and intestinal epithelial barrier function. However, the cells were collected 2 h after LPS addition to proceed to the RNA extraction and the subsequent analysis of the mRNA expression of different genes.

The effects of AFC or dexamethasone on cell viability were also assessed using the CellTiter 96[R] AQueous One Solution Cell Proliferation Assay from Promega (Madison, WI, USA) according to the suggested protocol. Briefly, the cells were seeded into 96-well plates and incubated for 24 h with AFC (1, 10, and 100 [micro]g/ml) followed by the addition of 100 ng/ml LPS. 24 h later, the [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium solution was added to each well and incubated for l-4h, and the absorbance of the solution was measured at 490 nm. The cellular viability was determined from the absorbance value and compared with that of the untreated control cells.

Analysis of gene expression by RT-PCR

Total RNA (from CMT93 cells and mouse colon) was extracted using TRI Reagent[R], following the manufacturer's instructions. All RNA samples were quantified with the Thermo Scientific NanoDrop 2000 Spectrophotometer (ThermoFisher, Waltham, MA, USA), and 2 [micro]g of RNA was reverse transcribed using oligo (dT) primers (Promega, Southampton, UK). Real time quantitative PCR amplification and detection was performed on optical-grade 48 well plates in Eco Real time PCR System (Illumina, San Diego, CA, USA) with 20 ng of cDNA, the KAPA SYBR[R] FAST qPCR Master Mix (Kapa Biosystems, Wilmington, MA, USA) and specific primers at their annealing temperature (Table 1). To normalize mRNA expression, the expression of the housekeeping gene glyceraldehydes 3phosphate dehydrogenase (GAPDH) was measured for comparative reference. The mRNA relative quantitation was calculated using the [DELTA][DELTA]Ct method.

Statistical analysis

All data were expressed as mean [+ or -] 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) with a statistical significance set at P < 0.05.

Results

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), met value, characteristic UV absorption maxima, and main MS/MS fragments. Therefore, the characterization of the alkaloids was based on the UV-Vis spectra, the generation of the molecular formulae thanks to accurate mass measurements and the comparison of MS/MS fragmentation pattern with those of literature and the information retrieved from chemical databases. In this way, a total of 23 isoquinoline were identified in our tentative to characterize the total alkaloid extract (Fig. 1 and Table 2). They belonged to the following types of alkaloids: morphinandienone (1), spirobenzylisoquinoline (2, 8, 9, and 19), benzylisoquinoline (3, 4, and 7), aporphine (6 and 10), protoberberine (11-12, 14-16, 20, 21-23), and protopine (17 and 18). Interestingly, although most of these alkaloids were previously described in F. capreolata, the analytical methodology applied here enables us to also find other minor alkaloids that were reported in other Fumarioideae spp. (e.g., compounds 3, 9, 12, 13, 16, and 23, consult KNApSAcK and Reaxys databases).

Parfumine (8), isoboldine (10), coreximine (11), cheilanthifoline (14), dehydrocheilanthifoline (15), protopine (18), fumariline (19), stylopine (20), coptisine (21), and corysamine (22) were the most abundant based on MS information (Table 2). Since alkaloids are related to the biological activity of Fumaria spp. (Chlebek et al., 2016; Rathi et al., 2008), the latter ones were chosen as analytical markers in order to standardize the present extract and relatively quantify them with respect to protopine (Table 3). The amount of these alkaloids was slightly higher or in the range of those found in other Fumaria extracts (SouSek et al., 1999; Suau et al., 2002b; Rathi et al., 2008).

Effects of AFC administration on DNBS-induced colitis in mice

DNBS-induced colitis in control mice was characterized by body weight loss after the colonic insult, which reached up to 15% after 5 days (Fig. 2). AFC treatment of colitic mice ameliorated the body weight loss, which was statistically improved from day 2 at the doses of 50 and 100 mg/kg in comparison with the untreated control group. After mice sacrifice, colonic inflammation was evidenced since the weight/length ratio was 2.4 times greater in the control colitic group when compared to the non-colitic group (P < 0.01; Fig. 2). However, the administration of the different doses of AFC decreased two times this ratio, thus revealing the intestinal anti-inflammatory properties of the AFC, although no differences were observed among the three doses assayed (Fig. 2). The administration of dexamethasone also had a positive impact on colonic inflammation since a significant reduction in the weight/length ratio (32%) was observed in comparison with untreated colitic mice (P < 0.05 vs. colitic group), although it was significantly higher than that observed in non-colitic mice (P < 0.05).

The intestinal anti-inflammatory effect of AFC was confirmed after the histological assessment of the colonic samples, since the treatment promoted the recovery of the colonic histology. This was evidenced by a significant reduction in the microscopic scores in comparison with the untreated DNBS colitic control group (Fig. 3). In this group, the inflammatory process was characterized by epithelial ulceration that affected almost 50% of the surface in most of the animals. The colon showed areas of massive infiltration of granulocyte cells, predominately neutrophils, and mononuclear cells in the lamina propria. The presence of oedema was also evidenced in the majority of the specimens. Consequently, this group was assigned a microscopic score expressed as median (range) of 12.5 (11.0-15.0). The treatment with AFC attenuated the impact of the DNBS-induced colonic damage, and an improvement of the intestinal tissue compared with the control group was observed, being the reduction of the microscopic score statistically significant when compared with the control colitic group at all the doses assayed, showing values of 7 (6-8) (25 mg/kg), 7 (5-8) (50mg/kg) and 5 (3-7) (100 mg/kg) (P < 0.05 vs. colitic control) (Fig. 2). Thus, this recovery of the mucosa was apparent, and the ulceration affected less than 25% of the mucosal surface in all samples, showing a complete restoration in most of the cases. Although the leukocyte inflammatory infiltrate also occurred, this was considered as slight in most of the samples, being neutrophils, again, the predominant cell type (Fig. 3). Of note, the administration of dexamethasone to colitic mice showed a similar effect on colonic histology to that observed in AFC-treated mice (Fig. 3).

The intestinal inflammatory process induced by DNBS in mice was also characterized by an altered expression of the different colonic markers evaluated. Thus, a significant increase in the expression of different pro-inflammatory cytokines, including IL-1[beta] (5-fold), TNF[alpha] (45-fold), IL-6 (4.5-fold), IL-12 (40-fold) and IL-17 (16.5-fold), was obtained in comparison with the non-colitic group (P < 0.05 vs. colitic control), derived from the deregulated immune response induced by DNBS. The AFC treatment was associated with a significant reduction in the expression of all these cytokines (P < 0.05 vs. colitic control), showing the highest efficacy at doses of 50 and 100mg/kg, which were able to produce up to 14-, 27-, 3-, 9- and 8-fold decrement of the expression of these mediators, respectively (Fig. 4). Also, the up-regulated expressions of the two enzymes iNOS (5-fold) and MMP-9 (4-fold), as well as the adhesion molecule ICAM-1 (19-fold), were observed in control colitic mice (Fig. 5). Similarly to what occurs with the pro-inflammatory cytokines, the treatment with the different doses of AFC significantly reduced the expression of all these proteins involved in the inflammatory process by up to 8, 5 and 12 folds, respectively (P < 0.05 vs. colitic control) (Fig. 5). When MUC-2 was evaluated, colitic mice from control group showed a 50% reduction in its expression in comparison with normal mice (P < 0.05), thus revealing an impairment of the epithelial barrier function due to the colonic inflammatory process induced by DNBS (Fig. 5). The treatment with the different doses of AFC restored the colonic expression of this mucin to similar values to those obtained in the non-colitic mice (Fig. 5). Similarly, most of these markers were significantly ameliorated after treatment with dexamethasone, except when the expression of the mucin MUC-2 was considered, in which the glucocorticoid did not show any significant effect when compared with control colitic mice without treatment (Figs. 4 and 5).

Effects of AFC on CMT93 cell activity

The incubation of the epithelial cell line CMT93 with different concentrations of AFC for 24 h revealed that the survival rate was greater than 70% at all concentrations assayed, and only the highest one, 100 [micro]g/ml, induced a significant cell toxicity. In these assays, the incorporation of LPS to the culture media resulted in a significant reduction of the cell survival rate, which was not significantly modified after the incorporation of the different concentrations of AFC to the culture media in these experimental conditions (Fig. 6). No significant effect was observed on cell viability after incorporation of dexamethasone in the cell culture, either in absence or in presence of LPS (Fig. 6).

The beneficial effects exerted by AFC on immune response in colitic mice were supported by the in vitro assays on CMT93 cells. Cell stimulation with LPS resulted in an increased production and release of the pro-inflammatory cytokines TNF[alpha] and IL-6 (Fig. 7). The cytokine levels were significantly diminished by AFC in a concentration dependent manner, ranging from 93% (1 [micro]g/ml) to 100% (100 [micro]g/ml) inhibition of TNF[alpha] and from 34% to 97% inhibition of IL-6, respectively (Fig. 7). The inhibitory effects observed at the different concentrations of AFC on cytokine production could be correlated to the reduced mRNA expressions of both cytokines when evaluated by RT-qPCR in LPS-stimulated CMT93 cells, which was 77-96% for TNF[alpha] and 70-80% for IL-6 (P < 0.05 vs. LPS) (Fig. 7). When dexamethasone was assayed, a clear inhibition on TNF[alpha] production (54%) and expression (70%) was observed (P < 0.05 vs. LPS); however, although the glucocorticoid was able to significantly reduce the production of IL-6 (54%) (P < 0.05 vs. LPS), no significant decrease was obtained when the expression of this cytokine was considered (Fig. 7). Similarly, AFC and dexamethasone strongly suppressed ICAM-1 mRNA expression in these intestinal epithelial cells after LPS stimulation (more than 90% and 85%, respectively) (P < 0.05 vs. LPS) (Fig. 8). Furthermore, the expression of two proteins involved in epithelial integrity, MUC-2 and ZO-1, was reduced (83% and 71%, correspondingly P < 0.05 vs. control) after cell stimulation with LPS. The incorporation of AFC to the cell culture media had a positive effect since it was able to significantly increase this reduced expression of these proteins induced in CMT93 cells after incubation with LPS, similarly to that observed when dexamethasone was assayed. However, and surprisingly, the lowest doses (1 and 10 [micro]g/ml) were the most active in restoring these altered expressions and bringing them back to basal levels (Fig. 8).

Discussion

At present, 1BD 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 compounds for preventing or treating IBD. In particular, phytochemicals can be considered as naturally derived alternative therapy for IBD, with a promising future since they can combine efficacy and safety (Hur et al., 2012). Furthermore, the extracts obtained from medicinal plants possess a combination of constituents, which may involve different actions simultaneously, thus sometimes offering synergic beneficial effects (Dharmasiri et al., 2003; Gilani and Rahman, 2005). However, and before studying their beneficial effects in humans, it is necessary to evaluate the efficacy and safety of these plant-derived extracts in experimental models to confirm their potential use in these intestinal conditions.

The results obtained in the present study reveal that an alkaloid extract of F. capreolata (AFC) containing 1.3% stylopine and 0.9% protopine, used as analytical markers, possess anti-inflammatory effect in the DNBS experimental model of mouse colitis, a well characterized model of colonic transmural inflammation similarly to what occurs in human Crohn's disease (Martin et al., 2014). The beneficial effect showed by AFC was already evidenced in the course of the experiment by a reduced weight loss in those colitic mice treated with different doses of the extract, thus revealing an improvement in the health status of the animals, which is clearly compromised in experimental colitis. Also, AFC administration to colitic mice significantly reduced colonic weight/length ratio, thus ameliorating the typical tissue oedema. This anti-inflammatory effect was clearly associated with an improvement of the altered immune response associated with the DNBS-induced colonic damage. In the intestine, the activity of the different immune cells is finely regulated by several mechanisms, and the development of IBD is associated with an impairment of the immune function (Jurjus et al., 2004; Kayama and Takeda, 2012). Following acute mucosal inflammation, the production of pro-inflammatory cytokines, like IL-1[beta], TNF[alpha] and IL-6, are drastically enhanced due to the activation of different cells involved in the innate immune response of the intestine, including epithelial cells, macrophages or dendritic cells. The subsequent sustained inflammatory response is associated with the increased expression of cytokines like IL-12 and IL-17, which is indicative of the imbalance in Th1 and Th17 responses (Neurath, 2014). IL-17 has been described to play a key role in the development of chronic intestinal inflammation, since it contributes to neutrophil migration and their subsequent activation, enhances dendritic cell maturation, T cell priming and the production of inflammatory mediators from different cell types. IL-17 also leads to the induction of many pro-inflammatory factors, including TNF[alpha], IL-6, and IL-1[beta], suggesting an important role for IL-17 in localizing and amplifying inflammation, thus being essential for the maintenance of the inflammatory response in the intestine. These cytokines appear to be interlinked in a cascade, being produced serially by cells during an inflammatory response (Algieri et al., 2013; Sanchez-Munoz et al., 2008). The treatment of colitic mice with the different doses of AFC resulted in an amelioration of most of the colonic pro-inflammatory cytokines evaluated, thus revealing the contribution of the immunomodulatory properties of the different alkaloids present in the extract to the intestinal anti- inflammatory effect observed.

AFC composition has been properly characterized by LCDAD-MS and -MS/MS and revealed the presence of a mixture of isoquinoline alkaloids, already reported as the major secondary metabolites in F. capreolata (Suau et al., 2002a; Maiza-Benabdesselam et al., 2007). Two components were identified, stylopine and protopine, which have been described to exert immunomodulatory properties that could justify the intestinal anti-inflammatory properties evidenced by this alkaloid fraction in the present study. In this sense, in vitro studies have shown that stylopine was able to suppress the major macrophage-derived inflammatory mediators IL-1[beta], TNF[alpha] and IL-6, and to interfere with inducible enzyme activities like COX-2 and iNOS Qang et al., 2004). Similarly, protopine was found to reduce NO production, as well as to inhibit COX-2 activity and prostaglandin [E.sub.2] production, by blocking phosphorylation of mitogen-activated protein kinases and activation of NF-[kappa]B (Bae et al., 2012; Park et al., 2011).

Of note, it was reported previously that this alkaloid fraction showed a very low toxicity when administered orally to mice or when assayed in vitro on erythrocytes (Bribi et al., 2013). The in vitro experiments performed in the present study with the intestinal epithelial cell line CMT93 have confirmed these observations. AFC did not significantly affect cell viability when compared to the corresponding controls after LPS stimulation. However, AFC affected this cell activity when stimulated with LPS, since the alkaloid mixture reduced the mRNA expression and production of the pro-inflammatory cytokines TNF[alpha] and IL-6, thus confirming the results obtained in the in vivo experiments, in which an modulation of the altered immune response contributes to the beneficial effects observed in the experimental model of colitis.

The impact of AFC on the epithelial cell activity, together with the above commented effects of their main alkaloids on macrophages, can be of special importance in the intestinal anti-inflammatory effect observed in the in vivo experiments. In fact, a defect in the intestinal barrier function, associated with an aberrant response from epithelial cells, has been considered to play a key role in the pathogenesis of IBD (Salim and Soderholm, 2011). Thus, it has been proposed that, in these intestinal conditions, commensal bacteria abnormally activates pattern recognition receptors in epithelial cells thereby triggering the transcription factor NF-[kappa]B signalling, which leads to increased levels of inflammatory factors, such as TNF[alpha] and IL-6 (Chen et al., 2014). These cytokines promote the disruption of the epithelial integrity, thus facilitating the antigen access to the submucosa, and the activation of the resident immune cells, like macrophages. Also, the cytokines enhance leucocyte migration and infiltration to the inflamed tissue, subsequently promoting both Th1 and Th17 responses, which collaborate to maintain the inflammatory response (Soderholm et al., 2004). It is well reported that the adhesion molecule ICAM-1 that is expressed on leucocytes, dendritic cells, fibroblasts, epithelial cells and endothelial cells may have a prominent role in the intestinal inflammatory process, which may be considered as an interesting target in IBD therapy (Lobaton et al., 2014). In fact, AFC was able to reduce ICAM-1 expression in LPS-stimulated CMT93 cells, as well as in colonic tissue from DNBS-colitic mice, which can account for the intestinal anti-inflammatory effects showed by this alkaloid mixture. Furthermore, the inhibitory effect of AFC on the production and release of these pro-inflammatory cytokines in CMT93 cells was associated with the increased expression of both MUC-2 and ZO-1, which may collaborate in the preservation of the barrier function that is altered in inflammatory conditions. The in vivo studies support this, since the epithelial restoration was confirmed by the histological studies, in accordance with the increased colonic expression of the mucin MUC-2, therefore restoring the colonic mucus layer that also contributes to keep the epithelial integrity (Kim and Ho, 2010). Moreover, the beneficial effects of the AFC treatment were related to a down-regulation of the matrix metalloproteinase MMP-9 and the inducible enzyme iNOS, which are mainly synthesized by inflammatory cells, particularly T cells, macrophages and polymorphonuclear leucocytes, being considered all of them key pathogenic players in intestinal inflammation (Camuesco et al., 2004). MMPs are proteases that participate in the different phases of the inflammatory process, including cell migration and infiltration, cytokine activation, tissue damage, remodelling and repair (Parks et al., 2004). Therefore, they have been also involved in the pathogenesis of IBD, and an inhibition of MMP activation has been shown to improve experimental colitis (O'Sullivan et al., 2015). The increased activity of MMPs in intestinal inflammation accelerates the degradation of connective tissue, thus promoting epithelial damage and subsequent increased mucosal permeability. Consequently, the down-regulation of the expression of these enzymes could be helpful for the recovery of the epithelial integrity and the restoration of the altered immune response, as observed in the present study. Similarly, the reduction of the colonic iNOS expression induced by AFC in colitic mice can also contribute to their beneficial effects, since this could result in the inhibition of the secretion of inflammatory mediators such as nitrogen reactive species produced after induction of iNOS expression. In this way, the reaction between NO and superoxide anions is inhibited, and the subsequent production of peroxynitrite, a potent oxidizing agent that can prompt changes in the structure and function of proteins (Virag et al., 2003) is avoided, thus facilitating the recovery of the damaged colonic tissue.

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 proinflammatory mediators, as well as to improve epithelial barrier integrity. This observation was supported by the results obtained in vitro in LPS-stimulated CMT93 cells, where AFC also downregulated some inflammatory mediators, such as, TNF[alpha], IL-6, IL-17, and (IL)-1[beta], and up-regulated the expression of proteins involved in the maintenance of the intestinal barrier function, like the mucin MUC-2 and ZO-1. Considering all the above, the total alkaloid fraction of F. capreolata could have a potential use in the management of IBD due to its anti-inflammatory activity.

http://dx.doi.org/10.1016/j.phymed.2016.05.003

ARTICLE INFO

Article history:

Received 26 November 2015

Revised 4 May 2016

Accepted 10 May 2016

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 Andalucia (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 C1BER-EHD; M. d. M. Contreras is a postdoctoral fellow of the Excellence Project P11-CTS-7625. The CIBER-EHD is funded by the Instituto de Salud Carlos III. The authors would like also to thank the Ministry of Higher Education and Scientific Research, Algeria, for its support of the stay of Noureddine Bribi in Spain.

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Noureddine Bribi (a,b), Francesca Algieri (b), Alba Rodriguez-Nogales (b), Teresa Vezza (b), Jose Garrido-Mesa (b), Maria Pilar Utrilla (b), Maria del Mar Contreras (c,d), Fadila Maiza (a), Antonio Segura-Carretero (c,d), Maria Elena Rodriguez-Cabezas (b,1), Julio Galvez (b,1),*

(a) Laboratoire de Biotechnologies Vegetaies et Ethnobotanique, Faculte des Sciences de la Nature et de la Vie, Universite de Bejaia, 06000 Bejaia, Algeria

(b) CIBER-EHD, Department of Pharmacology, ibs.GRANADA, Center for Biomedical Research (CIBM), University of Granada, Avenida del Conocimiento s/n 18016-Armilla, Granada, Spain

(c) Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avenida Fuentenueva s/n, 18071- Granada, Spain

(d) Research and Development Centre for Functional Food (CIDAF), Health-Science Technological Park, Avenida del Conocimiento 37, 18016-Granada, Spain

Abbreviations: AFC, total alkaloid fraction extracted from the aerial parts of Fumaria capreolata: ANOVA, one-way analysis of variance; BPC, base peak chromatogram; DAD, diode array detection; DNBS, dinitrobenzenesulphonic acid; EIC, extracted ion chromatogram; ESI, electrospray ionization; GAPDH, 3-phosphate dehydrogenase; 1BD, inflammatory bowel disease; ICAM, intercellular adhesion molecule; IFN-[gamma], interferon-[gamma]; IL, interleukin; iNOS, inducible nitric oxide synthase; LC, liquid chromatography; LPS, lipopolysaccharide; MMP, metalloproteinase; MS, mass spectrometry; RT, retention time; TGF, transforming growth factor; TNF[alpha], tumour necrosis factor [alpha]: TOF, time-of-flight; ZO, zonula occludens.

* Corresponding author. Fax: +34958248964.

E-mail address: jgalvez@ugr.es (J. Galvez).

(1) Both authors contributed equally to the supervision of the study.

Table 1
Primer sequences used in real-time PCR assays in CMT93 cells and
colonic tissue.

Gene         Sequence (5'-3')                Annealing T ([degrees]C)

IL-6         FW -CTTCCCTACTTCACAACTC         60
             RV -CTCCATTAGGAGAGCATTG
IL-12        FW -CTGGTGCAAAGAAACATGGA        60
             RV -TGGTTTGATGATGTTCCTGA
IL-17        FW -CCTGGGTGAGCCGACAGAAGC       60
             RV -CCACTCCTGGAACCTAAGCAC
IL-1[beta]   FW -TGATGAGAATGACCTCTTCT        55
             RV -CTtCrrCAAAGATGAACGAAA
TNF[alpha]   FW -AACTAGTGGTGCCAGCCGAT        56
             RV -CTTCACAGAGCAATGACTCC
iNOS         FW -GTTGAAGACTGAGACTCTGG        56
             RV -GACrAGGCTACTCCGTCGA
ICAM-1       FW -GAGGAGGTGAATGTATAAGTTATG    60
             RV -GGATGTGGAGGAGCAGAG
MMP-9        FW -TGGGGGGCAACTCGGC            60
             RV -GGAATGATCTAAGCCCAC
MUC-2        FW -GCAGTCCTCAGTGGCACCTC        60
             RV -CACCGTGGGGCTACTGCAGAG
ZO-1         FW -GGGGCCTACACTGATCAAGA        56
             RV -TGGAGATGAGGCTTCTGCTT
GADPH        FW -CCATCACCATCTTCCAGGAG        60
             RV -CCTGCTTCACCACCTTCTTG

Table 2
Alkaloids characterized in the total alkaloid fraction from Fumaria
capreolata (AFC) using LC/DAD/MS and -MS/MS.

No    RT       Molecular           Experimental      Error   m[sigma]
      (min)    formula             m/z               (ppm)
                                   ([[M+H].sup.+]/
                                   [M.sup.+])

1     12.16    C19H21N04           328.1548          -1.5    6.0

2     12.83    C20H21NO5           356.1502          1       8.2

3     13.88    C17H19N03           286.1435          0.8     96.1

4     14.05    C18H21N03           300.1602          -2.6    11.1

5     14.26    C19H21N04/          328.1546          -0.7    18.2
               [C19H22N04.sup.+]

6     14.75    [C20H24NO4.sup.+]   342.1699          0.4     63

7     15.49    C19H23N04           330.1701          -0.2    1.3

8     15.57    C20H19NO5           354.1350          -0.2    4.5

9     15.89    C21H21N05           368.1486          1.9     30.9

10    16.02    C19H21N04           328.1543          0       3.4

11    16.32    C19H21N04           328.1548          -1.4    2.1

12    16.76    C20H23NO4           342.1698          0.6     8.5

13    17.17    C20H23NO4           342.1697          0.8     20.2

14    17.17    C19H19N04           326.1395          -2.4    1.4

15    18.38    [C19H16N04.sup.+]   322.1078          -1.2    5.3

16    18.88    [C19H18N04.sup.+]   324.1235          -1.4    26.8

17    19.05    C21H23N05           370.1641          2.1     15.9

18    19.63    C20H19NO5           354.1351          -4.3    17.6

19    20       C20H17NO5           352.1188          1.5     42.7

20    20.7     C19H17N04           324.1243          -3.8    14

21    20.9     [C19H14N04.sup.+]   320.0931          -4.4    13.6

22    22.11    [C20H16NO4.sup.+]   334.1085          -3.2    5.2

23    23.91    C22H21N06           396.1428          33      4.2

No    UV max.         Main MS/MS fragments
      (nm)

1     282             297.1051, 265.0834, 251.0635, 243.0954,
                      237.0846, 211.0704, 192.0962

2     283             338.1313, 277.0788, 249.0847, 137.0541

3     286             269.1131, 237.0864, 209.0921, 175.0723,
                      145.0631, 143.0457, 107.0460

4     282             269.1105, 237.0846, 209.0902, 177.0489,
                      175.0692, 145.0232, 107.0445

5     ND              297.1074, 282.0847, 265.0817, 237.0868,
                      209.0923, 191.0823

6     268, 305        297.1081, 282.0853, 265.0828, 237.0915,
                      191.0832

7     281             192.0990, 175.0719, 143.0460, 137.0564

8     271, 372        336.1191, 323.0927, 305.0774, 295.0930,
                      279.0611, 179.0912, 137.0567

9     290             323.0859, 305.0756, 293.0754,
                      261.0502, 193.1053, 137.0556

10    279, 304        297.1125, 282.0840, 265.0826, 237.0877,
                      233.0559, 205.0610

11    282             313.1226, 178.0803, 151.0693, 119.0436,
                      91.0493

12    282             327.1431, 192.0989, 177.0754, 151.0728,
                      137.0573

13    280             192.0976, 177.0743, 151.0712

14    285             311.1157, 192.0988, 178.0829, 151.0721,
                      119.0461, 91.0517

15    267, 355, 458   307.0755, 294.1039, 279.0808,
                      264.0663, 250.0778, 222.0839

16    276, 355        309.1003, 294.0765, 266.0814, 210.0910

17    283             291.1008. 222.1115, 204.1006, 190.0853,
                      165.0898, 165.0536, 149.0582

18    288             206.0780. 189.0755. 188.0678, 165.0518,
                      149.0603

19    270, 372        334.1026, 309.0703, 279.0601, 263.0653,
                      177.0740, 135.0404

20    288             176.0678, 149.0563, 119.0459, 91.0515

21    265, 357, 459   305.0668, 292.0912. 277.0725,
                      262.0855, 249.0779. 234.0905

22    268, 345, 449   320.0909, 306.1083, 291.0843, 276.0974,
                      261.0741, 248.1019

23    288             322.1019, 176.0671, 149.0557, 119.0452,
                      91.0507

No    Proposed alkaloid         Previous report in F.   Relative
                                capreolata              %

1     Pallidine                 Tanahashi and Zenk,     0.25
                                  1985

2     Fumaritine                SouSek et ai. 1999      0.29

3     Codaurine                                         0.06

4     N-Methylcoclaurine        Tanahashi and Zenk,     0.25
                                  1985

5     Not identified                                    0.07

6     Magnoflorine              Tanahashi and Zenk,     0.59
                                  1985

7     Reticuline                Tanahashi and Zenk,     0.60
                                  1985

8     Parfumine                 Suau et al" 2002a,b     6.94

9     Parfumidine                                       0.27

10    Isoboldine                Tanahashi and Zenk,     9.79
                                  1985

11    Coreximine                Tanahashi and Zenk,     5.31
                                  1985

12    Methylcoreximine 1                                0.63

13    Methylcoreximine 2                                0.26

14    Cheilanthifoline          Forgacs et al. 1986     6.61

15    Dehydrocheilanthifoline   Tanahashi and Zenk,     2.01
                                  1985

16    Demethyleneberberine/                             0.50
      jatrorubine

17    Cryptopine                Sousek et al. 1999      0.19

18    Protopine                 Suau et al. 2002b;      16.99
                                  Sousek et al. 1999

19    Fumariline                Suau et al., 2002a,b;   3.18
                                  Sousek et al. 1999

20    Stylopine                 Suau et al., 2002a,b;   22.67
                                  Tanahashi and Zenk,
                                  1985

21    Coptisine                 Tanahashi and Zenk,     19.78
                                  1985; Sousek et al.
                                  1999

22    Corysamine                                        2.46

23    Impatien Cl                                       030

Table 3
Main alkaloids content in the total alkaloid
fraction from Fumaria capreolata extract (AFC).

Alkaloid                     Content (mg/g)

Parfumine                  3.05 [+ or -] 0.25
Isoboldine                 4.46 [+ or -] 0.07
Coreximine                 2.26 [+ or -] 0.11
Cheilanthifoline           2.93 [+ or -] 0.21
Dehydrocheilanthifoline    0.68 [+ or -] 0.02
Protopine                  8.95 [+ or -] 0.64
Fumariline                 1.16 [+ or -] 0.03
Stylopine                 12.61 [+ or -] 0.12
Coptisine                 10.90 [+ or -] 0.27
Corysamine                 0.86 [+ or -] 0.03


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Title Annotation:Original Article
Author:Bribi, Noureddine; Algieri, Francesca; Rodriguez-Nogales, Alba; Vezza, Teresa; Garrido-Mesa, Jose; U
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Date:Aug 15, 2016
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