Spray-drying Microencapsulation of an Extract from Tilia tomentosa Moench Flowers: Physicochemical Characterization and in Vitro Intestinal Activity.

Silver linden (Tilia tomentosa Moench, TtM) flowers possess several health-promoting properties, especially at the neurological level, such as intestinal relaxation activity associated with specific flavonols, particularly quercetin and kaempferol derivatives. However, such molecules are susceptible to degradation upon different triggers like heat, light and extreme pH values. To overcome the scarce stability of TtM flowers bioactive molecules and make them suitable for developing functional food and supplements, we applied microencapsulation. Spray-drying microencapsulation of TtM flowers extract was performed using three starch-derived wall materials: maltodextrin 12 DE (MD12) and 19 DE (MD19), and OSA-modified starch (OSA-S). The stability of total phenols, flavanols, and antioxidant capacity was monitored for 70 days under accelerated stress conditions (40 °C/70% RH) by HPLC and spectrophotometric methods, and the intestinal contractile activity was tested in a murine model. In comparison to MD12 and MD19, OSA-S stood out for the higher encapsulation efficiency of quercetin and kaempferol glycosides (+ 36-47% compared to MD12 and + 18-24% compared to MD19) and stability thereof (half-life on average + 30% compared to MD12 and + 51% compared to MD19). The intestinal contractile activity of OAS-S powders resulted comparable to the original extract, indicating that flavonols were biologically active and accessible. Our results underly the potential advantages of OSA-S encapsulated formulation as a functional ingredient for the development of nutraceutical products.

Small intestine neuromuscular dysfunction in a mouse model of dextran sulfate sodium-induced ileitis: Involvement of dopaminergic neurotransmission.

AIMS: Anomalies in dopaminergic machinery have been shown in inflammatory bowel disease (IBD) patients and preclinical models of IBD. Thus, we aimed to evaluate the impact of dextran sodium sulfate (DSS)-induced ileitis on enteric dopaminergic pathways. MATERIALS AND METHODS: Male C57/Bl6 mice (10 ± 2 weeks old) received 2% DSS in drinking water for 5 days and were then switched to regular drinking water for 3 days. To measure ileitis severity inflammatory cytokines (IL-1ß, TNFα, IL-6) levels were assessed. Changes in ileal muscle tension were isometrically recorded following: 1) cumulative addition of dopamine on basal tone (0.1-1000 µM); ii) 4-Hz electric field stimulation (EFS) in the presence of 30 µM dopamine with/without 10 µM SCH-23390 (dopamine D1 receptor (D1R) antagonist) or 10 µM sulpiride (D2R antagonist). Immunofluorescence distribution of the neuronal HuC/D protein, glial S100ß marker, D1R, and dopamine transporter (DAT) were determined in longitudinal-muscle-myenteric plexus whole-mounts (LMMPs) by confocal microscopy. D1R and D2R mRNA transcripts were evaluated by qRT-PCR. KEY FINDINGS: DSS caused an inflammatory process in the small intestine associated to dysmotility and altered barrier permeability, as suggested by decreased fecal output and enhanced stool water content. DSS treatment caused a significant increase of DAT and D1R myenteric immunoreactivity as well as of D1R and D2R mRNA levels, accompanied by a significant reduction of dopamine-mediated relaxation, involving primarily D1-like receptors. SIGNIFICANCE: Mouse ileitis affects enteric dopaminergic neurotransmission mainly involving D1R-mediated responses. These findings provide novel information on the participation of dopaminergic pathways in IBD-mediated neuromuscular dysfunction.

Influence of Tilia tomentosa Moench Extract on Mouse Small Intestine Neuromuscular Contractility.

Functional gastrointestinal disorders (FGIDs) are characterized by abdominal pain, bloating and bowel disturbances. FGID therapy is primarily symptomatic, including treatment with herbal remedies. Flower extract of Tilia tomentosa Moench (TtM) is occasionally used as an anti-spasmodic in popular medicine. Since its effect on intestinal response is unknown, we evaluated the influence of TtM extract on small intestine contractility. Ileal preparations from C57BL/6J mice were mounted in organ baths to assess changes in muscle tension, following addition of TtM extract (0.5-36 µg/mL) or a vehicle (ethanol). Changes in contractile response to receptor- and non-receptor-mediated stimuli were assessed in ileal preparations pretreated with 12 µg/mL TtM. Alterations in the enteric nervous system neuroglial network were analyzed by confocal immunofluorescence. Increasing addition of TtM induced a marked relaxation in ileal specimens compared to the vehicle. Pretreatment with TtM affected cholinergic and tachykininergic neuromuscular contractions as well as K+-induced smooth muscle depolarization. Following incubation with TtM, a significant reduction in non-adrenergic non-cholinergic-mediated relaxation sensitive to Nω-Nitro-L-arginine methyl ester hydrochloride (pan-nitric oxide synthase inhibitor) was found. In vitro incubation of intestinal specimens with TtM did not affect the myenteric plexus neuroglial network. Our findings show that TtM-induced intestinal relaxation is mediated by nitric oxide pathways, providing a pharmacological basis for the use of TtM in FGIDs.

Involvement of Enteric Glia in Small Intestine Neuromuscular Dysfunction of Toll-Like Receptor 4-Deficient Mice.

Enteric glial cells (EGCs) influence nitric oxide (NO)- and adenosine diphosphate (ADP)- mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4-/- mice. Ileal segments from male TLR4-/- and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4-/- ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO- and ADP- mediated relaxation in the TLR4-/- mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100ß and GFAP immunoreactivity in TLR4-/- myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4-/- mice.