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This study investigates the gut anti-inflammatory activity of a plant sterol (PS) food supplement (PS-FS), alongside PS-enriched milk-based fruit beverage and PS-enriched rye bread. A co-culture model based on a dual-chamber system with differentiated intestinal-like Caco-2 cells (apical) and RAW264.7 macrophages (basolateral) was used. The bioaccessible fractions (BF) of the samples were obtained after INFOGEST 2.0 simulated gastrointestinal digestion. The BF were added to the apical part (diluted 1/20 v/v with culture medium to avoid cytotoxicity) for 90 min, followed by stimulation with lipopolysaccharide (LPS) (1 μg mL -1 , 24 h) on the basolateral side. The pharmacological interaction between samples and budesonide (1 μM, 90 min) was evaluated. Results indicate that PS-FS significantly attenuated LPS-induced secretion of IL-8 (28%) by Caco-2 cells, and TNF-α (9%) and IL-6 (54%) by RAW264.7 macrophages, whereas PS-enriched beverage and bread did not exhibit protective effects. Additionally, PS-FS demonstrated an improvement in oxidative status in Caco-2 cells, evidenced by reduced levels of reactive oxygen species (47%), iNOS protein expression (27%), and nitrite/nitrate secretion (27%). Mechanistically, PS-FS inhibited the NF-κB-COX-2-PGE 2 signaling pathway in macrophages, resulting in decreased NF-κB p65 nuclear translocation (39%), COX-2 protein expression (32%), and PGE 2 production (27%). Co-treatment with budesonide and PS-FS displayed an antagonistic effect (combination index 0.38-0.63). This study demonstrates the potent intestinal anti-inflammatory activity of a PS-FS, positioning it as a promising nutraceutical product for the management of inflammatory bowel diseases. However, the food matrix of the milk-based fruit beverage and rye bread appear to interfere with the anti-inflammatory activity of PS.

Sterols can be metabolized by gut microbiota. The cholesterol metabolites have been proposed as promoters of colorectal cancer (CRC), while the effect of plant sterol metabolites is unknown. This study aimed to evaluate the cytotoxicity of metabolites from cholesterol (coprostanol, cholestanol, coprostanone and cholestenone) and β-sitosterol (ethylcoprostanol) on human colon tumor (Caco-2) and non-tumor (CCD-18Co) cells at physiological concentrations (9-300 μM) and exposure time (24 h). Ethylcoprostanol reduced the tumor cell proliferation (MTT), showing in flow cytometry assays induction of apoptosis via production of reactive oxygen species (ROS) and ceramide. Transcriptomic analysis (qPCR) showed activation of the intrinsic apoptosis pathway ( BAX / BCL2 ratio and CASP9 increased), accompanied by downregulation of the p21 gene. Cholesterol metabolites, mainly the most hydrophobic, induced apoptosis and G 0 /G 1 phase arrest in non-tumor cells through overproduction of ROS. Both the intrinsic and extrinsic ( CASP8 increased) apoptosis pathways occurred. In turn, a reduction in the expression of the cyclin E 1 gene confirmed the cell cycle arrest. In addition, ethylcoprostanol protected non-tumor cells from the most cytotoxic cholesterol metabolite (cholestenone). In conclusion, ethylcoprostanol is a promising candidate as a therapeutic adjuvant in CRC, while cholesterol metabolites could act as CRC promoters through their cytotoxicity.

The prevention of cardiovascular disease using foods fortified with plant sterols (PS), with a hypocholesterolemic effect, is important for the elderly population. This study aimed at identifying the different PS present in PS-enriched wholemeal rye bread (WRB) and in the ingredient source of PS, to evaluate their bioaccessibility in WRB by simulated static digestion. The gastrointestinal conditions of the elderly were adapted, and the results were compared with the adult population. Nine PS were identified, and a total amount of 2.18 g/100 g WRB was determined. Bioaccessibility was reduced in the elderly model with gastrointestinal adaptation vs. the adult model (11.2 vs. 20.3%), but no differences were observed when adapting only the gastric phase. Even though there was lower bioaccessibility of PS in the elderly, they could benefit from the consumption of WRB as it has a good nutritional profile. Further investigation including in vivo assays is needed to strengthen the results.

Bioaccessibility of plant sterols (PS) in an enriched wholemeal rye bread was evaluated, for the first time, using the INFOGEST protocol without gastric lipase (GL) and cholesterol esterase (CE), with GL or GL + CE. Moreover, human chewing and an in vitro oral phase (simulated salivary fluid and α-amylase) were evaluated for this purpose. The addition of GL decreased the bioaccessibility of total PS (from 23.8 to 18.5%), whereas the use of GL + CE does not significantly affect PS bioaccessibility. The in vitro oral phase resulted in an ineffective homogenization of the fresh vs partially dried and milled bread, reducing the bioaccessibility of total (from 20.2 to 12.8%) and individual PS. The INFOGEST digestion including the use of GL and CE, as well as an oral phase with human chewing, is proposed for the assessment of PS bioaccessibility in a solid matrix such as wholemeal rye bread since it more closely approximates the in vivo situation.

This study evaluates the influence of increasing bile salts and the addition of key enzymes of the lipidic metabolism in the INFOGEST digestion method on sterol bioaccessibility from a plant sterol (PS)-enriched beverage. The assayed modifications were increasing concentration of bovine bile salts (10 vs. 17.5 mM), and addition of gastric lipase (GL) (60U/mL), cholesterol esterase (CE) (0.075 or 2U/mL) or both. Compared to the original method (10 mM bile salts without enzymes), the assayed conditions significantly reduced bioaccessibility of individual (from 11.3 to 19.7 to 5.1-16.6%) and total PS (13.7 to 6.9-8.0%), and cholesterol (52.8 to 20.9-26.1%), except only when CE is added not allowing cholesterol quantification. The bioaccessibility achieved when lipolytic enzymes were tested was similar for all sterols. For a more physiological approach to in vivo conditions, incorporation of bile salts (10 mM), GL (60U/mL) and CE (0.075U/mL) to the INFOGEST method is proposed, although it increases the cost compared to the established method., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Elderly people suffer from a higher cardiovascular risk. Thus, the fortification of foods with plant sterols (PSs), which have a cholesterol-lowering function, could be of great interest for this target group. To date, no studies have analyzed how the gastrointestinal conditions of the elderly affect PS bioaccessibility. Therefore, this study evaluated the impact of the adaptation of the gastric phase alone and in combination with the intestinal phase on sterol bioaccessibility. For this purpose, the standardized INFOGEST 2.0 method previously adapted for sterol bioaccessibility evaluation in healthy adults was applied to PS-enriched milk-based fruit beverages, examining changes in enzyme activity, incubation time, agitation and pH, based on elderly physiology. The results suggest that the specific gastrointestinal conditions of the elderly could increase absorption of PSs, since their bioaccessibility (%) in a PS-enriched milk-based fruit beverage was significantly increased compared with that in adults (14.95 ± 0.33 vs . 7.96 ± 0.26), also indicating that these conditions increase the bioaccessibility of the beverage's own cholesterol (61.25 ± 2.91 vs. 20.86 ± 2.79). These data support the recommendation of foods of this type for the elderly who can benefit from the increase in bioaccessibility of PSs to have an improved potential cholesterol lowering effect, thus decreasing their risk of cardiovascular disease. However, the performance of subsequent in vivo tests to confirm these results is necessary.

Plant sterols (PSs) have reported benefits in alleviating colitis in mice, but the mechanisms involved require further investigation. This study aimed to evaluate the effect of a dietary supplement containing PSs (PS-DS) on inflammation biomarkers in a mice model of chronic ulcerative colitis induced by dextran sulfate sodium (DSS). C57BL/6J mice (n = 34) were exposed to 1.5% DSS in drinking water for three 5-day periods, with 10-day rest intervals in between. The mice received daily PS-DS (35 mg PS/kg) or placebo by oral gavage, either simultaneously (treatment) or 30 days prior (pre-treatment) to DSS exposition. After euthanasia, myeloperoxidase (MPO) activity and the levels of pro- (TNF-α and IL-6) and anti-inflammatory cytokines (IL-10) in the colonic tissue were analyzed. PS-DS treatment reduced (vs. DSS + placebo) myeloperoxidase (MPO) activity (2.6 ± 0.2 vs. 2.1 ± 0.1-fold change) and levels of TNF-α (85 ± 11 vs. 39 ± 7 pg/mg protein) and IL-6 (214 ± 26 vs. 128 ± 18 pg/mg protein), increasing the levels of IL-10 (46 ± 5 vs. 136 ± 16 pg/mg protein). PS-DS pre-treatment provided a greater inhibition (vs. treatment) of MPO activity (2.1 ± 0.3 vs. 1.3 ± 0.1-fold change) and a greater increase in IL-10 levels (50 ± 9 vs. 178 ± 30 pg/mg protein). These findings suggest that PS-DS has the potential to alleviate colitis in mice by modulating the inflammatory response and reducing oxidative stress. However, studies in humans are required to validate and fully understand its anti-inflammatory effect. [ABSTRACT FROM AUTHOR]