Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells.

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl-/HCO3- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

Multilevel Proteomic Profiling of Colorectal Adenocarcinoma Caco-2 Cell Differentiation to Characterize an Intestinal Epithelial Model.

Emergent advancements on the role of the intestinal microbiome for human health and disease necessitate well-defined intestinal cellular models to study and rapidly assess host, microbiome, and drug interactions. Differentiated Caco-2 cell line is commonly utilized as an epithelial model for drug permeability studies and has more recently been utilized for investigating host-microbiome interactions. However, its suitability to study such interactions remains to be characterized. Here, we employed multilevel proteomics to demonstrate that both spontaneous and butyrate-induced Caco-2 differentiations displayed similar protein and pathway changes, including the downregulation of proteins related to translation and proliferation and upregulation of functions implicated in host-microbiome interactions, such as cell adhesion, tight junction, extracellular vesicles, and responses to stimuli. Lysine acetylomics revealed that histone protein acetylation levels were decreased along with cell differentiation, while the acetylation in proteins associated with mitochondrial functions was increased. This study also demonstrates that, compared to spontaneous differentiation methods, butyrate-containing medium accelerates Caco-2 differentiation, with earlier upregulation of proteins related to host-microbiome interactions, suggesting its superiority for assay development using this intestinal model. Altogether, this multiomics study emphasizes the controlled progression of Caco-2 differentiation toward a specialized intestinal epithelial-like cell and establishes its suitability for investigating the host-microbiome interactions.

Gingipain-carrying outer membrane vesicles from Porphyromonas gingivalis cause barrier dysfunction of Caco-2 cells by releasing gingipain into the cytosol.

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.

Enantioselective analysis of the pesticide imazamox after in vitro permeability study in Caco-2 cells.

Imazamox (IMX), a chiral herbicide used in cereals and oilseed crops to control weeds, is commonly sold as a racemic mixture. Its enantiomers, being chiral compounds, may exhibit unique properties when exposed to chiral environments. While IMX enantiomers have been reported to degrade differently in soil and be toxic to some species, their effects on human systems remain poorly understood. This study utilized Caco-2 (human colon adenocarcinoma cell line) cells to assess the in vitro permeability of a racemic mixture of IMX and its isolated enantiomers. Additionally, the study aimed to evaluate whether the metabolite imazamox-O-desmethyl (IMX-D) forms during the permeability process. An enantioselective chromatographic method was developed, fully validated, and the apparent permeability values were obtained. The apparent permeability of rac-IMX, (+)-IMX, and (-)-IMX was determined to be 4.15 × 10-5, 5.78 × 10-5, and 7.33 × 10-5 cm s-1, respectively. These findings suggest that IMX exhibits high intestinal permeability, with an enantioselective absorption for (-)-IMX as compared to (+)-IMX. Finally, the permeability study in Caco-2 cells revealed that the metabolite IMX-D was not generated.

Effect of soybean proteins and peptides on the growth and adhesive ability of Limosilactobacillus Reuteri DSM17938.

Limosilactobacillus reuteri DSM17938 is one of the most pivotal probiotics, whose general beneficial effects on the intestinal microbiota are well recognized. Enhancing their growth and metabolic activity can effectively regulate the equilibrium of intestinal microbiota, leading to improved physical health. A common method to promote the growth of Lactobacillus is the addition of prebiotics. Current research suggests that proteins and their hydrolysates from different sources with potential prebiotic activity can also promote the growth of probiotics. In this study, soybean proteins and peptides were effective in promoting the growth, organic acid secretion, and adhesive properties of Limosilactobacillus reuteri DSM17938 to Caco-2 cells. These results illustrate the feasibility of soybean proteins and peptides as prebiotics, providing theoretical and practical advantages for their application.

The Enterococcus secretome inhibits the growth of vancomycin-resistant Enterococcus faecalis V853 with their antiproliferative properties and nanoencapsulation effects.

In our study, the secretome of the clinical isolate Enterococcus faecalis HY7 displayed antibacterial activity against the vancomycin-resistant Enterococcus faecalis V853. These bacteriocin-like substances showed thermal stability at a wide range of temperatures up to 121 °C, while proteinase K treatment resulted in a total loss of their activity. PCR-based screening for bacteriocin biosynthetic genes revealed that Enterococcus faecalis HY7 harbored multiple enterocin-producing genes, including ent A, avc A, and as-48. The production kinetics demonstrated the highest levels of bacteriocins production at 16 h, whereas the activity was diminished after 32 h of microbial growth. Notably, the partially purified bacteriocins exhibited anti-proliferative activity on the colon cancer cells, Caco2, with an IC50 value of 172.8 µg/mL. Remarkably, the nanoencapsulation of our bacteriocins in liposome showed a fourfold increase in its anti-vancomycin-resistant Enterococcus activity, which is the first report of liposome encapsulation with anti-vancomycin resistant Enterococcus bacteriocin.

Molecular mechanisms of extracellular-ATP-mediated colorectal cancer progression: Implication of purinergic receptors-mediated nucleocytoplasmic shuttling of HuR.

One of the leading causes of cancer-related deaths worldwide is colorectal cancer (CRC). Extracellular ATP (e-ATP) and purinergic receptors (P2R) play a central role in CRC proliferation and progression. Human antigen R (HuR) is becoming more and more understood to be essential for the expression of genes linked to cancer. The current study demonstrates that ATP can mediate CRC (Caco-2 cells) progression via induction of HuR nucleocytoplasmic shuttling and subsequent expression of cancer-related genes, a consequence mostly mediated via the P2R receptor. It was also noted that suppression of HuR activity by using dihydrotanshinone I (DHTS) prevents cancer-related gene expression and subsequent CRC (Caco-2 cells) progression induced by ATP. The expression of cyclin A2/cyclin-dependent kinase 2 (CDK2), Bcl-2, ProT-α, hypoxia-inducible factor1-α (HIF1-α), vascular endothelial growth factor A (VEGF-A), transforming growth factor-ß (TGF-ß) and matrix metallopeptidase 9 (MMP-9) induced by ATP were highly reduced in the presence of either PPADS (non-selective P2R antagonist) or DHTS. In addition, e-ATP-induced Caco-2 cell proliferation as well as cell survival were highly reduced in the presence of either PPADS or DHTS or selective CDK-2 inhibitor (Roscovitine) or selective Bcl-2 inhibitor (ABT-263). Furthermore, it was found that MMP-9 is critical for Caco-2 cells migration induced by e-ATP as demonstrated by a clear reduction in cells migration in the presence of a selective MMP-9 inhibitor (Marimastat). Collectively, these data demonstrate that ATP through P2R activation can induce HuR nucleocytoplasmic shuttling that could be translated into an increase in cancer-related genes expression and subsequent, cell proliferation and progression.

Clinical Analysis and in Vitro Correlation of BCRP-Mediated Drug-Drug Interaction in the Gastrointestinal Tract.

Breast cancer resistance protein (BCRP) is a drug efflux transporter expressed on the epithelial cells of the small intestine and on the lateral membrane of the bile duct in the liver; and is involved in the efflux of substrate drugs into the gastrointestinal lumen and secretion into bile. Recently, the area under the plasma concentration-time curve (AUC) of rosuvastatin (ROS), a BCRP substrate drug, has been reported to be increased by BCRP inhibitors, and BCRP-mediated drug-drug interaction (DDI) has attracted attention. In this study, we performed a ROS uptake study using human colon cancer-derived Caco-2 cells and confirmed that BCRP inhibitors significantly increased the intracellular accumulation of ROS. The correlation between the cell to medium (C/M) ratio of ROS obtained by the in vitro study and the absorption rate constant (ka) ratio obtained by clinical analysis was examined, and a significant positive correlation was observed. Therefore, it is suggested that the in vitro study using Caco-2 cells could be used to quantitatively estimate BCRP-mediated DDI with ROS in the gastrointestinal tract.

Qualification of In Vitro Dissolution Absorption System 2 (IDAS2) with Caco-2 and MDCK Cell Monolayers: Dose Sensitivity Study Using BCS Class I and III Drugs.

This study aimed to validate the In vitro Dissolution Absorption System 2 (IDAS2) containing a biological barrier of Caco-2 or Madin-Darby canine kidney (MDCK) cell monolayer through dose sensitivity studies. Metoprolol and propranolol were selected as Biopharmaceutics Classification System (BCS) Class I model drugs, and atenolol as a Class III model drug. The IDAS2 is comprised of a dissolution vessel (500 mL) and two permeation chambers (2 × 8.0 mL) mounted with Caco-2 or MDCK cell monolayer. One or two immediate-release tablet(s) of the model drug were added to the dissolution vessel, and the time profiles of dissolution and permeation were observed. Greater than 85% of metoprolol and propranolol (tested at two dosing concentrations) were dissolved by 15 min, and all drugs were fully dissolved by 30 min. All three drugs were more permeable across Caco-2 cells than MDCK cells with a linear increase in permeation across both cells at both dose concentrations. Thus, the dose sensitivity of the IDAS2 was demonstrated using both cell barriers. These results indicate a successful qualification of IDAS2 for the development/optimization of oral formulations and that MDCK cells can be utilized as a surrogate for Caco-2 cells.

Exploring the Hypocholesterolemic Potential of a Fucus vesiculosus Extract: Omic Insights into Molecular Mechanisms at the Intestinal Level.

High blood cholesterol levels are a major risk factor for cardiovascular diseases. A purified aqueous extract of Fucus vesiculosus, rich in phlorotannins and peptides, has been described for its potential to inhibit cholesterol biosynthesis and intestinal absorption. In this work, the effect of this extract on intestinal cells' metabolites and proteins was analysed to gain a deeper understanding of its mode of action on lipids' metabolism, particularly concerning the absorption and transport of exogenous cholesterol. Caco-2 cells, differentiated into enterocytes, were exposed to the extract, and analysed by untargeted metabolomics and proteomics. The results of the metabolomic analysis showed statistically significant differences in glutathione content of cells exposed to the extract compared to control cells, along with an increased expression of fatty acid amides in exposed cells. A proteomic analysis showed an increased expression in cells exposed to the extract compared to control cells of FAB1 and NPC1, proteins known to be involved in lipid metabolism and transport. To the extent of our knowledge, this study is the first use of untargeted metabolomics and a proteomic analysis to investigate the effects of F. vesiculosus on differentiated Caco-2 cells, offering insights into the molecular mechanism of the extract's compounds on intestinal cells.

Transcriptomics Analysis of the Immune Effects of Okadaic Acid on Caco-2 Cells.

Okadaic Acid, a type of diarrhetic shellfish poison, is widely distributed and harmful, causing symptoms such as diarrhea, vomiting, and more in humans. Recent studies have demonstrated that OA can lead to various toxicities such as cytotoxicity, neurotoxicity, embryotoxicity, and hepatotoxicity. In order to investigate the immunotoxicity of OA on intestinal cells, a transcriptome analysis was conducted to compare the differences in the Caco-2 cell transcriptional group before and after administration. The CCK-8 experiment demonstrated that OA had a detrimental effect on the activity of Caco-2 cells, with an IC50 value of 33.98 nM. Transcriptome data revealed changes in immune-related genes between the experimental and control groups, including inflammatory factors, heat shock proteins, and zinc finger proteins. The analysis of the results suggests that OA can induce the production of inflammatory factors and apoptosis in cells, and may also affect cell ferroptosis. These findings indicate that OA has a significant impact on intestinal immunity, providing valuable insights for the study of immune toxicity associated with OA.

Simultaneous assessment of absorption and pharmacokinetic characteristics of four active flavonoids from Chimonanthus nitens Leaf Granules using LC-MS determination: in vivo and in vitro.

A sensitive UPLC-HRMS method was developed and validated for simultaneous quantification of four active flavonoids from Chimonanthus nitens Leaf Granules (CNLG) in biological matrix. The method was utilized in pharmacokinetic study of the four flavonoids in rats as well as other evaluation assays in vitro. It was revealed that rutin, nicotiflorin, and astragalin had poor oral bioavailability in rats possibly due to low intestinal permeability and metabolism in intestinal flora. Kaempferol underwent rapid glucuronidation and sulphation in rat plasma with medium permeability coefficient. The results provided valuable data for future research and development of CNLG flavonoids.

Impact of radish seeds (Semen Raphani) on the absorption and transportation of ginsenosides in the Caco-2 cell model: a UPLC-ESI-MS analysis.

This study examined the impact of Semen raphani on the absorption of ginsenosides from Panax ginseng C.A. Meyer (ginseng) using a Caco-2 cell model and Ultra-High-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (UPLC-ESI-MS). Six primary ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rd) were quantified. Results showed that Semen Raphani increased the efflux rate of ginsenosides, particularly at higher concentrations, suggesting it inhibits their absorption. The research elucidates the intestinal absorption process of ginsenosides and the antagonistic mechanism of Semen Raphani against ginseng.

Effects of Daidzein, Tempeh, and a Probiotic Digested in an Artificial Gastrointestinal Tract on Calcium Deposition in Human Osteoblast-like Saos-2 Cells.

Adequate calcium intake is crucial for the prevention and treatment of bone-related issues. Developing a nutritional source of readily bioavailable calcium is particularly significant for individuals deficient in this essential element and at risk of developing osteoporosis. This research aimed to evaluate the impact of tempeh (T), daidzein (D), and Lactobacillus acidophilus (LA) within a simulated intestinal environment consisting of Caco-2 epithelial and Saos-2 cells, focusing on their implications for bone mineralization mechanisms. In the initial phase, calcium bioaccessibility from calcium citrate (CaCt), LA, D, the daidzein combination D-CaCt-LA (D1:1:1), and the tempeh combination T-CaCt-LA (T1:1:1) was assessed through digestion simulation. The calcium content of both untreated and digested samples was determined using atomic absorption spectrometry (AAS). In the subsequent stage, the digested samples were used to induce intestinal absorption in differentiated enterocyte-like Caco-2 cells. The permeable fractions were then evaluated in a culture of osteoblast-like Saos-2 cells. Preliminary cellular experiments employed the MTT assay to assess cytotoxicity. The results indicated that the analyzed products did not influence the deposition of extracellular calcium in Saos-2 cells cultured without mineralization stimulators. The combined formulations of permeable fractions of digested CaCt, LA, D, and T demonstrated the capacity to enhance the proliferation of Saos-2 cells. In Saos-2 cells, D, D1:1:1, and LA showed no discernible impact on intracellular calcium accumulation, whereas T and T1:1:1 reduced the calcium deposits. Additionally, mRNA transcripts and alkaline phosphatase (ALP) activity levels in Saos-2 cells cultured without mineralization induction were unaffected by the analyzed products. An examination of the products revealed no discernible effect on ALP activity or mRNA expression during Saos-2 cell differentiation. Our findings suggest that tempeh, daidzein, and L. acidophilus did not positively impact cellular calcium deposition in Saos-2 cells. However, tempeh, daidzein and its combination, and L. acidophilus might enhance the process of osteogenic differentiation in Saos-2 cells. Nevertheless, this study did not identify any synergistic impact on calcium deposition and the process of osteogenic differentiation in Saos-2 cells of isoflavones and probiotics.

Additive Cytotoxic and Colony-Formation Inhibitory Effects of Aspirin and Metformin on PI3KCA-Mutant Colorectal Cancer Cells.

Human malignancies are one of the major health-related issues throughout the world and are anticipated to rise in the future. Despite huge investments made in anticancer drug development, limited success has been obtained and the average number of FDA approvals per year is declining. So, an increasing interest in drug repurposing exists. Metformin (MET) and aspirin (ASP) possess anticancer properties. This work aims to test the effect of these two drugs in combination on colorectal cancer (CRC) cells in vitro. The effects of MET and/or ASP on cell proliferation, viability, migratory ability, anchorage-independent growth ability (colony formation), and nutrient uptake were determined in two (HT-29 and Caco-2) human CRC cell lines. Individually, MET and ASP possessed antiproliferative, cytotoxic, and antimigratory effects and reduced colony formation in HT-29 cells (BRAF- and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PI3KCA)-mutant), although MET did not affect either 3H-deoxy-D-glucose or 14C-butyrate uptake and lactate production, and ASP caused only a small decrease in 14C-butyrate uptake. Moreover, in these cells, the combination of MET and ASP resulted in a tendency to an increase in the cytotoxic effect and in a potentiation of the inhibitory effect on colony formation, although no additive antiproliferative and antimigratory effects, and no effect on nutrient uptake and lactate production were observed. In contrast, MET and ASP, both individually and in combination, were almost devoid of effects on Caco-2 cells (BRAF- and PI3KCA-wild type). We suggest that inhibition of PI3K is the common mechanism involved in the anti-CRC effect of both MET, ASP and their combination and, therefore, that the combination of MET + ASP may especially benefit PI3KCA-mutant CRC cases, which currently have a poor prognostic.

The Influence of Mesotrione on Human Colorectal Adenocarcinoma Cells and Possibility of Its Toxicity Mitigation by Cichoric Acid.

Mesotrione, as a widely used herbicide, is present in the environment in detectable amounts, causing serious damage. Here, we aimed to investigate the effect of mesotrione on Caco-2 cells and the possibility of its toxicity mitigation by cichoric acid. Therefore, we analyzed the cytotoxicity of both these compounds and the selected oxidative stress parameters, apoptosis and interaction of both the tested compounds with the cell membrane and their accumulation within the cells. In cytotoxicity studies, the stimulating activity of mesotrione was observed, and simultaneously, the inhibitory effect of cichoric acid was noticed. This effect was related to the results of oxidative stress analysis and apoptosis measurements. The activity level of key enzymes (glutathione peroxidase, catalase and superoxide dismutase) in Caco-2 cells exposed to cichoric acid was higher as compared to that of the control. The treatment with mesotrione did not induce apoptosis in the Caco-2 cells. The penetration of the studied compounds into the Caco-2 cells was measured by using an HPLC methodology, and the results indicate mesotrione's high penetration capacity. The distribution of charge on the surface of the cell membranes changed under the influence of both compounds. Considering the mutual interactions of beneficial and potentially toxic food ingredients, it should be noted that, despite the observed favorable trend, cichoric acid is not able to overcome the toxic and cancer-stimulating effects of this pesticide.

Synthetic Derivatives of Natural ent-Kaurane Atractyligenin Disclose Anticancer Properties in Colon Cancer Cells, Triggering Apoptotic Cell Demise.

The antitumor activity of different ent-kaurane diterpenes has been extensively studied. Several investigations have demonstrated the excellent antitumor activity of synthetic derivatives of the diterpene atractyligenin. In this research, a series of new synthetic amides and their 15,19-di-oxo analogues obtained from atractyligenin by modifying the C-2, C-15, and C-19 positions were designed in order to dispose of a set of derivatives with different substitutions at the amidic nitrogen. Using different concentrations of the obtained compounds (10-300 µM) a reduction in cell viability of HCT116 colon cancer cells was observed at 48 h of treatment. All the di-oxidized compounds were more effective than their alcoholic precursors. The di-oxidized compounds had already reduced the viability of two colon cancer cells (HCT116 and Caco-2) at 24 h when used at low doses (2.5-15 µM), while they turned out to be poorly effective in differentiated Caco-2 cells, a model of polarized enterocytes. The data reported here provide evidence that di-oxidized compounds induced apoptotic cell death, as demonstrated by the appearance of condensed and fragmented DNA in treated cells, as well as the activation of caspase-3 and fragmentation of its target PARP-1.

A View on the Chemical and Biological Attributes of Five Edible Fruits after Finishing Their Shelf Life: Studies on Caco-2 Cells.

We studied five common perishable fruits in terms of their polyphenols dynamic, minerals distribution, scavenger activity and the effects of 50% ethanolic extracts on the viability of Caco-2 cells in vitro, over a period of time between T = 0 and T = 5/7 days, typically the end of their shelf life. Altogether, there were few changes found, consisting of either an increase or a decrease in their chemical and biological attributes. A slow decrease was found in the antioxidant activity in apricot (-11%), plum (-6%) and strawberry (-4%) extracts, while cherry and green seedless table grape extracts gained 7% and 2% antioxidant potency, respectively; IC50 values ranged from 1.67 to 5.93 µg GAE/µL test extract. The cytotoxicity MTS assay at 24 h revealed the ability of all 50% ethanol fruit extracts to inhibit the Caco-2 cell viability; the inhibitory effects ranged from 49% to 83% and were measured at 28 µg GAE for strawberry extracts/EES, from 22 µg to 45 µg GAE for cherry extracts/EEC, from 7.58 to 15.16 µg GAE for apricot extracts/EEA, from 12.50 to 25.70 µg GAE for plum extracts/EEP and from 21.51 to 28.68 µg GAE for green table grape extracts/EEG. The MTS anti-proliferative assay (72 h) also revealed a stimulatory potency upon the Caco-2 viability, from 34% (EEA, EEG) and 48% (EEC) to 350% (EES) and 690% (EEP); therefore fruit juices can influence intestinal tumorigenesis in humans.

Differences in Metabolite Profiles of Dihydroberberine and Micellar Berberine in Caco-2 Cells and Humans-A Pilot Study.

We investigated the pharmacokinetic pathway of berberine and its metabolites in vitro, in Caco-2 cells, and in human participants following the administration of dihydroberberine (DHB) and micellar berberine (LipoMicel®, LMB) formulations. A pilot trial involving nine healthy volunteers was conducted over a 24 h period; blood samples were collected and subjected to Ultra High-Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) analyses to quantify the concentrations of berberine and its metabolites. Pharmacokinetic correlations indicated that berberrubine and thalifendine follow distinct metabolic pathways. Additionally, jatrorrhizine sulfate appeared to undergo metabolism differently compared to the other sulfated metabolites. Moreover, berberrubine glucuronide likely has a unique metabolic pathway distinct from other glucuronides. The human trial revealed significantly higher blood concentrations of berberine metabolites in participants of the DHB treatment group compared to the LMB treatment group-except for berberrubine glucuronide, which was only detected in the LMB treatment group. Similarly, results from in vitro investigations showed significant differences in berberine metabolite profiles between DHB and LMB. Dihydroberberine, dihydroxy-berberrubine/thalifendine and jatrorrhizine sulfate were detected in LMB-treated cells, but not in DHB-treated cells; thalifendine and jatrorrhizine-glucuronide were detected in DHB-treated cells only. While DHB treatment provided higher blood concentrations of berberine and most berberine metabolites, both in vitro (Caco-2 cells) and in vivo human studies showed that treatment with LMB resulted in a higher proportion of unmetabolized berberine compared to DHB. These findings suggest potential clinical implications that merit further investigation in future large-scale trials.

Whey Protein Sodium-Caseinate as a Deliverable Vector for EGCG: In Vitro Optimization of Its Bioaccessibility, Bioavailability, and Bioactivity Mode of Actions.

Epigallocatechin gallate (EGCG), the principal catechin in green tea, exhibits diverse therapeutic properties. However, its clinical efficacy is hindered by poor stability and low bioavailability. This study investigated solid particle-in-oil-in-water (S/O/W) emulsions stabilized by whey protein isolate (WPI) and sodium caseinate (NaCas) as carriers to enhance the bioavailability and intestinal absorption of EGCG. Molecular docking revealed binding interactions between EGCG and these macromolecules. The WPI- and NaCas-stabilized emulsions exhibited high encapsulation efficiencies (>80%) and significantly enhanced the bioaccessibility of EGCG by 64% compared to free EGCG after simulated gastrointestinal digestion. Notably, the NaCas emulsion facilitated higher intestinal permeability of EGCG across Caco-2 monolayers, attributed to the strong intermolecular interactions between caseins and EGCG. Furthermore, the emulsions protected Caco-2 cells against oxidative stress by suppressing intracellular reactive oxygen species generation. These findings demonstrate the potential of WPI- and NaCas-stabilized emulsions as effective delivery systems to improve the bioavailability, stability, and bioactivity of polyphenols like EGCG, enabling their applications in functional foods and nutraceuticals.