Caco-2 Cell Absorption Research Articles

Milk osteopontin mediates zinc uptake in intestinal cells in the presence of phytic acid

The bioavailability and the intestinal absorption of the essential mineral zinc are challenged by the food matrix and especially the content of zinc chelating antinutrients. Many milk proteins and peptides affect zinc bioavailability. Osteopontin (OPN) is an acidic and highly phosphorylated whey protein that has been shown to bind calcium, magnesium and iron. Here we report the zinc binding properties of OPN, and the effect of OPN-Zn complexes on zinc absorption in Caco-2 cells. By isothermal titration calorimetry milk OPN was shown to bind approximately 36 zinc ions (KD of ∼70 μM). The binding was mainly mediated by the phosphorylations in the protein. OPN retained zinc bound after in vitro simulated gastrointestinal transit. Interestingly the OPN-Zn complex enhanced zinc bioavailability in the presence of phytic acid, compared to inorganic zinc salts. Zinc uptake mediated by OPN significantly upregulated the gene expression of MT1G and ZnT1 which are crucial proteins involved in preserving enterocyte zinc homoeostasis. These results indicate that OPN could be incorporated into functional foods and infant formulas to increase zinc bioavailability and uptake.

Characterization and Mechanism of a Novel Rice Protein Peptide (AHVGMSGEEPE) Calcium Chelate in Enhancing Calcium Absorption in Caco-2 Cells.

Rice protein peptides (RPP) are a potentially valuable source of high-quality calcium chelating properties. However, there is a lack of information regarding the calcium-absorption-promoting effect of RPP and its underlying mechanism. The present study adopted molecular docking methodologies to analyze the 10 most potent peptide segments from RPP. Results revealed that the peptide AHVGMSGEEPE (AHV) displayed optimal calcium binding properties (calcium-chelating capacity 55.69 ± 0.66 mg/g). Quantum chemistry analysis revealed that the AHV peptide effectively binds and forms stable complexes with calcium via the carbonyl oxygen atoms in valine at position 3 and the carbonyl of the C-terminal carboxyl group of glutamate at position 11. The spectral analysis results indicated that AHV may bind to calcium through carboxyl oxygen atoms, resulting in a transition from a smooth surface block-like structure to a dense granular structure. Furthermore, this study demonstrated that the 4 mmol/L AHV-Ca chelate (61.75 ± 13.23 μg/well) significantly increases calcium absorption compared to 1 mM CaCl2 (28.57 ± 8.59 μg/well) in the Caco-2 cell monolayer. In terms of mechanisms, the novel peptide-calcium chelate AHV-Ca derived from RPP exerts a cell-level effect by upregulating the expression of TRPV6 calcium-ion-channel-related genes and proteins (TRPV6 and Calbindin-D9k). This study provides a theoretical basis for developing functional foods with the AHV peptide as ingredients to improve calcium absorption.

Natural Product – Drug Interactions and Their Evaluation Using a Caco-2 Cell Culture Model

Natural products, such as herbal medicinal products, food supplements, and functional food, are widely used to support well-being and for promoting health. In general, the effects of using these products are desired and beneficial, but unexpected adverse effects might also occur, especially when natural products are used with medication. Consumers do not often even know that they are exposed to bioactive compounds that might interact with the body and have effects on their well-being. One of the objectives of the World Health Organization is to promote herb–drug interaction monitoring, and it is important to know how marketed preparations and compounds from common foods interact when they are absorbed. In this review, we describe how a Caco-2 cell absorption model has been used to study how natural products, such as flaxseed, rapeseed, purple loosestrife, pine, echinacea, certain berries and herbs, anthranoid laxatives, and traditional medicinal plants, affect the absorption of co-administered drugs. We discuss the types of interactions and adverse effects that might occur and their possible reasons. Overall, we conclude that the Caco-2 cell absorption model is a useful tool for studying the absorption of natural products with drugs; and that to enable the safe use of natural products with medicines, concomitant use should be studied.

Characterization, cytotoxicity, and stability evaluation of novel nanocurcumin functionalized cream powder under accelerated storage conditions

Cellular-uptake and storage stability of nanocurcumin formulations remains a challenge due to loss of their functional properties over time. In this work, a novel nano-curcumin functionalized milk cream powder (CP) was developed and analyzed for the stabilityand retention of physico-fucntional properties in an accelerated storage conditions. The powder was prepared through microfluidization of milk-cream emulsion, and spray-dried assisted encapsulation with sodium caseinate (N) and gum arabic (G) as carriers, followed by storage at 25 and 50 °C for 60 days. Results showed that NCP exhibited higher curcumin stability and antioxidant-activity during storage as compared to the control, even at 50 °C after 60 days. TEM of NCP revealed that curcumin particles were still in nano-form (50–150 nm) after in-vitro digestion and storage. Curcumin bioavailability did not change after accelerated storage period as 86.57% (day 60) absorption in Caco-2 cells versus 95.01% (day 0) in NCP was observed. In-vitro cytotoxicity assay confirmed safety of the developed nanocurcumin formulations even after 60 days of storage in accelerated conditions.

Curcumin protects against high-fat diet-induced nonalcoholic simple fatty liver by inhibiting intestinal and hepatic NPC1L1 expression via down-regulation of SREBP-2/HNF1α pathway in hamsters

Niemann-pick C1-like 1 (NPC1L1) mediates cholesterol absorption and plays a key role in the pathogenesis of nonalcoholic simple fatty liver (NASFL). Our previous study showed that curcumin reduced NPC1L1 expression and cholesterol absorption in Caco-2 cells. This study aimed to investigate whether curcumin could inhibit intestinal and hepatic NPC1L1 expression through suppressing sterol regulatory element binding protein-2 (SREBP-2) / hepatocyte nuclear factor 1α (HNF1α) pathway, then exert anti-NASFL effects. Six-week hamsters were fed high-fat diet (HFD) with or without 0.1% curcumin for 12 weeks. Curcumin supplementation lowered blood total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol levels (20.2%, 48.7%, and 36.5%), and reduced liver TC and TG contents (26.1% and 26.5%). Oil Red O staining demonstrated that curcumin significantly alleviated HFD-induced liver fat accumulation and hepatic steatosis, which was accompanied by reduced intestinal and hepatic NPC1L1, SREBP-2 and HNF1α expression (P < .05) and increased fecal neutral sterol excretion (114.5%). Furthermore, curcumin decreased cholesterol absorption in Caco-2 cells and HepG2 cells (49.2 % and 52.7 %). The inhibitory effects of curcumin on NPC1L1 expression and cholesterol absorption could be prevented by blockade of the SREBP-2 and HNF1α pathway. These findings indicated that curcumin protected against HFD-induced NASFL by inhibiting intestinal and hepatic NPC1L1 expression via down-regulation of SREBP-2/HNF1α pathway, thus reducing intestinal cholesterol absorption and hepatic biliary cholesterol reabsorption, consequently alleviating liver cholesterol accumulation and steatosis. Our study provides evidence for curcumin as a potential nutritional therapy for NASFL by regulating NPC1L1 and enterohepatic circulation of cholesterol.

Effect and Mechanism of Theaflavins on Fluoride Transport and Absorption in Caco-2 Cells.

This paper investigated the effect and mechanism of theaflavins (TFs) on fluoride (F-) uptake and transport in the Caco-2 cell model through structural chemistry and transcriptome analysis. The results showed that the four major TFs (TF, TF3G, TF3'G and TFDG) at a 150 μg/mL concentration could all significantly decrease F- transport in Caco-2 cells after 2 h of treatment and, at 2 μg/mL F- concentration, the F- transport was more inclined to efflux. During transport, the F- retention in Caco-2 cells was significantly increased by TF3G while it was clearly decreased by TF. The interaction between TFs and F- was analyzed by Raman spectroscopy and isothermal titration calorimetry, and F- was shown to affect the π bond vibration on the benzene ring of TFs, thus influencing their stability. Additionally, F- showed weak binding to TF3G, TF3'G and TFDG, which may inhibit F- transport and absorption in the Caco-2 cell line. Transcriptome and RT-PCR analysis identified three key differentially expressed genes related to cell permeability, and TFs can be assumed to mediate F- transport by regulating the expression of permeability-related genes to change cell monolayer permeability and enhance cell barrier function; however, this needs to be further elucidated in future studies.

Curcumin suppresses cell proliferation and reduces cholesterol absorption in Caco-2 cells by activating the TRPA1 channel

BackgroundCurcumin (Cur) is a bioactive dietary polyphenol of turmeric with various biological activities against several cancers. Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths. Intestinal cholesterol homeostasis is associated with CRC. Chemotherapy for CRC is related to varied adverse effects. Therefore, natural products with anti-cancer properties represent a potential strategy for primary prevention of CRC.MethodsThe present study used Cur as a therapeutic approach against CRC using the Caco-2 cell line. The cells were treated with different concentrations of Cur for different duration of time and then the proliferation ability of cells was assessed using Cell Counting Kit-8 and 5-Ethynyl-2′-deoxyuridine assays. Oil red O staining and cholesterol assay kit were used to evaluate cellular lipid content and cholesterol outward transportation. Finally, the protein expressions of cholesterol transport-related protein and signal transduction molecules were assessed using Western blot assay.ResultsCur inhibited cell proliferation in Caco-2 cells in a dose- and time-dependent manner by activating the transient receptor potential cation channel subfamily A member 1 (TRPA1) channel. Activation of the TRPA1 channel led to increased intracellular calcium, peroxisome proliferator-activated receptor gamma (PPARγ) upregulation, and the subsequent downregulation of the specificity protein-1 (SP-1)/sterol regulatory element-binding protein-2 (SREBP-2)/Niemann-Pick C1-like 1 (NPC1L1) signaling pathway-related proteins, and finally reduced cholesterol absorption in Caco-2 cells.ConclusionsCur inhibits cell proliferation and reduces cholesterol absorption in Caco-2 cells through the Ca2+/PPARγ/SP-1/SREBP-2/NPC1L1 signaling by activating the TRPA1 channel, suggesting that Cur can be used as a dietary supplement for the primary prevention of CRC.Graphical In Caco-2 cells, Cur first stimulates calcium influx by activating the TRPA1 channel, further upregulates PPARγ and downregulates SP-1/SREBP-2/NPC1L1 signaling pathway, and finally inhibits the absorption of cholesterol. TRPA1, transient receptor potential cation channel subfamily A member 1; NPC1L1, Niemann-Pick C1-like 1; PPARγ, peroxisome proliferator-activated receptor gamma; SP-1, specificity protein-1; SREBP-2, sterol regulatory element-binding protein-2; Cur, curcumin.

The stability and absorption of naturally occurring cAMP by its weak interactions with jujube polysaccharides were greatly improved

Cyclic adenosine monophosphate (cAMP) has received considerable attention because it naturally occurs in jujube fruit and possesses many activities such as antitumor activity. However, its poor membrane permeability limits its applications in food industry. In this study, we found that polysaccharides likewise derived from jujube (ZJP) are able to increase the membrane permeability of cAMP for the first time. To elucidate the mechanism, the interactions of cAMP and ZJP were determined by spectroscopic, thermodynamic and molecular docking methods. Results showed that acidic polysaccharide (ZJPA) owned a stronger affinity to cAMP as compared to neutral polysaccharide (ZJPN). Consequently, the binding of ZJPA to cAMP existed a better synergistic antioxidant capacity and also protected ZJPA against pH-induced degradation. More importantly, the formed ZJPA-cAMP complexes exhibited much better bio-accessibility than unbound cAMP as was approved by Caco-2 cell absorption experiment. These new findings are beneficial for designing polysaccharide-based nanocarriers for cAMP. • Jujube polysaccharides do bind to cyclic adenosine monophosphate. • Acidic polysaccharides bind to cyclic adenosine monophosphate more strongly. • Charge interaction, H-bonding and hydrophobic effect contribute to binding. • Cyclic adenosine monophosphate enhances bioactivity of Polysaccharides. • Polysaccharides promote absorption of cyclic adenosine monophosphate by Caco-2 cells.

Structural degradation and uptake of resveratrol-encapsulated liposomes using an in vitro digestion combined with Caco-2 cell absorption model

Resveratrol (RES), a polyphenol with strong antioxidant capacity but poor bioavailability and light instability, urgently needs an effective delivery technique to overcome its drawbacks. As it is a highly biocompatible delivery system, liposomes were used to carry RES to form resveratrol-encapsulated liposomes (RES-LPS). Results showed that the diameter of RES-LPS was 333 ± 50 nm and the encapsulation efficiency was 84.69 ± 0.02 %, with a spherical shape and double-layered structure. Morphology showed that RES-LPS, could maintain an intact membrane structure during stomach digestion, as well as while under hydrolysis, mimicking intestinal conditions, before releasing RES. Moreover, Caco-2 cells uptake study also demonstrated that the digesta of RES-LPS resulted in a better cell absorption efficiency and a stronger ability to reduce reactive oxygen species when compared with free RES. Thus, these results indicate that liposomes play a key role in improving the bioavailability of RES, demonstrating the promising role of liposomes as a delivery system for food supplements.

Immunomodulatory role of edible bone collagen peptides on macrophage and lymphocyte cell cultures

ABSTRACT Immunonutrition or modulation of immune capacity through food and supplements has been gaining significant importance. Hydrolysed collagen has long been used as a functional ingredient, showing multiple physiological activities, including enhancement of immune functions. However, how collagen peptides may affect the immune system still needs further research. This study investigates bone collagen peptides (BCP) immunomodulatory activity on human monocytic THP-1 and human Jurkat T lymphocyte cell lines, using cytokine mRNA expressions as biomarkers. In vitro, gastrointestinal digestion and Caco-2 cell absorption allow obtaining digested and absorbed BCP fractions, respectively, which are tested on immune cells. Results show: (1) Immunostimulatory effect on M0 macrophages, but not on M1 macrophages (lipopolysaccharide (LPS)-activated), (2) Significant T lymphocyte proliferation after incubation with absorbed BCP fraction. (3) Significant increase of anti-inflammatory IL-10 cytokine biomarker. These results suggest that BCP could act as an immunonutrient, modulating the immune response and inflammatory processes. Abbreviations: ATCC: American Type Culture Collection BCP: bone collagen peptide DMSO: dimethyl sulphoxide FBS: fetal bovine serum ECM: extracellular matrix GRAS: Generally recognized as safe; IFN: interferon IL: interleukin, LPS: lipopolysaccharide SEM: standard error of the mean TEER: transepithelial electrical resistance

Curcumin Supplementation Ameliorates Bile Cholesterol Supersaturation in Hamsters by Modulating Gut Microbiota and Cholesterol Absorption.

Curcumin is a polyphenol that has been shown to have prebiotic and cholesterol-lowering properties. This study aimed to investigate the impact of curcumin on bile cholesterol supersaturation and the potential mechanistic role of intestinal microbiota and cholesterol absorption. Male hamsters (n = 8) were fed a high-fat diet (HFD) supplemented with or without curcumin for 12 weeks. Results showed that curcumin significantly decreased cholesterol levels in the serum (from 5.10 to 4.10 mmol/L) and liver (from 64.60 to 47.72 nmol/mg protein) in HFD-fed hamsters and reduced the bile cholesterol saturation index (CSI) from 1.64 to 1.08 due to the beneficial modifications in the concentration of total bile acids (BAs), phospholipids and cholesterol (p < 0.05). Gut microbiota analysis via 16S rRNA sequencing revealed that curcumin modulated gut microbiota, predominantly increasing microbiota associated with BA metabolism and short-chain fatty acid production, which subsequently up-regulated the expression of hepatic cholesterol 7-alpha hydroxylase and increased the synthesis of bile acids (p < 0.05). Furthermore, curcumin significantly down-regulated the expression of intestinal Niemann–Pick C1-like protein 1(NPC1L1) in hamsters and reduced cholesterol absorption in Caco-2 cells (p < 0.05). Our results demonstrate that dietary curcumin has the potential to prevent bile cholesterol supersaturation through modulating the gut microbiota and inhibiting intestinal cholesterol absorption.

Co-encapsulation of Egg-White-Derived Peptides (EWDP) and Curcumin within the Polysaccharide-Based Amphiphilic Nanoparticles for Promising Oral Bioavailability Enhancement: Role of EWDP.

The comprehensive utilization of food-derived nutraceuticals with different polarities has been extremely restricted by their poor bioavailability and coexistence in a single system. This study aimed to fabricate a self-assembly of amphiphilic nanoparticles (NPs) for the hydrophilic EWDP and hydrophobic curcumin based on the carboxymethyl chitosan (CMCS) shell and γ-cyclodextrin (γ-CD) core. Notably, EWDP could cooperate with CMCS to yield superior colloidal properties with an excellent curcumin aqueous solubility and co-encapsulation capacity (>10%) for the NPs (pH 2.0-7.0). This phenomenon was mainly ascribed to the additional hydrogen-bonding network and hydrophobic interaction introduced by EWDP. Besides, the overall antioxidant activity, bioaccessibility, gastrointestinal stability, and Caco-2 cell absorption properties were significantly improved in the presence of EWDP (>20% increase). Therefore, EWDP could function as both a potential affinity agent and a nutrition enhancer to expand the co-delivery applications for diverse nutraceuticals with promising oral bioavailability enhancement in food and pharmaceutical areas.

Flaxseed-derived peptide, IPPF, inhibits intestinal cholesterol absorption in Caco-2 cells and hepatic cholesterol synthesis in HepG2 cells.

Flaxseed peptides reduced serum cholesterol levels in Sprague-Dawley rats fed with a high-cholesterol diet. However, the mechanism of this action remains unclear. Flaxseed-hydrolyzed proteins were separated through ultrafiltration. The fifth fraction (FP5 , ≤ 1kDa) had the highest cholesterol micelle solubility inhibition rate (CMSR) of 72.39% among the other fractions. Eleven peptides were identified from FP5 . Ile-Pro-Pro-Phe (IPPF), which had the highest CMSR of 93.47%, was selected for further analyses. IPPF substantially reduced the cholesterol transported content in Caco-2 cells and the total cholesterol content in HepG2 cells. Moreover, IPPF modulated the protein levels of NCP1L1 and ABCG5/8 (cholesterol transporters) in Caco-2 cells and reduced the mRNA levels of Srebp-2 and Hmgcr (cholesterol synthesis enzymes) in HepG2 cells. IPPF inhibits cholesterol intestinal absorption by modulating the cholesterol transporters expression and reduces hepatic cholesterol synthesis by inhibiting the SREBP2-regulated mevalonate pathway. IPPF is a new food-derived cholesterol-lowering nutritional supplement. PRACTICAL APPLICATIONS: We isolated active peptides with cholesterol-lowering properties from flaxseed protein, a by-product of industrial oil production, which greatly improved the economic and medicinal value of flaxseed protein. According to our research, IPPF can be used as a new food-derived type of cholesterol intestinal absorption inhibitor to reduce dietary cholesterol absorption and cholesterol synthesis inhibitor (same pharmacological mechanism as statins). IPPF provide a nutritional therapy component for hypercholesterolemia and prevent atherosclerosis. Our research provides theoretical basis for development and utilization of new nutritional supplements and plant proteins.

Traditionally Used Plants in the Treatment of Diabetes Mellitus: Screening for Uptake Inhibition of Glucose and Fructose in the Caco2-Cell Model.

The traditional use of plants and their preparations in the treatment of diseases as a first medication in the past centuries indicates the presence of active components for specific targets in the natural material. Many of the tested plants in this study have been traditionally used in the treatment of Diabetes mellitus type 2 and associated symptoms in different cultural areas. Additionally, hypoglycemic effects, such as a decrease in blood glucose concentration, have been demonstrated in vivo for these plants. In order to determine the mode of action, the plants were prepared as methanolic and aqueous extracts and tested for their effects on intestinal glucose and fructose absorption in Caco2 cells. The results of this screening showed significant and reproducible inhibition of glucose uptake between 40 and 80% by methanolic extracts made from the fruits of Aronia melanocarpa, Cornus officinalis, Crataegus pinnatifida, Lycium chinense, and Vaccinium myrtillus; the leaves of Brassica oleracea, Juglans regia, and Peumus boldus; and the roots of Adenophora triphylla. Furthermore, glucose uptake was inhibited between 50 and 70% by aqueous extracts made from the bark of Eucommia ulmoides and the fruit skin of Malus domestica. The methanolic extracts of Juglans regia and Peumus boldus inhibited the fructose transport between 30 and 40% in Caco2 cells as well. These findings can be considered as fundamental work for further research regarding the treatment of obesity-correlated diseases, such as Diabetes mellitus type 2.

Bioaccessibility and Cellular Uptake of Carotenoids Extracted from Bactris gasipaes Fruit: Differences between Conventional and Ionic Liquid-Mediated Extraction.

Currently, on an industrial scale, synthetic colorants are used in many fields, as well as those extracted with conventional organic solvents (COSs), leading to several environmental issues. Therefore, we developed a sustainable extraction and purification method mediated by ionic liquids (IL), which is considered an alternative high-performance replacement for COSs. Carotenoids are natural pigments with low bioaccessibility (BCT) and bioavailability (BV) but with huge importance to health. To investigate if the BCT and cellular uptake of the carotenoids are modified by the extraction method, we conducted a comparison assay between both extraction procedures (IL vs. COS). For this, we used the Amazonian fruit Bactris gasipaes, a rich source of pro-vitamin A carotenoids, to obtain the extract, which was emulsified and subjected to an in vitro digestion model followed by the Caco-2 cell absorption assay. The bioaccessibility of carotenoids using IL was better than those using COS (33.25%, and 26.84%, respectively). The cellular uptake of the carotenoids extracted with IL was 1.4-fold higher than those extracted using COS. Thus, IL may be a feasible alternative as extraction solvent in the food industry, replacing COS, since, in this study, no IL was present in the final extract.

High Concentrations of Folic Acid Are Associated with Decreased Hemin Absorption in Caco-2 Cells

BackgroundFolic acid (FA) is the synthetic form of folate, commonly used in vitamin supplements, food fortification and therapy. Currently, there is concern about the possibility of potential adverse effects of high doses of FA. In Brazil, daily use of FA 5 mg is recommended for several population groups, including pregnant women and patients with hemolytic anemia. This dose is five times greater than the tolerable upper intake level for healthy people. Both FA and heme iron are absorbed into duodenal enterocytes by the proton coupled folate transporter/heme carrier protein 1 (PCFT/HCP1). This transporter has a higher affinity for FA than heme iron, and we therefore surmised that high doses of FA might interfere with heme iron absorption.ObjectiveThe aim of this study was to test the effects of high levels of FA on the absorption of hemin (source of heme iron) using Caco-2 cell monolayers as an in vitro model of intestinal absorption.Material and methodsCells grown in 6-well plates were incubated with 25 µM hemin and varying FA concentrations (0, 5, 10, 31.25, 62.50 and 125 µM) for 12 and 24 h. Intracellular iron was measured by atomic absorption spectrometry and total protein was measured by the Bradford method.ResultsAt both time points, cells incubated with 125 μM of FA showed lower concentrations of intracellular iron compared with cells without FA in the medium (0 µM). Cells incubated for 12 h with 62.5 μM and 125 μM of FA had 1.50 and 1.56-fold lower intracellular iron concentrations, respectively, than cells incubated without FA (125 μM: p= 0.002; 62.5 μM: p=0.007). Decrease of intracellular iron in Caco-2 cells was greater at 24 h than at 12 h of incubation. After 24 h, cells incubated with 125 μM of FA showed an intracellular iron concentration 3.08-fold lower than cells with 0 µM FA (p = 0.047).ConclusionsHigh levels of FA simulating intraluminal intestinal concentrations of FA following 5 mg FA ingestion were associated with lower intracellular iron content in Caco-2 cells due to decreased hemin absorption. These changes indicate that high doses of FA may impair heme iron absorption through their shared uptake via the PCFT/HCP1 transporter.Financing: CNPq 4826412012-6, CNPq 401586/2014-6, CNPq 208204/2015-6 and FAPESP 2012/12912-1 DisclosuresNo relevant conflicts of interest to declare.

Novel milk casein–derived peptides decrease cholesterol micellar solubility and cholesterol intestinal absorption in Caco-2 cells

This study sought to assess the cholesterol-lowering activity of peptides obtained from milk casein hydrolyzed with neutrase. The bioactive peptides were separated using a Sephadex G-10 chromatographic column (Amersham Pharmacia Biotech, Uppsala, Sweden) after ultrafiltration using a 1-kDa molecular mass cutoff membrane. Via ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, we determined that peptides Thr-Asp-Val-Glu-Asn [TDVEN; β-casein f(143-147)], Leu-Gln-Pro-Glu [LQPE; β-casein f(103-106)], Val-Ala-Pro-Phe-Pro-Glu [VAPFPE; αS1-casein f(40-45)], and Val-Leu-Pro-Val-Pro-Gln [VLPVPQ β-casein f(185-190)] reduced micellar cholesterol solubility. After Caco-2 cells were treated with LQPE, VLPVPQ, and VAPFPE, the Niemann-Pick C1-Like 1 (NPC1L1) protein levels decreased by (means ± SEM) 19.33 ± 2.47%, 52.1 ± 3.77%, and 23.09 ± 8.52%, respectively, compared with the control group. Treatment with each peptide induced significant upregulation of ATP binding cassette subfamily G member 8 antibody (ABCG8) mRNA expression by 398.1 ± 23.27%, 86.4 ± 27.07%, and 92.8 ± 8.49%. We found that VLPVPQ and LQPE significantly upregulated ATP-binding cassette transporter A1 (ABCA1) transcription by 203.9 ± 8.44% and 220.8 ± 36.42% respectively, whereas VLPVPQ significantly decreased mRNA expression of acetyl-CoA-acetyltransferase 2 (ACAT2) and microsomal triacylglycerols (MTP). The cholesterol-lowering action of milk-derived peptides may be induced by suppression of micellar cholesterol solubility and affects the expression of cholesterol absorption-related proteins and enzymes in intestinal epithelial cells. This research discovers new milk-derived peptides with decreasing cholesterol micellar solubility and provides a theoretical basis of in vitro cholesterol-lowering effects of peptides.

Ultrasonication of Milky Stage Rice Milk with Bioactive Peptides from Rice Bran: Its Bioactivities and Absorption

Rice in early development stage contains peptides and various bioactive compounds. Rice protein hydrolysate was prepared from Riceberry bran protein hydrolysed with Alcalase® and trypsin. Protein hydrolysate from Alcalase® (MW < 3 kDa) was fractionated into 12 fractions using RP-HPLC and tested for antioxidant, ACE, and ROS-inhibitory activity. Peptide from RP-HPLC fraction 1 showed that the lowest ROS inhibition and highest antioxidant were identified by LC-MS/MS as Val-Leu-Ala-Ala-Gly-Cys-Pro-Leu. Peptide fraction 4 showed that the strongest in vitro ACE-inhibitory activity was identified as Ala-Met-Ser-Phe-Ala-Glu-Met. Young Riceberry milk was then enriched with Alcalase® protein hydrolysate (MW < 3 kDa) and sonicated at amplitude 20% for 30 min and 40% for 60 min. Particle size, transmittance, and Caco-2 cell absorption were measured. The highest peptide content and % absorption was found in young rice milk ultrasonicated at 40% for 60 min. Rice bran protein hydrolysate produced by Alcalase® had much smaller MW bioactive peptides and could be used as a potent functional food ingredient. The young rice milk fortified with bioactive peptides after high power ultra-sonication could increase the in vitro intestinal absorption.

Differences between phytosterols with different structures in regulating cholesterol synthesis, transport and metabolism in Caco-2 cells

Abstract Phytosterols have been shown to be effective in reducing blood cholesterol by suppressing intestinal cholesterol absorption in both humans and animals. In present study, the inhibition of cholesterol absorption in Caco-2 cells by ergosterol, ergosterol acetate, β-sitosterol, stigmasterol, campesterol and stellasterol were systematically assessed. Results showed that the phytosterols produced cholesterol-lowering effect in Caco-2 cells. This effect was largely mediated via reducing the biosynthesis of cholesterol (HMGCS1); meditating the key proteins in cholesterol transportation and metabolism (NPC1L1, ABCG5/ABCG8); interrupting the conversion of cholesterol to cholesterol esters (ACAT2). Results indicated that their cholesterol-lowering activity may be correlated with the functional group (hydroxyl or ester) on C-3 position (ergosterol stellasterol). Further researches are needed to confirm the cholesterol-lowering effects of phytosterols with functional group (methyl or ethyl) on C-24 and the functional groups on C-22 position.

Formation and Identification of Two Hydroxmethylfurfural-Glycine Adducts and Their Cytotoxicity and Absorption in Caco-2 Cells.

Our previous research showed that thioacetal and Schiff base formed between 5-hydroxymethylfurfural (HMF) and cysteine or lysine considerably decreased the cytotoxicity of HMF. In this study, two adol condensation adducts, named 2β-amino-3α-hydroxy-3-(5-(hydroxymethyl)furan-2-yl)propanoic acid (HGA) and 2α-amino-3β-hydroxy-3-(5-(hydroxymethyl)furan-2-yl)propanoic acid (HGB), were prepared from the reaction products of glycine and HMF, and their cytotoxicities were investigated in Caco-2 cells. Compared with HMF, HGA and HGB displayed lower cytotoxicities against Caco-2 cells with IC50 values of 36.50 and 43.47 mM, respectively, versus 16.11 mM (HMF). In contrast to our findings in thioacetal and Schiff base products, HGA and HGB underwent a very high metabolism rate (99%) in Caco-2 cells. HGA and HGB may degrade to other products instead of HMF since no extracellular or intracellular HMF was detected.