Xiu-Min Chen

Defining conditions for the co-culture of Caco-2 and HT29-MTX cells using Taguchi design

INTRODUCTION The co-culture of Caco-2 and HT29 cells for testing intestinal drug and nutrient transport and metabolism provides the presence of both absorptive and goblet cells, both of which have different culture requirements for optimal growth and function. The research on the co-culture of Caco-2 and HT29 cells is very limited in respect to refining specific conditions that reduce intra- and inter-laboratory variations. In the present study we reported conditions that enable reproducible results to be obtained for drug permeability using in vitro co-culture of Caco-2 and HT29-MTX based on Taguchi experimental design. METHODS The selection of four factors that specified cell culture conditions, namely culture medium, seeding time, seeding density, and Caco-2:HT29-MTX ratio on TEER value and individual permeability coefficients of propranolol, ketoprofen and furosemide was established. Based on the selected conditions for co-culture, we also confirmed the functionality of the final chosen culture condition using nitric oxide as an indicator of intestinal inflammation. RESULTS Choice of cell culture time and culture medium represented two of the most important factors that affected TEER values and the permeability coefficients of the model drugs. On the other hand, the seeding density and the Caco-2:HT29-MTX ratio exerted no significant influence on TEER values and the drug permeability coefficients. No absolute optimal cell culture condition could be obtained for all drugs; however subsequent confirmation experiments concluded that excellent precision for TEER values and drug permeability coefficients was obtained from the two operators using the following combination of conditions, namely an initial seeding density of 1 x 10(5) Caco-2 and HT29-MTX cells/cm(2) at a ratio of 9:1, followed by a 21day culture time in MEM medium. Finally, functionality of the co-culture model system using the above selected in vitro conditions resulted in comparable nitric oxide synthesis to that of a Caco-2 cell monolayer. DISCUSSION Taguchi experimental design enabled us to define a combination of in vitro culture conditions that resulted in excellent operator reproducibility for determining drug permeability coefficients in a Caco-2 and HT29-MTX co-culture system. Moreover, the selected conditions used in co-culture of absorptive and goblet intestinal cells did not compromise the synthesis of nitric oxide, an indicator of inflammation, measured in Caco-2 monolayers.

Demonstration of antioxidant and anti-inflammatory bioactivities from sugar-amino acid maillard reaction products.

Maillard reaction products (MRPs), both crude and fractionated, were assessed for antioxidant potential using cell-free, in vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, Fenton reaction induced deoxyribose degradation and oxygen radical absorbance capacity-fluorescein (ORACFL) chemical assays. All MRPs displayed various affinities to scavenge free radicals generated in different reaction media and using different reactive oxygen species (ROS) substrates. High molecular weight MRPs consistently showed the greatest (P < 0.05) antioxidant potential in chemical assays. Repeating these tests in Caco-2 cells with both reactive oxygen and nitrogen (RNS) intracellular assays revealed that the low molecular weight components (LMW) were most effective at inhibiting oxidation and inflammation. In particular, a glucose-lysine (Glu-Lys) mixture heated for 60 min had marked intracellular antioxidant activity and nitric oxide (NO) and interleukin-8 (IL-8) inhibitory activities compared to other MRPs (P < 0.05). Further studies employing ultrafiltration, ethyl acetate extraction, and semipreparative high-performance liquid chromatography (HPLC) produced a bioactive fraction, termed F3, from heated Glu-Lys MRP. F3 inhibited NO, inducible nitric oxide synthetase (iNOS), and IL-8 in interferon γ (IFN-γ)- and phorbol ester (PMA)-induced Caco-2 cells. F3 modified several gene expressions involved in the NF-κB signaling pathway. Two components, namely, 5-hydroxymethyl-2-furfural (HMF) and 5-hydroxymethyl-2-furoic acid (HMFA), were identified in the F3 fraction, with an unidentified third component comprising a major portion of the bioactivity. The results show that MRP components have bioactive potential, especially in regard to suppressing oxidative stress and inflammation in IFN-γ- and PMA-induced Caco-2 cells.

Antioxidant Activity and Chemical Properties of Crude and Fractionated Maillard Reaction Products Derived from Four Sugar–Amino Acid Maillard Reaction Model Systems

Antioxidant activity of Maillard reaction products (MRPs) derived from four sugar–amino acid Maillard reaction model systems (glucose [Glc] or ribose [Rib] reacted with glycine [Gly] and L‐lysine [Lys]) were examined in terms of chemical properties and molecular weight fractionation of reaction products. Rib–amino acid model systems produced MRPs with higher antioxidant activity than Glc–amino acid model MRPs (P < 0.05, Rib–Lys > Rib–Gly > Glc–Lys > Glc–Gly). In the same sugar or same amino acid model systems, antioxidant activity of MRPs was negatively related to the final pH, fluorescent intensity, and the content of dicarbonyl compounds. Antioxidant activity positively related to the production of late‐stage browning MRPs. Fraction I from the Glc–Lys model system separated by gel filtration chromatography had the highest oxygen radical absorbance capacity (ORAC) value (1736 μmol Trolox/g MRP). Fraction IV from the Rib–Lys model system had a higher ORAC value compared to Fraction III. This result indicated that high molecular weight MRPs do not necessarily have higher antioxidant activity compared to low molecular weight MRPs.

Determining conditions for nitric oxide synthesis in Caco-2 cells using Taguchi and factorial experimental designs

Intestinal inflammation correlates well with the increased synthesis of nitric oxide (NO), which is attributed mainly to the up-regulation of inducible nitric oxide synthase (iNOS). We optimized the use of interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), lipopolysaccharide (LPS), and phorbol myristate acetate (PMA) as inducers to stimulate NO synthesis in Caco-2 cells using a Taguchi design. The results indicated that IFN-gamma was the most important inducer of iNOS in Caco-2 cells. Treating Caco-2 cells with both IFN-gamma and PMA using an optimal mixture of 8000 U/ml IFN-gamma and 0.1 microg/ml of PMA resulted in a synergistic induction of NO synthesis. Further experiments using a 5-factor/2-level factorial design including Caco-2 growth conditions such as cell passage, culture medium composition, cell seeding time and density, and stimulation time were also performed. Cell seeding and stimulation times significantly (P<0.05) affected NO synthesis, whereas culture medium and seeding density did not appreciably affect NO synthesis in Caco-2 cells. Western blotting and RT-PCR findings confirmed that the optimal mixture of IFN-gamma and PMA effectively up-regulated iNOS mRNA and protein. The induced NO, iNOS mRNA, and protein were all inhibited by the iNOS selective inhibitor, aminoguanidine (AG).

Antioxidant and anti-inflammatory activities of Maillard reaction products isolated from sugar-amino acid model systems.

We investigated the antioxidant and anti-inflammatory activities of both crude and ultrafiltrated Maillard reaction (MR) products (MRPs) derived from sugar-amino acid MR models, comprising fructose (Fru), glucose (Glu) or ribose (Rib) reacted with glycine (Gly) or lysine (Lys), respectively. Crude MRPs derived from Glu-Lys showed the greatest capacity (P < 0.05) to inhibit nitric oxide (NO) and interleukin 8 (IL-8) production in interferon γ and phorbol ester-induced Caco-2 cells. Moreover, one ultrafiltrated fraction (MW < 1 kDa) recovered from Glu-Lys exhibited the greatest (P < 0.05) affinity to inhibit NO. This effect also corresponded to an inhibition of both iNOS transcription and translation. The NO and IL-8 inhibitory activities of crude MRPs were positively correlated with intracellular oxidation inhibitory activity. In conclusion, we have demonstrated an anti-inflammatory capacity of MRPs in inflamed Caco-2 cells that is specific to low molecular weight (MW < 1 kDa) Glu-Lys MRPs.

Correlating changes that occur in chemical properties with the generation of antioxidant capacity in different sugar-amino acid Maillard reaction models.

UNLABELLED We investigated the development of antioxidant activity relative to the change of pH, fluorescent intensity, ultraviolet (UV) absorbance (A294), browning (A420), and alpha-dicarbonyl compounds in sugar-amino acid Maillard reaction (MR) model systems comprising fructose, glucose, or ribose each with glycine (Fru-Gly, Glu-Gly, and Rib-Gly) or lysine (Fru-Lys, Glu-Lys, and Rib-Lys), respectively, which were heated at 121 °C for 5 to 90 min. For hexose models, the change in pH was shown to fit a second-order polynomial regression with A294 and A420. Antioxidant activity was significantly and positively correlated with UV absorbance (r = 0.905, P < 0.001) and browning products (r = 0.893, P < 0.001) rather than with fluorescent products or the alpha-dicarbonyl compounds. Type of sugar was most important in evoking a change in UV absorbance, browning, alpha-dicarbonyl compounds, and antioxidant activity of MR products (MRPs). In conclusion, the antioxidant activity of MRPs in six model systems was more closely associated with products derived at the intermediate-to-late stages of the reaction and influenced mostly by the type of sugar. PRACTICAL APPLICATION We report on the different factors and their interactions that are important for understanding the functional attributes of food components that comprise the generation of Maillard browning products and the associated antioxidant activities generated during high-temperature food processing.

Elucidation of the Chemical Structure and Determination of the Production Conditions for a Bioactive Maillard Reaction Product, [5-(5,6-Dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]methanol, Isolated from a Glucose–Lysine Heated Mixture

We previously isolated a bioactive molecule, named F3-A, from an aqueous glucose (Glc) and lysine (Lys) Maillard reaction (MR) model system. Herein, F3-A was verified as [5-(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]methanol (5) and was subsequently synthesized for confirmation of bioactivity. Using Taguchi and factorial designs, we determined that the conditions which best increased the yield of F3-A were at pH 6 with a sugar:amino acid ratio of 2:1 and heating time of 12 h at 100 °C. The MR mixtures containing glucose produced highest yield, compared to fructose, lactose, and sucrose. Both the F3-A recovered from Glc-Lys MR mixture and the synthesized product exhibited significant (P < 0.05), dose dependent, nitric oxide (NO) inhibitory activity in Caco-2 cells that was comparable to aminoguanidine (AG) and pyrrolidine dithiocarbamate (PDTC), respectively. Finally, an additional inhibitory effect of F3-A was determined when coincubated with AG in cytokine-induced Caco-2 cells. This bioactivity points to a potential role in preventing intestinal inflammation.

Chemical properties and reactive oxygen and nitrogen species quenching activities of dry sugar–amino acid maillard reaction mixtures exposed to baking temperatures

Maillard reaction products (MRPs) derived from 10 different, dry sugar-amino acid reaction model systems were examined for changes in color index (E), sugar loss, and formation of α-dicarbonyl compounds; the changes were correlated with relative activities to quench both reactive oxygen (ROS) and reactive nitrogen (RNS) species. Reducing sugars, xylose, ribose, fructose, glucose, and non-reducing sucrose were reacted with glycine (Xyl-Gly, Rib-Gly, Fru-Gly, Glc-Gly, and Suc-Gly), or lysine (Xyl-Lys, Rib-Lys, Fru-Lys, Glc-Lys, and Suc-Lys), respectively, at temperatures of 150°C and 180°C for time periods ranging from 5 to 60min. ROS quenching capacity was negatively correlated with color index (E) (r=-0.604, P<0.001), and positively correlated with sugar loss (r=0.567, P<0.001). MRPs also exhibited activity to quench RNS as assessed by nitric oxide (NO) inhibition in differentiated Caco-2 cells that were induced with interferon-γ (IFN-γ) and phorbol ester (PMA) cocktail. We also showed a correlation between RNS and color index, sugar loss, and ROS quenching activities for MR mixtures that were heated for a short time (e.g. 10min) at 150°C. MRP quenching of ROS was largely influenced by sugar type, whereas, RNS quenching was dependent more so on the interaction between reactants and reaction conditions used to generate MRPs.

Transepithelial transport across Caco-2 cell monolayers of angiotensin converting enzyme (ACE) inhibitory peptides derived from simulated in vitro gastrointestinal digestion of cooked chicken muscles

Korat-chicken breast and thigh were subjected to heating at 70, 100 or 121 °C for 30 min and simulated in vitro gastrointestinal digestion. At 70 or 100 °C heating, digests of breast possessed higher ACE inhibitory activity than those of thigh. The highest ACE inhibitory activity was found in the digest of breast cooked at 70 °C (B/H-70), whereas breast heated at 121 °C (B/H-121) exhibited the lowest. The 1-kDa permeate of the B/H-70 digest revealed higher permeability through colorectal adenocarcinoma monolayers and ACE inhibitory activity than did B/H-121. Among nine transported peptides, APP derived from myosin showed the highest ACE inhibition, with a non-competitive characteristic (Ki 0.93 μM). Molecular docking showed that APP interacts with ACE via hydrogen bonds, electrostatic and van der Waals interactions. In conclusion, mild thermal treatment of chicken breast resulted in a higher amount of transported peptides, exerting higher ACE inhibitory activity, which could lead to potential health benefits.

Effects of processing method and age of leaves on phytochemical profiles and bioactivity of coffee leaves

The use of coffee leaves as a novel beverage has recently received consumer interest, but there is little known about how processing methods affect the quality of final product. We applied tea (white, green, oolong and black tea) processing methods to process coffee leaves and then investigated their effects on phytochemical composition and related antioxidant and anti-inflammatory properties. Using Japanese-style green tea-processing of young leaves, and black tea-processing of mature (BTP-M) coffee leaves, produced contrasting effects on phenolic content, and associated antioxidant activity and nitric oxide (NO) inhibitory activity in IFN-γ and LPS induced Raw 264.7 cells. BTP-M coffee leaves also had significantly (P < .05) higher responses in NO, iNOS, COX-2, as well as a number of cytokines, in non-induced Raw 264.7. Our findings show that the age of coffee leaves and the type of processing method affect phytochemical profiles sufficiently to produce characteristic antioxidant and anti-inflammatory activities.