Motohiro Shima

Preparation of fine W/O/W emulsion through membrane filtration of coarse W/O/W emulsion and disappearance of the inclusion of outer phase solution

Abstract A water-in-oil-in-water (W/O/W) emulsion was prepared as a carrier system for the daily uptake of a bioactive compound using decaglycerol monolaurate and hexaglyceryl condensed ricinolate as hydrophilic and lipophilic surfactants, respectively. 1,3,6,8-Pyrenetetrasulfonic acid tetrasodium salt (PTSA) was used as a hydrophilic model compound of a bioactive substance. Membrane filtration of a coarse W/O/W emulsion prepared with a rotor/stator homogenizer produced a fine emulsion which had a mean oil-droplet diameter of 90%. Microscopic observations revealed that the fluorescent material in the outer-phase solution was included into the oil phase during rotor/stator homogenization. It was suggested that because the rotor/stator homogenizer was used to produce both O/W and W/O/W emulsions, some part of the outer phase solution would be included in the oil phase during the second homogenization. The fluorescence shown in the oil droplets of the coarse W/O/W emulsion was diminished after membrane emulsification. This phenomenon suggested that the included outer-phase solution was released to the outer-phase solution from the interior of the W/O/W oil droplets during the membrane emulsification.

Physico-chemical Properties of Fatty Acids for Assessing the Threshold Concentration to Enhance the Absorption of a Hydrophilic Substance

The effects of natural and non-natural fatty acids on enhancing the absorption of a hydrophilic marker through a human epithelial cell (Caco-2) monolayer were measured to elucidate the properties of the fatty acids. Fatty acids from C9 to C14 enhanced the absorption depending on the concentration and the carbon chain length. Those fatty acids with longer chains gave a higher permeability coefficient at low concentrations and a lower toxicity than those with shorter chains. The surface energy lowering coefficient (SELC), an intrinsic physico-chemical property, and the critical micellar concentration (CMC) were good criteria for identifying the threshold concentrations of a fatty acid to significantly enhance absorption.

Effects of Medium-Chain Fatty Acids on Intracellular Calcium Levels and the Cytoskeleton in Human Intestinal (Caco-2) Cell Monolayers

The effects of medium-chain fatty acids (MCFA) on intracellular calcium (Ca2+) levels and actin filaments in the Caco-2 monolayer were investigated. A site-dependent increase in intracellular Ca2+ levels caused by decanoic acid (C10) at 19 mM was observed by confocal laser scanning microscopy. The area in which the intracellular Ca2+ levels was increased was measured by image analysis, and increased to 11% of the total area of the monolayer within 1 minute. This was maintained for 5 minutes, and decreased thereafter. The other MCFAs did not significantly increase the intracellular Ca2+ levels. Obvious morphological changes of actin filaments were induced by only C10 among C8-C14. The area in which actin filaments were depleted was also quantified, and the increase in area became significant after 40 minutes. The area of the actin-depleted spot corresponded to the area occupied by 5 to 10 cells as well as that in which the intracellular Ca2+ level was increased. The effectiveness of only C10 suggested that the mechanism of the absorption enhancement by C10 would be different from that by the other MCFAs, or that C10 has some additional physiological functions although the mechanism of the enhancement is the same as for the other MCFAs.

Suppressive Effect of Alkyl Ferulate on the Oxidation of Linoleic Acid

The oxidation processes of linoleic acid in the presence of ferulic acid, and 1-pentyl, 1-hexyl and 1-heptyl ferulates were observed at various temperatures and different molar ratios of each additive to linoleic acid. The processes were analyzed based on a kinetic equation of the autocatalytic type to evaluate the oxidative rate constant, k, and the kinetic parameter, Y 0, by which the initiation period for the oxidation of linoleic acid was mainly governed. The k values for linoleic acid mixed with each of the alkyl ferulates were smaller than that for linoleic acid mixed with ferulic acid. The greater suppressive effect of the alkyl ferulates would be ascribable to their higher solubility in linoleic acid. Both the activation energy, E, and the frequency factor, k 0, for the oxidation of linoleic acid mixed with ferulic acid or pentyl ferulate decreased with increasing molar ratio of the additive to linoleic acid.

Recovery of Caco-2 Cell Monolayers to Normal from the Transport-enhanced State Induced by Capric Acid Sodium Salt and its Monoacylglycerol.

Caco-2 cell monolayers were used as a model of the intestinal epithelium to investigate the recovery profile from the transport-enhanced state induced by the transport enhancers, capric acid sodium salt (C10FANa) and capric acid monoacylglycerol (C10MG). The transepithelial electrical resistance, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay, and lactate dehydrogenase (LDH) release rate were investigated. The cell monolayer recovered depending on the concentration of the enhancer and on the exposure time. The MTT assay revealed that the cells recovered their mitochondrial dehydrogenase activity without proliferation. The cell monolayer exposed to C10FANa released LDH to both the apical and basolateral sides, but to C10MG, only to the apical side. The results were compared with those for SDS and taurocholic acid sodium salt, and the effect of C10FANa was found to be different. These results suggest that the damage by MCFA compounds is recoverable and that the recovery can be assessed by an MTT assay, but that the LDH-release behavior is different among the enhancers.

The Relationship between Transport-enhancement Effects and Cell Viability by Capric Acid Sodium Salt, Monocaprin, and Dicaproin

Using Caco-2 cell monolayers and MTT assay, the relationship between cell viability (a) and transport-enhancement effect of 1,2-dicaproin (C6DG), monocaprin (C10MG), and capric acid sodium salt (C10FANa) was examined. Transport enhancement effect was assessed by apparent permeability (P app) of penicillin V. There was a linear relationship between (P app-aS a) and (1-a) values, where S a was the apparent permeability for the viable cells. The apparent permeability for the damaged cells (S d) was evaluated from the slope of the line. Each of the enhancer compounds gave a different S d value 2.00×10(-4), 0.82×10(-4), and 0.10×10(-4) cm/s for C6DG, C10MG, and C10FANa, respectively, but the value was independent of its concentration for C10MG and C10FANa. C6DG would be the safest enhancer among the three compounds because of its high S d value at the low level of cell damage. S d could be used as a criterion for estimating the safety of enhancers.

Relationship between Structure and Permeability of Tryptophan Derivatives Across Human Intestinal Epithelial (Caco-2) Cells

ʟ-Trp and its derivatives were used as model compounds to clarify structural factors which influence the intestinal epithelial permeation and metabolism of amino-acid derivatives. Permeability of model compounds through Caco-2 cells was used as an in vitro absorption model for human intestinal epithelial cells. The influence of compound concentration, the effects of various transporter substrates on permeability coefficients, and pH dependency of permeability coefficients were investigated. The transcellular permeability of Trp and Trp-NH2 in the direction from the apical side to the basolateral side, in which nutrients and drugs were ordinarily absorbed, declined with increasing concentration and saturated at more than 1 and 0.4 mᴍ, respectively. The permeability coefficients for N-terminal protected Trp derivatives and Ac-Trp-NH2 showed similar and constant values in both from the apical-to-basolateral and basolateral-to-apical directions. In addition, significant inhibition of the apical-tobasolateral permeation of Trp by Leu and Phe was observed. The permeability coefficient ratio at pH 6.3 to that at pH 7.3 was explained by the ratio of the ionic form to the neutral form of the compounds. Based upon these results and the partition coefficients in the 1-octanol/water system, possible absorption mechanism of Trp and its derivatives across Caco- 2 cells was proposed

Autoxidation of Mono-, Di-, and Trilinoleoyl Glycerols at Different Concentrations

Mono-, di-, and trilinoleoyl glycerols were diluted with 1-undecanol or hexadecane to produce specific concentrations, and their oxidation processes were measured at 65 °C at 12% relative humidity. The rate constants for oxidation of the linoleoyl residue were proportional to the concentration for all substrates. This fact suggests that no intramolecular radical chain reaction between the linoleoyl residues occurred.

Trypsin-Catalyzed Synthesis of Oligopeptides Containing Hydrophilic and Essential Amino Acid, L-Lysine

The synthesis of peptides from N-(benzyloxycarbonyl)-L-lysine n-hexyl ester (Z-K-OHex) and L-lysine amide (K-NH2 was done in 65% (v/v) acetonitrile. We mainly got two dipeptides (Z-KK and Z-KK-NH2) and a tripeptide (Z-KKK-NH2). We had Z-KK-NH2 in the 90% yield but it rapidly decreased as the reaction proceeded. The decrease of the concentration of trypsin prolonged the period for which the yield was maintained at 90%. The yields of Z-KK and Z-KKK-NH2 increased when the concentration of K-NH2 increased. The above results were well simulated by a model of the reaction.