The mechanism of paracellular expansion by absorption enhancers, e.g., EDTA, sodium caprate (C10), and decanoylcarnitine (DC), was studied, the focus being on the process of actin microfilament contraction in the tight junction. The effects of various inhibitors such as KN-62 (a specific inhibitor of Ca2+/calmodulin dependent protein kinase), H7 (a protein kinase C (PKC) inhibitor), and W7 (a calmodulin antagonist) were examined on the paracellular expansion by the enhancers in Caco-2 cells. From the experimental results, the following mechanisms were suggested. EDTA activates PKC by depletion of extracellular calcium via chelation resulting in expansion of the paracellular route. C10 increases the intracellular calcium level by an interaction with the cell membrane independent of cell polarity resulting in contraction with actin microfilament. DC interacts specifically with the apical membrane to increase the intracellular calcium level, but the mechanistic details subsequent to the increase of calcium are not clear.
An important parameter used in physiologically based pharmacokinetic models is the partition coefficient (Kp), which is defined as the ratio of tissue drug concentration to the concentration of drug in the emergent venous blood of the tissue. Since Kp is governed by reversible binding to protein and other constituents in blood and tissue, an attempt was made here to estimate the Kp values for a model drug ethoxybenzamide (EB) by means of in vitro binding studies and to compare these Kp values to those obtained from in vivo kinetic parameters observed following the administration of EB by two different routes, i.e., i.v. bolus injection and constant rate infusion. The Kp values obtained by using these three different methods were in reasonably good agreement suggesting that binding data obtained in vitro can successfully be used to estimate in vivo distribution.
Garlic exerts various effects on the biological systems: e.g., inhibiting platelet aggregation (1-3), lowering serum and liver cholesterol levels (4, 5), and antibacterial activity (6). We have indicated that the aged garlic extract which was obtained by the ethanol-extraction of cracked garlic bulbs (Allium sativurn L.) protected biological membranes from lipid peroxidation (7). It is essential to clarify the component in garlic extract which has this antioxidant activity.We have tried to identify this component by fractionating the aged garlic extract. The garlic extract was fractionated through ethyl acetate-extraction and silica gel column chromatography with n-hexane as the eluent. The sulfide fractions thus obtained were further separated by high performance liquid chromatography [(HPLC; TSK gel ODS 8OTM (TOSOI-I), eluent: 90% ethanol]. All fractions obtained from these separation processes were subjected to the assay for antioxidant activity, using rat liver microsomes in the same way as described previously (7). Lipid peroxidation in rat liver microsomes was induced by ascorbic acid and Fe2 and was evaluated by the formation of thiobarbituric acid reactive substances (TBA-RS). As a result, five components were found to have high activities to inhibit lipid peroxidation. 'Fl-NMR, C-NMR, and the high resolution mass spectroscopy revealed these components to be the diallyl polysulfides [CH2 = CH -CH2 -(S) -CH2CH=CH2I (n = 3-7), i.e., diallyl trisulfide, diallyl tetrasulfide, diallyl pentasulfide, diallyl hexasulfide, and diallyl heptasulfide. Their inhibiting effects against lipid peroxidation were dose-dependent. A representative example is shown in Fig. 1. A remarkable inhibition against lipid peroxidation was observed at the concentration of 25g/ml diallyl heptasulfide. The inhibiting effects of all the diallyl polysulfides against lipid peroxidation were compared at a concentration of 25.tg/ml (Fig.2). Among them, in particular, four diallyl polysulfides, the diallyl tetra-, penta-, hexa-, and heptasulfides, exhibited much more potent antioxidant activities than diallyl trisulf'ide. The crude aged garlic extract showed an almost perfect protection against lipid peroxidation in rat liver microsomes at 40 mg/mI of garlic extract, as shown in the previous paper (7). In the same reaction systems, these four Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
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