A new metallofluorescent reagent (3-carboxy-2-naphthylamine-N, N-diacetic acid (CNDA)) was synthesized. Its possible application was discussed. CNDA was stable in 1% NaHCO3 solution. The fluorescence intensity of CNDA increased by the addition of Al, La (III) and Zn at pH 3-5 and Ca and Mg at pH 10. The acid dissociation constants (pKa) of CNDA were determined by the potentiometric method. The dissociation constants of carboxylic protons of CNDA were 2.44 for pKa1 and 3.23 for pKa2, and the pKa3 of ammonium proton was 7.67. The stability constants of CNDA Ca, Mg and Zn chelate were determined potentiometrically in accordance with the Bjerrum's method at 25± 0.2°C. The stability constants of CNDA Al and La (III) chelate were determined by spectrophotometric method at 25±0.5°C. The constitution of the CNDA chelate with each metal ion was 1 : 1, and the logarithmic stability constants of Al, Ca, La (III), Mg and Zn chelate were 11.83, 5.16, 8.25, 3.24 and 8.26, respectively. The relative quantum yields of CNDA were 0.04-0.05 at pH 5.0 or bellow and 0.09-0.10 at pH 7.0 or more. The relative quantum yields of the fluorescence of CNDA chelate were 0.06 (pH 3.0), 0.09 (pH 4.0) and 0.11 (pH 4.5) for Al, 0.08 (pH 5.0) for La (III), 0.10 (pH 4.5) for Zn, 0.18 (pH 7.0) and 0.27 (pH 10) for Ca, and 0.14 (pH 7.0) for Mg. Since CNDA can react with hard acid, such as Al, La (III) and Ca, CNDA should be a useful reagent for fluorophotometry and a metallofluorescent indicator for chelatometric titration. Using CNDA as a metallofluorescent indicator, the fluorophotometric titration of fluoride in p-fluorobenzoic acid and dexamethasone with Al by the oxygen flask method was performed. In titration, the recoveries were 99.3% or more, and the standard deviation were within 0.23%.
A rapid and simple method for the determination of allantoin in pharmaceuticals by reversed-phase ion-pair high-performance liquid chromatography using an ODS column was presented. In general, it is difficult to retain allantoin to the ODS column owing to its very low hydrophobicity. We solved these problems by the use of a Tris-HCl buffer (pH 7.5) containing tetra-n-hexylammonium bromide (THAB) as an ion-pair reagent for the mobile phase. Comparatively low concentrations of Tris-HCl buffer (0.9 mM) and THAB (0.5 mM) gave a high capacity factor (k'). As a results of the examination of the chromatographic behavior, it is comfirmed that the retention mechanism of allantoin to the ODS column on the present method was not the ion-pair mode, but the ion-exchange mode. Calibration curves for allantoin showed a good linearity in the range of 10 to 400 μg/ml (r=0.9999). The reproducibility (R.S.D., n=6) was invariably good (0.37%). The lowest concentration of allantoin for the determination was 200 ng per 20 μl of injection. The present method was successfully applied to the determination of allantoin in commercial eyedrops with good recovery (99.4%). It was found that allantoin in pharmaceuticals could be determined by the present method in short time and without any complicated derivatization.
We examined the effect of a carbon tetrachloride (CCl4)-induced hepatic injury on the stereoselective N-demethylation of RS-(±)-chlorpheniramine (Chp) by cytochrome P450 (CYP) 2C11 isozyme. In the non-treated rat liver microsomes, the stereoselective N-demethylation of racemic Chp was observed. However, in the CCl4-treated (0.5 ml/kg, i.p.) rat liver microsomes, the N-demethylation activities of S-(+)- and R-(-)-Chp decreased continuously up to the third day after the treatment with CCl4, and reached about 9 and 13% of control values, respectively, and the stereoselective N-demethylation of Chp was not observed. Moreover, in the liver microsomes at the 7th day after the treatment with CCl4, the N-demethylation activities of both enantiomers recovered to an original level, and the stereoselective N-demethylation of Chp was again observed. The addition of 30μl of the anti-rat CYP2C11 serum to the reaction mixture containing 1 mg of microsomal protein inhibited the formation of monodesmethylchlorpheniramine (DMChp) from both enantiomers to 74 and 57% of the control values for S-(+)- and R-(-)-Chp, respectively. In the liver microsomes of a male rat at the 1st day after the treatment of CCl4, the addition of the anti-rat CYP 2C11 serum (30μl) also caused 25% inhibition of the formation of DMChp from S-(+)-Chp, but anti-rat CYP2C11 had no inhibitory effect on the rates of microsomal N-demethylation of R-(-)-enantiomer. On the other hand, in the liver microsomes of a male rat at the 7th day after the treatment with CCl4, the anti-rat CYP2C11 serum had an inhibitory effect on the rates of microsomal N-demethylation of either S-(+)- or R-(-)-enantiomers again. Moreover, it was confirmed by Western blotting analysis that the density of the stained bands of CYP2C11 in the liver microsomes from male rats at the 1st, 2nd and 3rd days after the treatment with CCl4, was thinner than that from non-treatment male rats. These results indicated that the changes of N-demethylation activities of Chp in the CCl4-induced hepatic injury were due to the variation of microsomal CYP2C11.
We examined sensitization and crossreaction by the guinea pig maximization test (GPMT) with phenolic compounds. Skin specimens were collected from earlobes of BALB/c mice on 24 h after elicitation phase of contact hypersensitivity reaction (CHR) with phenolic compounds. The expression of cytokines of skin specimens was examined by the reverse transcriptase/polymerase chain reaction (RT-PCR) method. Consequently, phenolic compounds which showed positive reaction in GPMT expressed IL-2 and IFN-γ on 24 h after elicitation, and some phenolic compounds showed marked crossreaction. Theirfore, it was found that on several phenolic compounds, dimerization of these compounds from monomer to dimer decrease sensitization.
The constituents of 13 species of bamboos and bamboo grasses were investigated. Almost all the species gave isoorientin and related flavone 6-C-glycosides as major constituents of leaves. Tricin 5-O-β-D-glucopyranoside and p-coumaric acid were also obtained as minor ones in some cases. (-)-Lyoniresinol 3a-O-β-D-glucopyranoside and (+)-lyoniresinol 3a-O-β-D-glucopyranoside were common constituents in culms of 4 species examined. 3, 4, 5-Trimethoxyphenol β-D-glucopyranoside, koaburaside, 2, 6-dimethoxy-p-benzoquinone, p-coumaric acid, sinapic aldehyde and ferulyl aldehyde occurred also in culms sporadically. Arbutin was a constituent of roots of a species examined.