Naoki Sugimoto

Marker Constituents of the Natural Antioxidant Eucalyptus Leaf Extract for the Evaluation of Food Additives

In order to establish the marker constituents of the natural antioxidant food-additive Eucalyptus leaf extract, the UV-absorbing constituents of two eucalyptus leaf extracts registered as food additives (eucalyptus A and B) were investigated. Several major peaks on the reversed-phase HPLC chromatogram of eucalyptus A were characterized as gallic acid, ellagic acid, 3-O-β-D-glucuronides of quercetin and kaempferol, and a hydrolyzable tannin dimer, oenothein B, by direct comparison with authentic specimens isolated from Eucalyptus globulus leaves. A new gallotannin was found in the E. globulus leaf extract, and its structure was found to be 1,2,3,6-tetra-O-galloyl-β-D-galactose. Two major peaks on the HPLC chromatogram of eucalyptus B were identified as gallic acid and ellagic acid, indicative of degradation products from hydrolyzable tannins in the leaves. Considering the evaluation of antioxidant activity by radical scavenging ability, a standardization of eucalyptus leaf extract, including the antioxidative polyphenol, oenothein B, is proposed.

Absolute quantification for benzoic acid in processed foods using quantitative proton nuclear magnetic resonance spectroscopy.

The absolute quantification method of benzoic acid (BA) in processed foods using solvent extraction and quantitative proton nuclear magnetic resonance spectroscopy was developed and validated. BA levels were determined using proton signals (δ(H) 7.53 and 7.98) referenced to 2-dimethyl-2-silapentane-5-sulfonate-d(6) sodium salt (DSS-d(6)) after simple solvent extraction from processed foods. All recoveries from several kinds of processed foods, spiked at their specified maximum Japanese usage levels (0.6-2.5 g kg(-1)) and at 0.13 g kg(-1) and 0.063 g kg(-1), were greater than 80%. The limit of quantification was confirmed as 0.063 g kg(-1) in processed foods, which was sufficiently low for the purposes of monitoring BA. The accuracy of the proposed method is equivalent to the conventional method using steam-distillation extraction and high-performance liquid chromatography. The proposed method was both rapid and simple. Moreover, it provided International System of Units traceability without the need for authentic analyte standards. Therefore, the proposed method is a useful and practical tool for determining BA levels in processed foods.

Application of 1H-NMR spectroscopy to validation of berberine alkaloid reagents and to chemical evaluation of Coptidis Rhizoma

Berberine, palmatine, and coptisine are major pharmacologically active protoberberine alkaloids in Coptidis Rhizoma, and have been used as indices for chemical evaluation of the crude drug. 1H-NMR spectroscopy was applied to determination of purities of commercial reagents of protoberberine alkaloids. The purities of the alkaloids were calculated from the ratios of the intensities of the H-13 singlet signal at about δ 8.7xa0ppm of target protoberberine alkaloids to integration of a hexamethyldisilane (HMD) signal at 0xa0ppm. The concentration of HMD was corrected with SI traceability using potassium hydrogen phthalate of certified reference material (CRM) grade. The purity of the reagent estimated by the 1H-NMR was, in general, lower than that claimed by the manufacturer, leading to over-estimation of the alkaloid contents of Coptidis Rhizoma when determined by HPLC. The present quantitative 1H-NMR method was also applicable to direct determination of protoberberine alkaloid contents in Coptidis Rhizoma.

Evaluation of gardenia yellow using crocetin from alkaline hydrolysis based on ultra high performance liquid chromatography and high‐speed countercurrent chromatography

Gardenia yellow is globally the most valuable spice and food color. It is generally a mixture of water-soluble carotenoid glycosyl esters which consist of crocetin bis(gentiobiosyl) ester as the main component. Crocetin is a natural carotenoid dicarboxylic acid that may be a candidate drug for pharmaceutical development, however, it is either present in trace amounts or is absent in natural gardenia yellow products. We here propose that crocetin produced by alkaline hydrolysis can be used to qualitatively evaluate gardenia yellow products using an ultra high performance liquid chromatographic assay. A useful and efficient isolation technique for isolating high-purity crocetin from gardenia yellow using high-speed countercurrent chromatography is described. High-speed countercurrent chromatographic fractionation followed by an ultra high performance liquid chromatographic assay showed that trans-crocetin is easily converted to about 15% cis-crocetin (85% trans-crocetin). Crocetin in gardenia yellow was quantitatively evaluated. Our approach is based on the hydrolysis process for converting crocetin glycosyl esters to crocetin before evaluation and isolation using the ultra high performance liquid chromatographic and high-speed countercurrent chromatographic methods. The combination of hydrolysis and chromatographic methods allows evaluation of the purity and quantity of crocetin in gardenia yellow.