Eun-Sun Yun

p38 MAPK activation is required for esculetin-induced inhibition of vascular smooth muscle cells proliferation.

The phenolic compound esculetin is known to inhibit the proliferation of vascular smooth muscle cells (VSMC). However, the signaling pathway by which esculetin mediates its molecular effects in VSMC remains to be identified. The present results suggest an unexpected role of the p38 MAPK signaling pathway in esculetin-induced inhibition of VSMC growth. Treatment of VSMC with esculetin resulted in significant growth inhibition and G1-phase cell-cycle arrest, which was followed by down-regulation of cyclins and cyclin-dependent kinase (CDK) expression. This G1-phase cell-cycle arrest was due to up-regulation of p21WAF1 expression. In addition, esculetin treatment activated p38 MAPK and ERK1/2. Pretreatment with SB203580, which is a p38 MAPK specific inhibitor, or expression of the dominant negative p38 MAPK (DN p38 MAPK) gene blocked esculetin-induced p38 MAPK activation and p21WAF1 expression. Finally, both the growth inhibition and the down-regulation of CDKs induced by esculetin were suppressed by either SB203580 or the DN p38 MAPK mutant gene. In conclusion, these results demonstrate that activation of p38 MAPK contributes to esculetin-induced p21WAF1 expression in VSMC by decreasing both the cyclin D1/CDK4 and cyclin E/CDK2 complexes. These novel results regarding the molecular mechanism of esculetin action suggest new preventive and therapeutic treatments for atherosclerosis.

Survey of Daily Caffeine Intakes from Children's Beverage Consumption and the Effectiveness of Nutrition Education

Seoul Metropolitan Government Research Institute of Public Health and Environment, Gyeonggi-do 137-734, KoreaAbstractThis study was conducted to identify daily caffeine intakes in beverages for elementary school children and to evaluate its effectiveness after nutrition education. The caffeine contents of 140 commercial beverages were analysed by high performance liquid chromatography-ultraviolet detector (HPLC-UV) and information about their consumption were obtained by surveying 267 children. Researchers gave nutrition education to the children, who were 6 to 11 years old and attended 9 classes of 3 elementary schools, by lecture, Powerpoint file and moving picture. Their preference and intake amount on beverages were investigated by questionnaire before and after nutrition education. The order on caffeine contents was coffee (33.8±2.4~49.1±5.6 mg/100 mL)> coffee milk (10.6 ±3.3 mg/100 mL)> cola (6.0 2.4 mg/100 mL)> green․black․oolong tea drink (6.0±2.4 mg/100 mL)> chocolate milk and chocolate drink (1.6±0.7~1.7 mg/100 mL)> black ice tea mix (1.3±1.7 mg/100 mL). The order on childrens preference was carbonated drink and fruit and vegetable drink (27%)> sports drink (26%)> processed cocoa mix (7%)> milk (6%)> vitamin & functional drink (3%)> green tea drink (2%)> black tea drink and coffee (1%). The average daily caffeine intakes except tea drink was 5.9±11.2 mg/person/day (0.17±0.32 mg/kg bw/day), ranged from 0.0~80.5 mg/person/day for children. The sources of caffeine were coffee 57% (3.4 mg/person/day), coffee milk 20% (1.2 mg/person/day), carbonated drink 15% (0.9 mg/person/ day), chocolate milk and chocolate drink 6% (0.4 mg/person/day), and vitamin & functional drink 2% (0.1 mg/ person/day). After nutrition education, the preference of carbonated drink, coffee, vitamin drinks & functional drink was decreased significantly (p<0.05, p<0.05, p<0.01) and the intakes of carbonated drink, chocolate milk & chocolate drink, and vitamin & functional drink were also decreased significantly (p<0.01, p<0.05, p<0.01). This study has shown that nutrition education influences the preference and the intake behavior of caffeinated beverages.Key words: caffeine, beverage, daily caffeine intakes, nutrition education

Determination of the esculetin contents of medicinal plants by liquid chromatography-tandem mass spectrometry.

We developed a LC-MS/MS method for the determination of esculetin contents in medicinal plants. The analysis was performed using multiple reaction monitoring in negative mode, and an XBridge™ C(18) column (2.1 × 100 mm, 3.5 µm) was used. Methanol and 0.1% formic acid were used for gradient analysis. The calibration curve showed good linearity (r(2)  > 0.9993). The limits of detection and quantitation were 0.02 and 0.07 ng/mL, respectively. The intra-day and inter-day precisions were 1.5-6.8 and 2.0-5.3%, respectively, and the accuracy was 102.0-110.2%. The contents of esculetin in 35 different plants were determined, and Fraxini Cortex showed the highest content of esculetin (761-5475 mg/kg). In Mori Folium and Artemisiae Capillaris Herba, 5.2-21.5 and 7.0-17.6 mg/kg of esculetin were found, respectively. In other medicinal plants, no esculetin was detected, or it was present at a concentration less than 10 mg/kg. The analysis method appears to be simple, sensitive and reproducible. Contrary to expectations based on traditional medical knowledge, although Artemisiae Capillaris Herba contains a large amount of esculetin, it appears from this study that Fraxini Cortex contains a greater amount. The pharmacological effects of esculetin isolated from medicinal plants should be investigated as part of new medicines development.

Monitoring of Forbidden Medicines as Adulterants in Dietary Supplements Marketed Online by HPLC and ESI-tandom Mass Spectrometry

It was carried out to detect anti-impotence drug-like compounds, sibutramine and their analogues in dietary supplements, which are doubtful whether they include illegal compounds. A total of 51 food products were bought online and have been investigated. The separation was achieved on a C18 column, with the mobile phase made up of water (5 mM sodium hexanesulfonate and 0.1% phosphoric acid) and 95% acetonitrile, at a flow rate of 1.2 mL/min with gradient elution using by HPLC-DAD. The UV signals were monitored at 220 nm and 291 nm. LC-ESI-tandom MS was utilized to confirm that detected compounds in samples are the same as the reference materials. Two nutrient supplement foods and ginseng products were found to contain 1.3-82.1 mg of sildenafil, dimethylthiolsildenafil and pseudovardenafil per serving size. In addition, two other processed products were detected to contain 1.7 and 2.2 mg of didesmethylsibutramine, derived from sibutramine per serving size.

Quality inspections of active components in oxidative hair coloring products by UPLC

To identify oxidative hair dyes in hair-coloring products, the thin-layer chromatography (TLC) screening method was used in accordance with Korean Quasi-drug Codex. However, the TLC method is not reliable when there are very small amount of materials to be tested or when values of several components are similar. In this study, Ultra Performance Liquid Chromatography (UPLC) with a rapid sample preparation method was developed for the reliable and sensitive identification of active components contained in oxidative hair-coloring products. Hexane-distilled water was used for the extraction of active components contained in the products prior to UPLC analysis. The limit of detection of active components was 6.7-77.9 , and the limit of quantitation was 22.3-259.7 . Except for -naphthol, the range of recovery ratio was 96.2-101.5%. From this study, we demonstrated that oxidative active hair-coloring components can easily be analyzed by rapid extraction method followed by UPLC analysis.