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Supplementary Data from Thiacremonone Augments Chemotherapeutic Agent–Induced Growth Inhibition in Human Colon Cancer Cells through Inactivation of Nuclear Factor-κB

Chemotherapeutic strategies commonly use multiple agents to overcome drug resistance and to lower drug toxicity. Activation of nuclear factor-κB (NF-κB) is implicated in drug resistance in cancer cells. Previously, we reported that thiacremonone, a novel sulfur compound isolated from garlic, inhibited NF-κB and cancer cell growth with IC50 values about 100 μg/mL in colon cancer cells. In the present study, we tested whether thiacremonone could increase susceptibility of cancer cells to chemotherapeutics through inactivation of NF-κB. Colon cancer cells were cotreated with thiacremonone (50 μg/mL, half dose of IC50) and lower doses of each chemotherapeutic agent (half dose of IC50) for 24 hours. NF-κB activity was completely abrogated in cells treated with a combination of thiacremonone and docetaxel, whereas thiacremonone on its own did not alter NF-κB activity. This combined drug effect was also found with other anticancer drugs in colon cancer and in other cancer cells. In good correlation with inhibition of cell growth and NF-κB activity, the combination treatment also regulated NF-κB target genes. Oral treatment of mice with thiacremonone (1 mg/kg) by administering it in drinking water for 4 weeks significantly augmented docetaxel (1 mg/kg, i.p., four times)–induced decrease of tumor growth accompanied with regulation of NF-κB activity and NF-κB target genes. These results warrant carefully designed clinical studies investigating the combination of thiacremonone and commonly used chemotherapeutic agents for the treatment of human cancers. (Mol Cancer Res 2009;7(6):870–9)

A high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry method was developed and validated for the determination of flavonoid glycosides, polymethoxyflavones, and coumarins in herbal medicines of Citrus fruits and peels and Poncirus fruits. The analytes were rutin, narirutin, naringin, hesperidin, neohesperidin, neoponcirin, poncirin, naringenin, sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, tangeretin, imperatorin, phellopterin, and isoimperatorin, and, together with puerarin (the internal standard), were separated in short run time using a gradient flow of acetonitrile and 0.1% formic acid. Mass spectrometry detection was performed in the multiple reaction monitoring mode. Ion detection was operated alternately between negative and positive ion modes in a single injection. The main fragmentation characteristics of the analytes were investigated to discriminate between isomeric pairs. Validation of the analytical method showed linearity, r > 0.999; accuracy,...

A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed for the determination of Grayanotoxin I (GTX I) and Grayanotoxin III (GTX III) in rat whole blood. Grayanotoxins (GTXs) and clindamycin as internal standard (IS) were extracted from rat blood via solid-phase extraction using PEP solid-phase extraction cartridges. Chromatographic separation of the analytes was achieved on a Kinetex C18 (100 × 2.1 mm, 2.6 µm) reversed-phase column using a gradient elution with the mobile phase of 1% acetic acid in water and methanol at a flow rate of 0.2 mL/min. Electrospray ionization mass spectrometry was operated in the positive ion mode with multiple reaction monitoring. The calibration curves obtained were linear over the concentration range of 1–100 ng/mL with a lower limit of quantification of 1 ng/mL for GTXs. The relative standard deviation of intra-day and inter-day precision was below 6.8% and accuracy ranged from 94.8 to 106.6%. The analytes were stable in the stability studies. The validated method was successfully applied to the quantification and toxicokinetic study of GTXs in rats for the first time after oral administration of 11.52 mg/kg mad honey and 0.35 mg/kg GTX III, respectively. Copyright © 2014 John Wiley & Sons, Ltd.

The ERETIC (Electronic REference To access In vivo Concentrations)2 method is a new qNMR experimental technique to measure analytes based on the signal of the reference compound without additional hardware equipment. In this study, ERETIC 2 method was validated, and we sought to identify whether it would be possible to apply this method to a specific compound analysis of metabolites in plant. The 90° pulse value (P1) and spin -lattice relaxation time ( T 1 ) of each compound were measured for ERETIC 2. The 9 1 H of 3-(trimethylsilyl) propionic -2,2,3,3 -d 4 acid (TSP) was used as a reference peak for ERETIC 2, and then, a suitable solvent and pulse sequence for each compound were selected. Under the NOESY -presat sequence, the relative accuracy e rror for quantitative analyses of primary metabolites was within the range of 5%, with the exception of glucose, which showed • 5% error due to saturation. It showed excellent results for the quantification of glucose by using a 30° pulse sequence, which did not suppress the water peak. In addition, the qua ntitative accuracy for secondary metabolites was extremely accurate, with DQ HUURU ” ZKHQ FRQVLGHULQJ WKH SXULW\ RI WKHstandard sample. The ERETIC 2 method showed outstanding linearity, precision, and accuracy. Keywords qNMR, ERETIC , quantification Introduction Nuclear magnetic resonance (NMR) is a qualitative and quantitative analytical technique. It is the only physical method used routinely that can provide quantitative valuable information at the molecular level. Therefore NMR has found various a pplications in other fields than chemistry, such as metabolomics methodology

In this study, quantitative and pattern recognition analyses were developed using HPLC/UV for the quality evaluation of Dipsaci Radix. For quantitative analysis, five major bioactive compounds were assessed. The separation conditions employed for HPLC/UV were optimized using ODS C18 column (250 × 4.6 mm, 5 μm) with a gradient of acetonitrile and water as the mobile phase at a flow rate of 1.0 mL/min and a detection wavelength of 212 nm. These methods were fully validated with respect to linearity, accuracy, precision, recovery, and robustness. The HPLC/UV method was applied successfully to the quantification of five major compounds in the extract of Dipsaci Radix. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of 17 Dipsaci Radix and four Phlomidis Radix samples. The results indicate that the established HPLC/UV method is suitable for quantitative analysis.

For a case study of suspected paraquat intoxication, we developed a simple and rapid method of qNMR to determine the mili-molar amount of paraquat in postmortem blood samples. There were no interfering signals from endogenous compounds in the chemical shift of paraquat and diquat (internal standard). The amount of sample used ranged from 0.25 mM to 10.0 mM. Diquat, which has similar physicochemical properties with paraquat, was chosen as an internal standard. The NMR experimental conditions, relaxation delay time and CPMG spin-echo pulse sequence were optimized. The developed method was validated in terms of specificity, accuracy, precision, matrix effect, recovery, limit of detection (LOD), and low limit of quantification (LLOQ). The proposed qNMR method provided a simple and rapid assay for the identification and quantification of the quaternary ammonium herbicide, "paraquat" in postmortem blood samples. This method was tested by using the blood from the heart of a man who was intoxicated with paraquat. In this particular case, the level of paraquat was 1.07 mM in the blood. For the determination of quaternary ammonium herbicides, qNMR could also be used to provide a better understanding of the currently available techniques.

A rapid and simple high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was developed for the determination of betaine from Lycii Fructus. Betaine was separated with an Atlantis hydrophilic interaction liquid chromatography silica column (4.6 × 150 mm, 5 μm, 100 Å) by isocratic elution using 30 mM ammonium acetate buffer and acetonitrile (20:80, v/v %) as the mobile phase. The flow rate was 1.0 mL/min, and the temperature for the spray chamber and drift tube was set at 30 and 50 °C, respectively. The method was fully validated with respect to linearity, precision, accuracy, stability and robustness. The HPLC/ELSD method was applied successfully to the quantification of betaine in the extract of Lycii Fructus. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of twenty-six L. barbarum L. from China (BC01-BC26), 3 L. barbarum L. (BJ27-BJ29) from Japan, 12 L. chinense Miller from China (CC30-CC41) and 51 L. chinense Miller samples (CK42-CK92) from Korea. The results indicate that the established HPLC/ELSD method is suitable for quality evaluation of Lycii Fructus.

Liquid chromatographic enantiomer separation of N-t-BOC and N-CBZ α-amino acid, methyl ester, and ethyl ester derivatives was performed on chiral stationary phases (CSPs) based on polysaccharide derivatives. Good resolution of N-t-BOC and N-CBZ α-amino acid derivatives was achieved on Chiralcel OD, Chiralcel OF, and Chiralpak AD, respectively. Enantioselectivites of N-CBZ protected derivatives were found better than those of N-t-BOC protected derivatives. Moreover, the results of liquid chromatography and computational chemistry suggest that L-form is more retained in the case of carboxylic group of enantiomer locating toward to the inside of grooves, on the other hand, D-form is more retained in the case of alkyl group of α-position of N-protected α-amino acid derivatives locating toward to the inside of grooves.

High-performance liquid chromatography was employed for the separation of five cationic surfactants and seven anionic surfactants. Detection method was compared with evaporative light scattering detector and charged aerosol detector. Acclaim surfactant and Capcell pak C18 column were selected for the study of separation efficiency. The elution behavior of surfactants on Acclaim surfactant column appeared as both hydrophobic and ionic interactions. In contrast, Capcell pak C18 column showed only hydrophobic interaction. In the case of cationic surfactants, good separation was achieved on an Acclaim surfactant column with methanol-0.1 M ammonium formate (pH 5) eluent. Additionally, anionic surfactants showed good separation results on Acclaim surfactant column with acetonitrile-0.1 M ammonium formate (pH 5) eluent. Optimum separation condition was applied to analysis of cationic surfactants as an antistatic agent in polybutyl acrylate adhesive. In the case of spiked calibration standards into polybutyl acry...

The evolved gas analysis-mass spectrometry (EGA-MS) method is described as a new approach for determining the degree of imidization (DOI) in polyimide (PI) films. Partially imidized PI films allowed water to release through the re-imidization process at a sufficiently high imidization temperature. Evolved water from the re-imidization process was quantitatively detected by EGA-MS. From the obtained water content, the number of moles of residual amic acid groups in repeating units of PI was found. Consequently, the DOI of the PI films could be found from the mole ratio of PI and the sum of the PI and the residual polyamic acid (PAA). A water content of 0.018% and a DOI of 99.85% can be measured from 40 mg of PI films using this method. In this study, it was found that rigid PIs showed fast imidization reactions at relatively lower temperatures, while flexible PIs were activated and showed fast imidization reactions at relatively higher temperatures. In addition, the end point of the imidization process in multi-layer PI films was determined by this method.

Thermal degradation behavior and its mechanism of rigid and soft polyurethanes (PUs) were investigated by evolved gas analysis-mass spectrometry (EGA-MS) and evolved gas analysis-gas chromatography/mass spectrometry (EGA-GC/MS). Four steps of thermal degradation processes were found in EGA-MS analysis and evolved gases from each thermal zone were analyzed by EGA-GC/MS. From the results, it is suggested that thermal degradation of PUs might be occurred by increased bond strain of polymer chain due to increasing temperature and the sequence of thermal degradation in polymer chain is from hard segment to soft segment through four steps of thermal degradations.

Enantiomeric separations of N-phthaloyl (N-PHT), N-tetrachlorophthaloyl (N-TCPHT), and N-naphthaloyl (N-NPHT) α-amino acids and their esters were examined on several kinds of polysaccharide-derived chiral stationary phases (CSPs). Resolution capability of CSPs was greater Chiralcel OF than the others for N-PHT and N-NPHT α-amino acids and their esters. In N-TCPHT α-amino acids and their esters, good enantioselectivities showed Chiralcel OG for N-TCPHT α-amino acids, Chiralpak AD for N-TCPHT α-amino acid methyl esters, and Chiralcel OD for N-TCPHT α-amino acid ethyl esters, respectively. From the results of liquid chromatography and computational chemistry, it is concluded that l-form is preferred and more retained with electrostatic interaction in case of interaction between N-PHT α-amino acid derivatives and Chiralcel OF, N-TCPHT α-amino acid derivatives and Chiralcel OD, and N-NPHT α-amino acid derivatives and Chiracel OF. On the other hand, d-form is preferred and more retained with van der Waals interaction in case of interaction between N-TCPHT α-amino acid ester derivatives and Chiralcel OG and Chiralpak AD. Chirality 24:1037–1046, 2012. © 2012 Wiley Periodicals, Inc.

Phellodendri Cortex is the bark of the stems of Phellodendron amurense Ruprecht or P. chinense Schneider (Rutaceae), which is orginated from periderm. The internal transcribed spacer sequences of 20 originated plants and identified samples were analyzed. The result showed that the 99% of the base sequences of P. amurense were identical to that of P. chinense, but the differentiation of P. amurense and P. chinense was difficult. In addition, the ribulose-1, 5-bisphospate carboxylase large subunit (rbcL) intergenic spacer sequences of specific parts produced the same result. However, when the analysis was carried out by using the RAPD (randomly amplification polymorphism DNA) analysis method, which utilizes 48 randomly primers, it allowed us to confirm the polymorphism of P. amurense and P. chinense in 12 primers. A high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous quantitation of berberine, palmatine and jatrorrhizine in a traditional herbal drug, Phellodendri Cortex. The HPLC method was applied successfully to the quantification of three constituents in the extract of twenty Phellodendri Cortex. The results indicated that the established HPLC and RAPD methods are suitable for the quantitative analysis and the quality control multi-simultaneous discrimination in Phellodendri Cortex.

This experiment investigated whether (-)-epigallocatechin-3-O-gallate (EGCG) (5-20 mg/kg, p.o.) has hypnotic effects and/or enhances pentobarbital-induced sleeping behaviors and whether these effects are mediated by γ-aminobutyric acid (GABA) receptors. EGCG prolonged sleeping time induced by pentobarbital (42 mg/kg, i.p.) and reduced sleeping latency induced by pentobarbital similarly to muscimol (0.2 mg/kg, i.p.), a GABA(A) receptor agonist in mice. EGCG also increased sleeping rate and sleeping time when co-administered with pentobarbital (28 mg/kg, i.p.) at a subhypnotic dosage. In addition, EGCG and pentobarbital increased chloride (Cl(-)) influx in primary cultured cerebellar cells. EGCG and pentobarbital decreased GABA(A) receptors α-subunit expression and had no effect on the expression of β- and γ-subunits and of glutamic acid decarboxylase in the hippocampus of rats. In conclusion, the EGCG enhancement of Cl(-) influx may play an important role in pentobarbital-induced sleeping behaviors.

A simple LC-MS/MS method has been developed and validated for the quantification of endogenous myo- and chiro-inositol in human urine. myo- and chiro-Inositol were completely resolved from other carbohydrates and there were no interference peaks in human urine. The correlation coefficient (n = 3) was greater than 0.9991 over the range 0.05-25.0 µg/mL with the weighted (1/C²) least square method. Precision (%RSD) and accuracy (%RE) were 0-10.0% and 0-6.0% for the intra-day assay (n = 5) and 0-14.3% and 0-10.0% for the inter-day assay (n = 5). myo- and chiro-Inositol have been shown to be stable in human urine stored at room temperature and for three freeze-thaw cycles.

‡§ In this study, quantitative and pattern recognition analysis for the quality evaluation of Cimicifugae Rhizoma using HPLC/UV was developed. For quantitative analysis, three major bioactive phenolic compounds were determined. The separation conditions employed for HPLC/UV were optimized using ODS C18 column (250 × 4.6 mm, 5 µM) with isocratic elution of acetonitrile and water with 0.1% phosphoric acid as the mobile phase at a flow rate of 1.0 mL/min and a detection wavelength of 323 nm. These methods were fully validated with respect to the linearity, accuracy, precision, recovery, and robustness. The HPLC/UV method was applied successfully to the quantification of three major compounds in the extract of Cimicifugae Rhizoma. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of twelve reference samples corresponding to five different species of Cimicifugae Rhizoma and seventeen samples purchased from markets. The results indicate that the established HPLC/UV method is suitable for the quantitative analysis and quality control of multi-components in Cimicifugae Rhizoma.

In this study, quantitative and pattern recognition analysis for the quality evaluation of Magnoliae Flos using HPLC/ UV was developed. For quantitative analysis, eleven major bioactive lignan compounds were determined. The separation conditions employed for HPLC/UV were optimized using ODS C 18 column (250 × 4.6 mm, 5 μm) with isocratic elution of acetonitrile and water with 1 % acetic acid as the mobile phase at a flow rate of 1.0 mL/min and a detection wavelength of 278 nm. These methods were fully validated with respect to the linearity, accuracy, precision, recovery, and robustness. The HPLC/UV method was applied successfully to the quantification of eleven major compounds in the extract ofMagnoliae Flos. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of twenty one reference samples corresponding to seven different species of Magnoliae Flos and nine samples purchased from market. The results indicate that the established HPLC/UV method is suitable for the quantitative analysis and quality control of multi-components in Magnoliae Flos.

Peroxisome proliferator-activated receptor (PPAR)-gamma agonists such as troglitazone, pioglitazone and thiazolidine have been shown to induce apoptosis in human colon cancer cells. The molecular mechanism of PPARgamma agonist-induced apoptosis of colon cancer cells, however, is not clear. Glycogen synthase kinase-3beta (GSK-3beta) is an indispensable element for the activation of nuclear factor-kappa B (NF-kappaB) which plays a critical role in the mediation of survival signals in cancer cells. To investigate the mechanisms of PPARgamma agonist-induced apoptosis of colon cancer cells, we examined the effect of troglitazone (0-16muM) on the activation of GSK-3beta and NF-kappaB. Our study showed that the inhibitory effect of troglitazone on colon cancer cell growth was associated with inhibition of NF-kappaB activity and GSK-3beta expression in a dose-dependent manner. Cells were arrested in G(0)/G(1) phase followed by the induction of apoptosis after treatment of troglitazone with concomitant decrease in the expression of the G(0)/G(1) phase regulatory proteins; Cdk2, Cdk4, cyclin B1, D1, and E as well as in the anti-apoptosis protein Bcl-2 along with an increase in the expression of the pro-apoptosis-associated proteins; Caspase-3, Caspase-9 and Bax. Transient transfection of GSK-3beta recovered troglitazone-induced cell growth inhibition and NF-kappaB inactivation. In contrast, co-treatment of troglitazone with a GSK-3beta inhibitor (AR-a014418) or siRNA against GSK-3beta, significantly augmented the inhibitory effect of troglitazone on the NF-kappaB activity, the cancer cell growth and on the expression of G(0)/G(1) phase regulatory proteins and pro-apoptosis regulatory proteins. These results suggest that the PPARgamma agonist, troglitazone, inhibits colon cancer cell growth via inactivation of NF-kappaB by suppressing GSK-3beta activity.

－ Thiacremonone, the main component isolated from heated garlic ( Allium sativum L.), is inter-ested for using as a cancer preventive or therapeutic agent since garlic has been known to be useful plant in the treatment of cancers. Nuclear factor kappaB (NF-κB) is constitutively activated in the prostate cancer and activation of NF-κB is implicated in drug resistance in cancer cells. Docetaxel, a semisynthetic analog of pa-clitaxel, is an antineoplastic drug widely used for advanced various cancer. In previous studies, we found that thiacremonone inhibited activation of NF-κB in cancer cells and marcrophages. In the present study, we in-vestigated whether thiacremonone could increase susceptibility of prostate cancer cells (PC-3 and DU145) to docetaxel via inactivation of NF-κB. We found that the combination treatment of thiacremonone (50 μg/ml) with docetaxel (5 nM) was more effective in the inhibition of prostate cancer cell growth and induction of apoptosis accompanied with the significant inhibition of NF-κB activity than those by the treatment of thiacre-monone or docetaxel alone. It was also found that NF-κB target gene expression of Bax, caspase-3 and cas-pase-9 was much more significantly enhanced, but the expression of Bcl-2 was also much more significantly inhibited by the combination treatment. These results indicate that thiacremonone inhibits NF-κB, and enhan-ces the susceptibility of prostate cancer cells to docetaxel. Thus, thiacremonone could be useful as an ad-juvant anti-cancer agent.Keywords: Thiacremonone, Docetaxel, NF-κB, Apoptotic cell death, Prostate cancer

Chemotherapeutic strategies commonly use multiple agents to overcome drug resistance and to lower drug toxicity. Activation of nuclear factor-κB (NF-κB) is implicated in drug resistance in cancer cells. Previously, we reported that thiacremonone, a novel sulfur compound isolated from garlic, inhibited NF-κB and cancer cell growth with IC50 values about 100 μg/mL in colon cancer cells. In the present study, we tested whether thiacremonone could increase susceptibility of cancer cells to chemotherapeutics through inactivation of NF-κB. Colon cancer cells were cotreated with thiacremonone (50 μg/mL, half dose of IC50) and lower doses of each chemotherapeutic agent (half dose of IC50) for 24 hours. NF-κB activity was completely abrogated in cells treated with a combination of thiacremonone and docetaxel, whereas thiacremonone on its own did not alter NF-κB activity. This combined drug effect was also found with other anticancer drugs in colon cancer and in other cancer cells. In good correlation with inhibition of cell growth and NF-κB activity, the combination treatment also regulated NF-κB target genes. Oral treatment of mice with thiacremonone (1 mg/kg) by administering it in drinking water for 4 weeks significantly augmented docetaxel (1 mg/kg, i.p., four times)–induced decrease of tumor growth accompanied with regulation of NF-κB activity and NF-κB target genes. These results warrant carefully designed clinical studies investigating the combination of thiacremonone and commonly used chemotherapeutic agents for the treatment of human cancers. (Mol Cancer Res 2009;7(6):870–9)

This paper describes a pattern recognition method of Magnoliae flos based on a gas chromatographic/mass spectrometric (GC/MS) analysis of the essential oil components. The botanical drug is mainly comprised of the four magnolia species (M. denudata, M. biondii, M. kobus, and M. liliflora) in Korea, although some other species are also being dealt with the drug. The GC/MS separation of the volatile components, which was extracted by the simultaneous distillation and extraction (SDE), was performed on a carbowax column (supelcowax 10; 30 m{\time}0.25 mm{\time}0.25{\mu}m$) using temperature programming. Variance in the retention times for all peaks of interests was within RSD 2% for repeated analyses (n = 9). Of the 74 essential oil components identified from the magnolia species, approximately 10 major components, which is -pinene, -pinene, sabinene, myrcene, d-limonene, eucarlyptol (1,8-cineol), -terpinene, p-cymene, linalool, -terpineol, were commonly present in the four species. For statistical analysis, the original dataset was reduced to the 13 variables by Fisher criterion and factor analysis (FA). The essential oil patterns were processed by means of the multivariate statistical analysis including hierarchical cluster analysis (HCA), principal component analysis (PCA) and discriminant analysis (DA). All samples were divided into four groups with three principal components by PCA and according to the plant origins by HCA. Thirty-three samples (23 training sets and 10 test samples to be assessed) were correctly classified into the four groups predicted by PCA. This method would provide a practical strategy for assessing the authenticity or quality of the well-known herbal drug, Magnoliae flos.

Ginsenoside Rg3, the main constituent isolated from Panax ginseng, has been of interest for use as a cancer preventive or therapeutic agent. We investigated here whether Rg3 can inhibit the activity of NF-kappaB, a key transcriptional factor constitutively activated in colon cancer that confers cancer cell resistance to chemotherapeutic agents. To investigate whether RG3 can suppress activation of NF-kappaB, and thus inhibit cancer cell growth, we examined the susceptibility of colon cancer cells (SW620 and HCT116) to treatment with Rg3 (25, 50, 75, 100 microM) and RG3-induced activation of NF-kappaB. RG3 dose-dependently inhibited cancer cell growth through induction of apoptosis and decreased NF-kappaB activity. In a further study of compounds in colon cancer, we used half of the IC(50) dose, values in combined treatments of Rg3 (50 microM) with conventional agents - docetaxel (5 nM), paclitaxel (10 nM) cisplatin (10 microM) and doxorubicin (2 microM). Compared to treatment with Rg3 or chemotherapy alone, combined treatment was more effective (i.e., there were synergistic effects) in the inhibition of cancer cell growth and induction of apoptosis and these effects were accompanied by significant inhibition of NF-kappaB activity. NF-kappaB target gene expression of apoptotic cell death proteins (Bax, caspase-3, caspase-9) was significantly enhanced, but the expression of anti-apoptotic genes and cell proliferation marker genes (Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP), Cox-2, c-Fos, c-Jun and cyclin D1) was significantly inhibited by the combined treatment compared to Rg3 or docetaxel alone. These results indicate that ginsenoside Rg3 inhibits NF-kappaB, and enhances the susceptibility of colon cancer cells to docetaxel and other chemotherapeutics. Thus, ginsenoside Rg3 could be useful as an anti-cancer or adjuvant anti-cancer agent.

This study was undertaken to investigate whether honokiol could enhance the pentobarbital- induced sleeping behaviors through γ-aminobutyric acid (GABA) receptor Cl - channel activation. Thirty minutes after the oral administration of honokiol, mice were received sodium pentobarbital (42 mg/kg, i.p.). The time elapsed from pentobarbital injection to the loss of the righting reflex was taken as sleeping latency. The time elapsed between the loss and voluntary recovery of the righting reflex was considered as the total sleeping time. Western blot technique and Cl - sensitive fluorescence probe were used to detect the expression of GABA A receptor subunits and Cl - influx in the primary cultured cerebellar granule cells. Honokiol (0.1 and 0.2 mg/kg) prolonged the sleeping time induced by pentobarbital (42 mg/kg) in a dosage-dependent manner. Honokiol (20 and 50 µM) increased Cl - influx in primary cultured cerebellar granule cells, and selectively increased the GABA A receptor α-subunit expression, but had no effect on the abundance of β or γ-subunits. Chronic treatment with 20 µM honokiol in primary cultured cerebellar neurons did not affect the abundance of GAD65/67. The results suggested that honokiol could potentiate pentobarbital-induced sleeping through GABA A receptor Cl - channel activation.

A comprehensive analytical method was established for the separation of polyethylene glycol (PEG) stearates according to the distribution of ethylene oxide (EO) and subsequent determination of the surfactants in cosmetic samples by using a high-performance liquid chromatography-evaporative light scattering detection. Separation of the PEG stearates comprising approximately up to 82 EO adducts was performed on a reversed-phase YMC-Pack C(8) column using water-acetonitrile gradient elution. The PEG oligomers were separated in order of the increasing number of EO adducts. Quantitation of the PEG fatty acid esters, which was separated as single peak per each component, was performed by chromatography on a reversed-phase Wakosil 10 C(18) column using water-methanol gradient elution. The standard curve to quantify the PEG stearates was constructed by the log-log plot, which showed good linearity with the correlation coefficients (R(2)) 0.998 and more. Working range, repeatability, limit of detection and recovery were acceptable for analysis of the surfactants in cosmetic products. The analytical methods were applied to characterize the PEG stearates according to the EO distributions, then to quantify the surfactants in cosmetic products.

Activity-directed isolation of the methylene chloride fraction of the roots of Rubia cordifolia L. resulted in the identification of a new epoxymollugin (3) and eight known compounds (1, 2, 4-9). The structures of the compounds were elucidated from chemical and spectroscopic evidence. In addition, their topoisomerase I and II inhibitory activities and cytotoxicities were measured.

Molecular mechanisms underlying epothilone-induced apoptotic cell death were investigated in SW620 human colon cancer cells. Treatment with epothilone B and D at different concentrations (1–100 nmol/L) dose-dependently inhibited cell growth and caused cell cycle arrest at G2-M, which was followed by apoptosis. Consistent with this induction of apoptotic cell death, epothilone B and D enhanced the constitutional activation of nuclear factor-κB (NF-κB) via IκB degradation through IκB kinase (IKKα and IKKβ) activation, and this resulted in p50 and p65 translocation to the nucleus. Moreover, cells treated with sodium salicylic acid, an IKK inhibitor, or transiently transfected with mutant IKKα and β did not show epothilone-induced cell growth inhibition or p50 translocation, although p65 was still translocated to the nucleus. Treatment with epothilone B and D also enhanced β-tubulin polymerization and the formation of p50/β-tubulin complex. However, β-tubulin polymerization was not inhibited in the cells treated by sodium salicylic acid or transiently transfected with mutant IKKα and β. Moreover, epothilone B and D increased the expressions of NF-κB–dependent apoptotic cell death regulatory genes, i.e., Bax, p53, and the active form of caspase-3, but reduced Bcl-2 expression, and these actions were partially reversed by salicylic acid. In addition, caspase-3 inhibitor reduced epothilone B–induced cell death and NF-κB activation. These findings suggest that the activation of NF-κB/IKK signals plays an important role in the epothilone-induced apoptotic cell death of SW620 colon cancer cells in a tubulin polymerization–independent manner. [Mol Cancer Ther 2007;6(10):2786–97]

An immunoglobulin (IgG) preparation with micro-amount of histamine fixed on the active protein fraction has been used to increase the resistance to allergic reactions. However, excessive histamine may cause hypo- or hypertension, headache, or anaphylactic shock and so the histamine content of the drug is strictly controlled by a regulation: 0.15 microg of histamine dihydrochloride is allowed for 12 mg of immunoglobulin. In this study, a liquid chromatographic method to determine micro-amount of histamine in the pharmaceutical was developed and validated. This method include a sample cleanup by a solid phase extraction (SPE) using a polystyrene-divinyl benzene (PS-DVB) polymeric sorbent and high-performance liquid chromatography after precolumn fluorescent labeling of the histamine with o-phthaldialdehyde. The drug sample was loaded to the SPE cartridge after adjusting to pH 9.5. After successive washings of the cartridge with water and 30% aqueous methanol, histamine was then eluted with 100 mM sodium acetate (pH 9.5)-methanol (20:80, v/v). An aliquot from the eluate was labeled with o-phthaldialdehyde-mercaptoethanol (OPA-ME) for fluorescence detection at the excitation maximum of 340 nm and emission maximum of 450 nm. HPLC analysis was performed on a phenyl-hexyl column with an acetonitrile-phosphate buffer (pH 6.8; 50 microM) (35:65, v/v) as the mobile phase. The retention times of histamine and 3-methylhistamine (IS) were approximately 7.2 and 9.5 min, respectively. The quantitation range was between 0.01-0.2 mg/mL of histamine showing good linearity (r=0.9996). This analytical method would provide a potential mean for the strict control of histamine content in the pharmaceutical product.

Four known germacranolide sesquiterpene lactones, 2alpha,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-isobutyloxy-germacran-8alpha,12-olide (1), 2alpha,5-epoxy-5,10-dihydroxy-6alpha,9beta-diangeloyloxy-germacran-8alpha,12-olide (2, divaricin B), 2alpha,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-(2-methylbutyloxy)-germacran-8alpha,12-olide (3) and 2alpha,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-(3-methylbutyloxy)-germacran-8alpha,12-olide (4), were isolated from the chloroform-soluble fraction of the whole plants of Carpesium triste var. manshuricum. Their chemical structures were determined using spectroscopic methods, including 2D-NMR. All the isolates showed significant cytotoxicities (ED50 value: 4.3-16.8 microM) against five human tumor cell lines; A549, SK-OV-3, SK-MEL-2, XF498 and HCT15.

1-Methyl-2-undecyl-4(1H)-quinolone (1) was previously isolated as a selective MAO-B inhibitor from the Evodiae Fructus. Further bioassay-guided purification led to the identification of five known quinolone alkaloids, 1-methyl-2-nonyl-4(1H)-quinolone (2), 1-methyl-2-[(Z)-6-unde-cenyl]-4(1H)-quinolone (3), evocarpine (4), 1-methyl-2-[(6Z,9Z)-6,9-pentadecadienyl]-4(1H)-quinolone (5), and dihydroevocarpine (6). All the isolates showed more potent inhibitory effects against MAO-B compared to MAO-A. The most MAO-B selective compound 5 among the isolates inhibited MAO-B in a competitive manner, according to kinetic analyses by Lineweaver-Burk reciprocal plots.

Preclinical studies are currently underway to examine the potential antitumor effects of a 1:1 mixture of acriflavine (ACF; CAS 8063-24-9) and guanosine. Guanosine potentiates the anti-cancer activity of some compounds. However, the effects of guanosine on the pharmacokinetics of ACF in mammals are unknown. Therefore, this study investigated the effects of guanosine on the pharmacokinetics of ACF after administering a 1:1 mixture of ACF and guanosine in rats. The rats were given either 10 mg/kg of the mixture or 5 mg/kg ACF via an intravenous bolus injection; or 30 mg/kg of the mixture or 15 mg/kg ACF intramuscularly. An HPLC-based method, which was validated in this laboratory, was used to analyze the levels of trypaflavine (TRF) and proflavine (PRF) in the plasma, bile, urine, and tissue homogenates. It was found that TRF and PRF were rapidly cleared from the blood and transferred to the tissues after the i.v. bolus or i.m. injection of the combination mixture. Both TRF and PRF were found to be most highly concentrated in the kidneys after the i.v. bolus or i.m. injection, followed by slow excretion to the bile or urine. Guanosine had no effect on the plasma disappearance of TRF or PRF after the i.v. bolus injection. However, guanosine led to a prolongation of the plasma levels of PRF after the i.m. administration of the combination mixture, resulting in a 2 fold increase in the bioavailability (BA) of PRF. The concentrations of TRF and PRF in all the tissues examined were similar in the groups given the mixture and ACF. However, guanosine led to a prolongation of the biliary and urinary excretions of both TRF and PRF after the i.v. bolus (1.25 fold) or i.m. (1.5-2.4 folds) injection. These prolonged effects of guanosine on the plasma disappearance or urinary excretion of TRF and PRF might be one reason for the enhanced antitumor effects of ACF. However, more study will be needed to further examine this potential mechanism.

We investigated whether the snake venom toxin (SVT) from Vipera lebetina turanica inhibits cell growth of human prostate cancer cells by inducing apoptosis and also studied possible signaling pathways involved in this cell death. SVT inhibited growth of PC-3 and DU145 cells, androgen-independent prostate cancer cells, but not LNCaP cells, a human androgen-dependent prostate cancer cell. Cells were arrested in the G2-M phase by SVT with a concomitant decrease in the expression of the G2-M phase regulatory protein cyclin B1 and were also arrested in the G1-S phase with decreasing expression of cyclin-dependent kinase 4, cyclin D1 and cyclin E. In addition to the growth-inhibitory effect, SVT increased the induction of apoptotic cell death. Untreated PC-3 cells show high DNA binding activity of nuclear factor κB (NF-κB), an antiapoptotic transcriptional factor, but this was inhibited by SVT and accompanied by a significant inhibition of p50 translocation into the nucleus, as well as phosphorylation of inhibitory κB. Consistent with the induction of apoptosis and inhibition of NF-κB, this toxin increased the expression of proapoptotic proteins such as p53, Bax, caspase-3, and caspase-9, but down-regulated antiapoptotic protein Bcl-2. However, SVT did not show an inhibitory effect on cell growth and caspase-3 activity in cells carrying mutant p50 and inhibitory κB kinase plasmids. Confocal microscopy analysis showed that SVT is taken up into the nucleus of the cells. These findings suggest that a nanogram concentration range of SVT from V. lebetina turanica could inhibit hormone-refractory human prostate cancer cell growth, and the effect may be related to NF-κB signal–mediated induction of apoptosis. [Mol Cancer Ther 2007;6(2):675–83]

Cinnamaldehyde derivatives isolated from Cinnamomum cassia have been widely used for treating dyspepsia, gastritis, and inflammatory disease as well as cancer. To investigate the anti-tumor activities of several cinnamaldehyde derivatives, we compared the inhibitory effect of cinnamaldehyde derivatives on cell growth and AP-1 transcriptional activity in SW620 human colon cancer cells since AP-1 is a transcriptional factor implicated to control cancer cell growth. Among the derivatives, 2’-hydroxycinnamaldehyde (HCA) most significantly inhibited cancer cell growth and AP-1 transcriptional activity in a dose-dependent manner with an IC50 value of 12.5 and 9 µg/ml, respectively. In further studies on the mechanism, we found that consistent with the inhibitory effect on cell growth, HCA dose-dependently (0 – 20 µg/ml) inhibited DNA binding activity of AP-1 accompanied with down regulation of c-Jun and c-Fos expressions. HCA also induced apoptotic cell death as well as expression of the apoptosis-regulating gene caspase-3, but inhibited the anti-apoptosis regulating gene bcl-2 in a dose-dependent manner. These results suggested that HCA has the most potent inhibitory effect against human colon cancer cell growth, and AP-1 may be an important target of HCA. Keywords:: 2’-hydroxycinnamaldehyde (HCA), AP-1, colon cancer, cell growth inhibition

Background : The aim of our study was to optimize and establish erythropoietin (EPO) enzyme linked immunosorbent assay (ELISA) system. Methods : We prepared several monoclonal and polyclonal antibodies specific to human-EPO. The best combinations of antibodies for coating and detecting antibodies were selected for the establishment of ELISA. We tested several methods such as a competitive EIA and a sandwich ELISA. Results : The best sandwich ELISA was optimized compared to competitive EIA when purified polyclonal antibody (PoAb) was used as a coating antibody and biotinylated PoAb as a detecting antibody. This sandwich ELISA easily detected EPO when PoAb pairs were used compared to the ELISA using monoclonal antibody and PoAb. There were no significant differences between the effects of various blocking solutions on the performance of sandwich ELISA using biotinylated antibody. The ELISA system using PBST containing 3% BSA as a blocking solution can sensitively detect EPO (10 mU/mL) in a broad range of EPO concentrations (10-2,000 mU/mL) and there were cross-reactions with other cytokines). Conclusions : EPO can be easily determined by using biotinylated PoAb as a detecting antibody and another PoAb as a coating antibody.

In the present study, we have investigated the bee venom (BV) and melittin (a major component of BV)-mediated antiproliferative effect and defined its mechanisms of action in cultured rat aortic vascular smooth muscle cell(s) (VSMC). BV and melittin ( approximately 0.4-0.8 microg/ml) effectively inhibited 5% fetal bovine serum-induced and 50 ng/ml platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation. The regulation of apoptosis has attracted much attention as a possible means of eliminating excessively proliferating VSMC. In the present study, the treatment of BV and melittin strongly induced apoptosis of VSMC. To investigate the antiproliferative mechanism of BV and melittin, we examined the effect of melittin on nuclear factor kappaB (NF-kappaB) activation, the PDGF-BB-induced IkappaBalpha phosphorylation, and its degradation were potently inhibited by melittin and whether DNA binding activity and nuclear translocation of NF-kappaB p50 subunit in response to the action of PDGF-BB were potently attenuated by melittin. In further investigations, melittin markedly inhibited the PDGF-BB-induced phosphorylation of Akt and weakly inhibited phosphorylation of extracellular signal-regulated kinase 1/2, upstream signals of NF-kappaB. Treatment of melittin also potently induced proapoptotic protein p53, Bax, and caspase-3 expression but decreased antiapoptotic protein Bcl-2 expression. These results suggest the antiproliferative effects of BV and melittin in VSMC through induction of apoptosis via suppressions of NF-kappaB and Akt activation and enhancement of apoptotic signaling pathway.

Erythropoietin (EPO), a hematopoietic factor, is also required for normal brain development, and its receptor is localized in brain. Our previous study showed that EPO promotes differentiation of neuronal stem cells into astrocytes. Since astrocytes have influence on the neuronal function, we investigated whether EPO-activated astrocytes could stimulate differentiation of neuronal stem cells into neurons. EPO did not promote neuronal differentiation of neuronal stem cells isolated from 17 day embryos, however, neuronal differentiation was promoted when the neuronal stem cells were co-cultured with astrocyte isolated from post neonatal (Day 1) rat brain. Moreover, neuronal differentiation was further promoted when the neuronal stem cells were cultured with astrocyte culture medium treated by EPO (10U/ml) showing increase of morphological differentiation, and expression of neuronal differentiation marker proteins, neurofilament, and tyrosine hydroxylase. The promoting effect of EPO-treated astrocyte medium was also found in the differentiation of PC12 cells. EPO-promoted morphological differentiation of neuronal stem cells as well as astrocytes was dose dependently reduced by treatment with anti-EPO receptor antibodies in culture with astrocyte culture medium. To clarify whether EPO itself or via production of well-known neurotropic factor could promote neuronal cell differentiation, we determined the level of neurotropic factors in the EPO-treated astrocytes. Compared to untreated astrocytes, EPO-treated astrocytes increased about 2-fold in beta-NGF and 3-4-fold in BMP2, but did not increase BNDF and NT-3 levels. Since the previous study showed that extracellular signal-regulated kinase (ERK) is involved in activation of astrocytes by EPO, we determined whether generation of neurotrophic factor may also be involved with the ERK pathway. In the presence of ERK inhibitor, PD98059, the generation of beta-NGF was diminished in a dose dependent manner consistent with the inhibiting effect on neuronal differentiation. These data demonstrate that EPO promotes neuronal cell differentiation through increased release of beta-NGF and BMP2 from astrocytes, and this effect may be associated with ERK pathway signals.

We developed a liquid chromatography‐tandem mass spectrometric (LC‐MS/MS) method for the determination of a ginseng saponin metabolite, IH901 or compound K (20‐O‐β‐D‐glucopyranosyl‐20(S)‐protopanaxadiol) in rat plasma. The method involves protein precipitation by acetonitrile and HPLC separation of the sample extracts on a reversed‐phase column (X‐terra™ RP18) with isocratic elution of 20 mM ammonium acetate and acetonitrile (30:70, v/v), at a flow rate of 0.2 mL/min. The MS analysis was performed by electrospray positive ionization mass spectrometry using multiple reaction‐monitoring mode. The mass transitions of IH901 and prednisolone (internal standard) were m/z 640→425 and 361→343, respectively. The standard curves for IH901 were linear over the concentration range of 2.0–500 ng/mL. The lower limit of quantification was 2 ng/mL and the limited of detection was 1 ng/mL for IH901. The coefficient of variation of intra‐ and inter‐day assays at five quality control levels ranged from 2.0 to 13.9%...

Based on the binding between peroxisome proliferators-activated receptor gamma (PPARgamma) and steroid receptor co-activator-1 (SRC-1), an enzyme-linked immunosorbent assay (ELISA) was used to screen new PPARgamma ligands from various benzylidinethiazole derivatives, which have anti-inflammatory activity. Among those derivatives, 5-(3,5-di-tert-butyl-4-hydroxybenzylidene) thiazolidine-2,4-dione (BTZD) increased the binding between PPARgamma and SRC-1. BTZD was found to induce adipogenesis and PPARgamma trans-activation in 3T3-L1 pre-adipocyte, and increased the binding between PPARgamma and SRC-1 in in vitro binding assay and complex consisting of PPARgamma and SRC-1 in the co-immunoprecipitaion. Chromatin immunoprecipitation (ChIP) analysis revealed that BTZD induced the binding of PPARgamma-SRC-1 complex to PPAR response element (PPRE) in the same pattern of other PPARgamma ligand. From these studies, we have identified and studied the function of a new PPARgamma ligand, BTZD. We suggest that BTZD can be used as a modulator of PPARgamma. This study applying ELISA and ChIP assay can offer new methods to screen PPARgamma ligand and understand the effects of PPARgamma ligands on inflammation.

Trimetazidine dihydrochloride has been used as an antianginal drug that possess protective properties against ischemia‐induced damage to heart. A simple and sensitive analytical method of trimetazidine dihydrochloride in human plasma by using high performance liquid chromatography (HPLC) was developed. The method employs a liquid‐liquid extraction for isolation and sample concentration, followed by reversed‐phase liquid chromatography (RPLC) analysis using ultraviolet (UV) detection at 207 nm. Analytes were extracted from plasma samples that previously were mixed with 300 µL saturated K2CO3 solution into an ethyl acetate phase. HPLC separation was accomplished at 40°C on a reversed‐phase column using a mobile phase, 15% acetonitrile in 50 mM potassium dihydrogen phosphate and phosphoric acid (pH=4.0), at a flow‐rate of 1.0 mL/min. The linear range of the method was between 10‐ and 150 ng/mL of tirmetazidine dihydrochloride in human plasma and the quantification limit was 10 ng/mL. The intra‐ and ...

We investigated the pharmacokinetic characteristics of 11-hydroxyaclacinomycin X (ID-6105), a novel anthracycline, after intravenous (i.v.) bolus administration in rats and beagle dogs. We developed an HPLC-based method to analyze ID-6105 levels in plasma, bile, urine, feces, and tissue homogenates and validated the method in a pharmacokinetic study. The plasma concentration of ID-6105 decreased to below the quantifiable limit (0.02 microg/ml) at 4 and 8 h after i.v. administration in rats at doses of 2 and 10 mg/kg, respectively (t(1/2,alpha) and t(1/2,beta) of 0.78 and 17.8 min at a dose of 2 mg/kg, 0.91 and 176 min at a dose of 10 mg/kg, respectively). The AUC increased with nonlinear pharmacokinetics following the dosage increase from 2 to 10 mg/kg in rats, while the pharmacokinetics were not significantly altered in beagle dogs following a dosage increase from 0.5 to 2.5 mg/kg. Of the various tissues tested, ID-6105 was mainly distributed in the lung, spleen, kidney, adrenal gland, and liver after i.v. bolus administration. ID-6105 levels in the lung or kidney 2 h after i.v. bolus administration were comparable to the initial plasma concentration. However, the ID-6105 concentrations in various tissues 48 h after i.v. bolus administration became too small to measure. The cumulative amounts of ID-6105 found in the bile 48 h after the administration of 2 and 10 mg/kg were calculated to be 26.7 and 18.5% of the initial dose, respectively. The corresponding values in the urine 72 h after i.v. administration were 4.33 and 3.07% of the initial dose, suggesting that ID-6105 is mostly excreted in the bile. In conclusion, our observations indicate that ID-6105 was rapidly cleared from the blood and transferred to tissues such as the lung, spleen, kidney, and liver 2 h after i.v. bolus administration. Moreover, the majority of ID-6105 appears to be excreted in the bile by 24 h after i.v. bolus administration.

Erythropoietin (EPO), a hematopoietic factor, is also required for normal brain development, and its receptor is localized in brain. Therefore, it is possible that EPO could act as a neurotropic factor inducing differentiation of neurons. In the present study, we investigated whether EPO can promote differentiation of neuronal stem cells into astrocytes. In primary culture of cortical neuronal stem cells isolated from post neonatal (Day 1) rat brain, EPO dose (0.1-10U/ml) dependently promoted initiation of morphological differentiation of astrocyte and expression of an astrocyte marker protein, glial fibrillary acidic protein (GFAP). Expression of EPO receptor was also increased during morphological differentiation of astrocytes. EPO-induced increased morphological differentiation of astrocytes and GFAP expression were reduced by treatment with anti-EPO and EPO receptor antibodies. Since our previous study showed that activation of MAPK family and transcription factors is differentially involved in neuronal cell differentiation, we further determined the activation of MAP kinase family and NF-kappaB during morphological differentiation of astrocytes. Concomitant with the progression of the morphological differentiation of astrocytes, ERK(2) but not JNK(1) and p38 MAPK as well as NF-kappaB were activated. However, in the presence of PD98,059, an inhibitor of ERK, and salicylic acid, an NF-kappaB inhibitor, the EPO-induced morphological differentiation of astrocytes and expression of FGAP and EPO receptor were reduced. Conversely, treatment with anti-EPO and EPO receptor antibodies also reduced EPO-induced ERK(2) and NF-kappaB activation. These data demonstrate that EPO can promote differentiation of neuronal stem cells into astrocytes in an EPO receptor dependent manner, and this effect may be associated with the activation of ERK kinase and NF-kappaB pathway.

A simple determination method of rebamipide in human plasma by using high performance liquid chromatography (HPLC) was developed. The method involves a single liquid–liquid extraction and reversed‐phase chromatography with fluorometric detection (excitation, 320 nm; emission, 380 nm). Analytes were extracted from plasma samples that contain an internal standard (ofloxacin) into ethylacetate with a high yield after adjustment to pH 2–3. Separation was accomplished at 60°C on a reversed‐phase column using a mobile phase of acetonitrile–water–acetic acid (30:70:5, v/v, pH 2.4), at a flow‐rate of 1.0 mL/min. The linear range of the assay was 2–500 ng/mL of the drug in plasma and the limit of quantitation was 2.0 ng/mL. The intra‐ and inter‐day relative standard deviation (RSD) were less than 10% and the accuracy of the assay was in the range of 97–104%. Analysis of the drug in human plasma indicates that the procedure can be carried out conveniently and quickly and, therefore, is suitable for obtaini...

The applicability of liquid chromatography‐atmospheric‐pressure chemical‐ionization‐mass spectrometry (LC‐APCI‐MS) for the determination of 13C‐urea in 13C‐urea/PEG capsules has been studied. It is essential to assess the stability of a newly developed low‐dose (38 mg) 13C‐urea/PEG capsule, which will be used for a 13C‐urea breath test (13C‐UBT) to detect Helicobacter pylori infection. A standard curve was linear over the concentration range 10–1000 µg/mL. Intra‐ and inter‐day variations were less than 2.75% in APCI‐MS. The detection limit was 10 pg when selected ion monitoring (SIM) was employed. The content of 13C‐urea in capsules was within the acceptable range between 95 and 105%. Therefore, it was established that 13C‐urea/PEG capsules were stable under an accelerated stability condition that was set at 40 ± 2°C with relative humidity of 75 ± 5%, during six months by using LC‐APCI‐MS. This research is the first report that describes LC‐APCI‐MS analysis of 13C‐urea capsules.

N-acetylsphingosine (C2-ceramide) is a synthetic water-soluble ceramide mimicking the activity of natural ceramides. By fixing chiral conformation on carbon numbers 2 and 3 in the ceramide structure, four chiral C2-ceramides naming d-erythro-, l-erythro-, d-threo- and l-threo C2-ceramide were synthesized. We have investigated the chiral effects of these C2-ceramides on the sphingolipid metabolism, particularly on both the sphingolipid biosynthetic pathway and on the degradation pathway. In both HL-60 and U937 cells, the chiral C2-ceramide (10 microM) showed sphingosine accumulation monitored fluoromatrically by a high performance liquid chromatographic separation of the sphingoid bases. Most importantly, in HL-60 cells, l-erythro C2-ceramide induced a 50 fold increase in sphingosine as compared to the control, while l-threo C2-ceramide exhibited a minimal 7-fold increase. In contrast, sphinganine, another sphingoid base, showed less accumulation by any chiral C2-ceramide tested under the same conditions. These results suggested that chiral C2-ceramide primarily acts on the sphingolipid degradation pathway rather than on the sphingolipid biosynthetic route. The strong G0/G1 phase arrest in the cell cycle by treatment of l-erythro C2-ceramide indicates that the blockade of the sphingolipid degradation pathway might be concomitantly involved in the dysfunction of the cell cycle. On the other hand, the fact that all chiral C2-ceramides tested failed to inhibit the activity of sphingosine kinase acting on the removal of sphingosine by producing sphingosine-l-phosphate demonstrates that chiral C2- ceramides may increase sphingosine by activating various ceramidases by which natural ceramides are divided into sphingosine and free fatty acids. However, the precise steps involved in this interaction are still unknown.

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of Grayanotoxin I (GTX I) and Grayanotoxin III (GTX III) in rat whole blood. Grayanotoxins (GTXs) and clindamycin as internal standard (IS) were extracted from rat blood via solid-phase extraction using PEP solid-phase extraction cartridges. Chromatographic separation of the analytes was achieved on a Kinetex C18 (100 × 2.1 mm, 2.6 µm) reversed-phase column using a gradient elution with the mobile phase of 1% acetic acid in water and methanol at a flow rate of 0.2 mL/min. Electrospray ionization mass spectrometry was operated in the positive ion mode with multiple reaction monitoring. The calibration curves obtained were linear over the concentration range of 1-100 ng/mL with a lower limit of quantification of 1 ng/mL for GTXs. The relative standard deviation of intra-day and inter-day precision was below 6.8% and accuracy ranged from 94.8 to 106.6%. The analytes were stable in the stability studies. The validated method was successfully applied to the quantification and toxicokinetic study of GTXs in rats for the first time after oral administration of 11.52 mg/kg mad honey and 0.35 mg/kg GTX III, respectively.

A rapid, accurate and sensitive liquid chromatography –tandem mass spectrometry method has been developed for the determination of a quaternary nitrogen muscle relaxant, rocuronium, in human blood. The procedure involves protein precipitation with chloroform and trichloroacetic acid, and purification using methanol. The chromatography was performed using a phenyl-hexyl column (150 3 2.0 mm i.d., 3 mm; Phenomenex) with a mobile phase consisting of 5 mM ammonium formate ( pH 3.0) and acetonitrile. Multiple reaction monitoring was used for quantification. The assay was linear over a concentration range of 4–500 ng/mL for rocuronium with R 2 � 0.998. The recoveries for this compound ranged from 96.0 to 109.1%. The intra-day and inter-day precision was less than 10.5% and the accuracy ranged from 106.6 to 114.9%. The validated method was applied to quantify the content of rocuronium in blood and a variety of tissues of a victim suspected of overdose. In conclusion, the method was successfully applied for the analysis of rocuronium in biological samples for forensic toxicology.

2,4-Bis(p-hydroxyphenyl)-2-butenal (Butenal), a tyrosine-fructose Maillard reaction product has been demonstrated as an effective compound for prevention of neuroinflammatory diseases. However, this compound was vulnerable to environmental factors. Our research has been continuously made to improve druggability of Butenal and identified 2,4-bis(4-hydroxyphenyl)but-2-enal diacetate (HPBD) as an alternative. Herein, to investigate potential anti-neuroinflammatory and anti-amyloidogenic effects of HPBD, we treated HPBD (0.5, 1, and 2 μg/ml) on the lipopolysaccharides (LPS) (1 μg/ml) stimulated astrocytes and microglial BV-2 cell. HPBD inhibited LPS-induced NO and ROS production, and LPS-elevated expression of iNOS, COX2, β-site APP-cleaving enzyme 1 (BACE1), C99, and Aβ1–42 levels as well as attenuation of β-secretase activities. The activation of nuclear factor-kappaB (NF-κB), signal transducer and activator of transcription1 (STAT1), and STAT3 was concomitantly inhibited by HPBD. Moreover, siRNA targeting STAT3 abolished HPBD-induced inhibitory effects on neuro-inflammation and amyloidogenesis. In addition, pull down assay and docking model showed interaction of HPBD with STAT3. These findings suggest that HPBD may be useful and potentially therapeutic choices for the treatment of neuroinflammatory diseases.

A gas chromatographic method is described for the determination of residual 2-(acryloyloxy)ethyl isocyanate (AOI) and 2-(methacryloyloxy)ethyl isocyanate (MOI) as curing agents in an ultraviolet curable adhesive. Pre-column derivatization was employed in the determination of AOI and MOI as a means of enhancing the response of the flame ionization detector. Urethane derivatives of AOI and MOI were derived using methanol for 30 min at room temperature. The accuracies (n = 5, three concentration levels) were in the range of 113.4 to 126.7%, and precisions (n = 5, three concentration levels) were in the range of 0.8 to 4.3% for AOI-OMe. Furthermore, the accuracies were in the range of 79.5 to 108.6% and the precisions were in the range of 1.0 to 2.4% for MOI-OMe. The correlation coefficients of six calibration standards were all greater than 0.9999 for AOI-OMe and greater than 0.9998 for MOI-OMe over the range from 10 to 100 µg/mL.