Ha-Jeong Kwon

Extracts from Citrus unshiu promote immune-mediated inhibition of tumor growth in a murine renal cell carcinoma model.

AIM OF THIS STUDY Citrus unshiu (Satsuma mandarin, SM) is a citrus fruit the peel of which has been used as a traditional Chinese medicine to treat common cold, relieve exhaustion, and cancer. In this study, we examined how effectively the content and peel extracts of SM can suppress cancer growth. The mechanism underlying cancer-suppressing properties of SM was investigated in tumor-bearing mice with renal carcinoma cell, Renca. MATERIALS AND METHODS Effectiveness of SM in tumor suppression was evaluated by measuring size of tumor mass in tumor-bearing mice treated with various doses of SM content and peel extracts. Proliferation of tumor cells and splenocytes was determined by MTT assay and [³H]TdR uptake, respectively. Relevant immunological mechanisms were chased by assaying cytokines including TGF-β, IL-6, IFN-γ, and TNF-α by ELISA. RESULTS The content and peel extracts of SM inhibited the growth of tumor cells in tumor-bearing mice. Especially, average tumor volume of two groups treated with 3 and 30 mg peel extracts per mouse weight (kg) were significantly decreased to 52.32% (p<0.05) and 68.72% (p<0.01), respectively. To identify tumor regression mechanism, anti-tumor cytokines measured in Con A-activated splenocytes from tumor-bearing mice. IFN-γ was increased in both of the peel extract-treated groups, while TNF-α, which had been decreased by tumor growth, was rescued to the normal level in SM content and peel extracts-treated groups. However, SM content and peel extracts did not inhibit proliferation and tumor-proliferative cytokines including TGF-β and IL-6 production of tumor cells. CONCLUSION These results indicate that SM content and peel extracts have anti-tumor properties in the tumor-bearing murine model. The mechanism underlying the anti-tumor effects of SM extracts is strongly suggested to be via boosting cytokines such as IFN-γ and TNF-α, enhancing immune-mediated anti-tumor properties.

A reversed-phase high-performance liquid chromatography method with pulsed amperometric detection for the determination of glycosides.

We have developed a reversed-phase high-performance liquid chromatography pulsed amperometric detection (RP-HPLC-PAD) method for the determination of glycosides. It is sensitive, repeatable, and selective without the pretreatment step. Ginsenosides were separated completely in 50 min using an water-acetonitrile gradient as the eluent and detected by PAD under NaOH alkaline conditions. The ginsenoside detection limit (S/N=3) was 0.02-0.07 ng and the quantification limit (S/N=10) was 0.1-0.2 ng. The coefficient of linear regression was 0.9984-0.9998 for concentrations between 1 and 50 microg/mL. The intra- and inter-day precision (RSD) was less than 6.35% in Ginseng Radix and Shy-jiun-tzyy-tang extracts. The average recoveries from Ginseng Radix and Shy-jiun-tzyy-tang extracts were 98.19-105.45% and 96.89-102.22%, respectively.

Determination of astragalin and astragaloside content in Radix Astragali using high-performance liquid chromatography coupled with pulsed amperometric detection

Astragalin and astragalosides were measured in Radix Astragali using reversed-phase chromatography coupled with pulsed amperometric detection. Because the target compounds showed poor stability in aqueous solutions, they were extracted in 100% methanol under reflux. All compounds were detected with high sensitivity under highly alkaline conditions using sodium hydroxide as a post-column eluent. The limits of detection and quantification of target compounds were 0.02-0.36 μg/mL and 0.06-1.09 μg/mL, respectively, and the linear regression coefficients were 0.9982-1.0000. The intra- and inter-day precisions were <0.92% in retention time and <4.78% in calculated contents. Average recoveries were >91.33%. Astragalin and astragaloside contents between Radix Astragali at different ages and in different parts were successfully determined without sample purification or concentration.

Dangguijakyak-san, a medicinal herbal formula, protects dopaminergic neurons from 6-hydroxydopamine-induced neurotoxicity

AIM OF THE STUDY Dangguijakyak-san (DJS) is a multi-herbal formula that has long been widely used in traditional Oriental medicine to treat gynecologic disorders, including neurological symptoms. Recent clinical and experimental studies have reported aging and anti-neurodegenerative effects of DJS. In this study, we evaluated the neuroprotective effects of DJS on dopaminergic (DA) neurons damaged by 6-hydroxydopamine (6-OHDA). MATERIALS AND METHODS To evaluate the protective effects of DJS, we analyzed viability in SH-SY5Y neuroblastoma cells and tyrosine hydroxylase (TH) staining in primary DA cells. To explore the possible mechanism(s) of neuroprotection, we assessed anti-oxidant activity by measuring reactive oxygen species (ROS) and glutathione (GSH) levels. To determine mitochondria-mediated apoptotic activity, we examined mitochondrial membrane potential, cytochrome c release, and caspase-3 activation. RESULTS DJS at 0.05-5 μg/mL significantly protected SH-SY5Y cells from 6-OHDA toxicity, dose-dependently, and attenuated 6-OHDA damage in primary DA cells. DJS reduced 6-OHDA-induced intracellular ROS production and GSH depletion and inhibited mitochondrial membrane instability, cytosolic cytochrome c release, and caspase-3 activation. CONCLUSIONS These results demonstrate that DJS has neuroprotective effects in DA neurons against 6-OHDA-induced toxicity through anti-oxidant and anti-mitochondrial-mediated apoptotic activities.

Simultaneous analysis method for polar and non-polar ginsenosides in red ginseng by reversed-phase HPLC-PAD.

The paper describes the development of a simultaneous determination method for polar and non-polar ginsenosides in red ginseng with a reversed-phase high-performance liquid chromatography-pulsed amperometric detection method. This method could be applied directly without any pretreatment steps and enabled the performance of highly sensitive analysis within 1h. The detection (S/N=3) and quantification (S/N=10) limits for the ginsenosides ranged 0.02-0.10 ng and 0.1-0.3 ng, respectively. The linear regression coefficients ranged 0.9975-0.9998. Intra- and inter-day precisions were <9.91%. The mean recoveries ranged 98.08-103.06%. The total amount of ginsenosides in the hairy root of red ginseng was higher than that in the main root.

HPLC with pulsed amperometric detection for sorbitol as a biomarker for diabetic neuropathy.

This study describes the optimal analytical conditions for sorbitol analysis by a high-performance anion-exchange chromatography-pulsed amperometric detection method. Its clinical utility as a diagnostic tool was established by measuring sorbitol in the sciatic nerves or salivary glands of diabetes mellitus-induced mice. Sorbitol was completely separated from other monosaccharides on an anion-exchange column with 100mM NaOH as eluent. The limit of detection (S/N=3) and limit of quantification (S/N=10) were 0.03 ng (3 ng/g) and 0.10 ng (10 ng/g), respectively. The linear dynamic range was 0.01-50.0 microg/g (r(2)=0.9997 and 0.9989 for sciatic nerves and salivary glands, respectively), and the mean recoveries for intra- or inter-day assays were in the range of 98.5-103.9%. This method easily identified diabetic and normal groups, making it a practical procedure for the rapid screening of diabetic neuropathy.

Analyses of organic acids and inorganic anions and their relationship in human saliva before and after glucose intake.

OBJECTIVES The goals of this study were to determine the content of organic acids and inorganic anions in human saliva by using an ion chromatography method, to compare the organic acid and inorganic anion concentrations before and after a sugar rinse, and to investigate the relationships between the levels of each compound. DESIGN Saliva samples were obtained from 37 subjects before and up to 60min after intake of a 10% glucose solution. Concentrations of seven organic acids (lactate, acetate, propionate, formate, butyrate, pyruvate, and valerate) and four inorganic anions (fluoride, chloride, sulphate, and phosphate) were determined via anion-exchange chromatography with an anion-suppressed conductivity detector. RESULTS The current analytical method showed good precision and accuracy. Organic acid levels increased after the sugar rinse and recovered to control levels within 20min. Acetate was the predominant organic acid detected in the saliva before the sugar rinse, and lactate was the predominant organic acid detected after the sugar rinse. The overall organic acid content generated by the sugar rinse was positively correlated with the chloride, sulphate, and phosphate concentration, but somewhat negatively correlated with the fluoride concentration. CONCLUSIONS Organic acid levels are increased in human saliva by glucose metabolism. Furthermore, the formation of organic acids following glucose intake is influenced by the prevailing anion content.

A pulsed amperometric detection method of galactose 1-phosphate for galactosemia diagnosis

Galactose 1-phosphate uridyltransferase deficiency causes the accumulation of galactose and galactose 1-phosphate (Gal 1-P) in the blood. We describe a new pulsed amperometric detection method for determining Gal 1-P levels as a pathognomic marker for the diagnosis of galactosemia. The method uses high-performance anion-exchange chromatography with pulsed amperometric detection. In an anion-exchange column, the analytes were separated in 5 min by the eluent mixture of 40 mM NaOH and 40 mM Na(2)CO(3). The detection limit (signal to noise ratio of 3) to Gal 1-P was 30 microg/dL. The linear dynamic range was 3.0-50 mg/dL (r(2)=0.9999). The mean recoveries of Gal 1-P for intra- and interday assays were 97.55-103.78%. This method clearly separated the type I galactosemia patients from the normal group and is a practical procedure for the rapid diagnosis of galactosemia.

Analysis of saikosaponins in Bupleuri Radix and Caihu-shugan-san using reversed-phase HPLC with pulsed amperometric detection

A simple and sensitive reversed-phase (RP) HPLC coupled with pulsed amperometric detection (PAD) method was developed to determine the saikosaponin content in Bupleuri Radix or Caihu-shugan-san. Four saikosaponins in Bupleuri Radix and Caihu-shugan-san were extracted with a 6:4 solution of 10 mM sodium phosphate buffer (pH 8)/100% ethanol. Pulsed amperometric detection of carbohydrates in four major saikosaponins was highly sensitive when used with a water-acetonitrile gradient on an alkaline RP column with a post-column delivery system. The limits of detection (S/N=3) and of quantification (S/N=10) of saikosaponins were 0.01-0.02 and 0.03-0.05 μg/mL, respectively. The intra- and inter-day precision (RSDs) were each <9.7% and the average recoveries were 95.0-97.6% in Bupleuri Radix. This method can be used to analyze saikosaponins in Bupleuri Radix and Caihu-shugan-san.

Simultaneous diagnostic method for phenylketonuria and galactosemia from dried blood spots using high-performance liquid chromatography-pulsed amperometric detection

We developed a simultaneous diagnostic method for phenylketonuria (PKU) and galactosemia through simultaneous determination of phenylalanine (Phe) and galactose (Gal) by high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD). The intra- and inter-day precisions were <5.8%, with satisfactory mean recoveries (98.2-105%). For all PKU-positive samples, Phe levels were above the cut-off value (>30.0 mg/L), but Gal levels were nearly zero. For 77% of galactosemia-positive samples, Phe levels were above the cut-off value, but Gal levels were above the cut-off value (>80.0 mg/L) for all samples. Our HPLC-PAD method can reduce the false-positive rate of misdiagnosis for PKU and galactosemia.