• BMB Reports
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• v.29 no.6
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• pp.500-506
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• 1996

We have isolated a water-extracted novel regulator for blood coagulation from an earthworm, Lumbricus rubellus. As a folk remedy, the earthworm has been known to facilitate blood circulation. After complete heat inactivation of endogenous proteases in the earthworm, an anticoagulant(s) was purified through ammonium sulfate fractionation and three consecutive gel permeation chromatography of Sephacryl S-300, Sephadex G-75, and G-150 by measuring activated partial thromboplastin time (APTT) The anticoagulant was further purified to 2,800 fold with a C4 reversed-phase HPLC This activity was stable under heat ($100^{\circ}C$ for 30 min) and acidic conditions (0.4 N HCl). The effects of this partially purified anticoagulant on thrombin were observed with various substrates such as N${\alpha}$-benzoyl-DL-arginine-p-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-L-arginine-p-nitroanilide (S-2238), N${\alpha}$-p-tosyl-L-arginine methyl ester (TAME), and fibrinogen as a natural substrate. Only TAME hydrolysis, due to an esterase activity of the enzyme, was inhibited among the chromogenic substrates. In addition, the anticoagulant not only inhibited the conversion of fibrinogen to fibrin but also prolonged the fibrin clot formation monitored with the in vitro coagulation test. Based on these observations, we suggest the significance of measuring the ability of antithrombotic drugs to inhibit the esterase activity of thrombin. In this report, it was also shown that the earthworm indeed contained a water-extractable, heat- and acid-stable anticoagulant which could be used as a novel antithrombotic agent.

• Archives of Pharmacal Research
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• v.29 no.11
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• pp.1049-1054
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• 2006

Activation of Sphingosine kinase (Sphk) increases a bioactive lipid, sphingosine 1-phosphate (S1P) and has been observed in a variety of cancer cells. Therefore, inhibition of Sphk activity was an important target for the development of anticancer drugs. As a searching tool for Sphk inhibitor, we developed fluorescent Sphk activity assay combined with high performance liquid chromatography (HPLC). Previously we established murine teraticarcinoma mutant F9-12 cells which lack S1P lyase and stably express Sphk1. By using F9-12 cells, optimal assay conditions were established as follows; $100\;{\mu}M\;of\;C_{17}-Sph\;and\;30\;{\mu}g$ protein of F9-12 cells lysate in 20 min. Sphingosine analog $C_{17}-Sph$ was efficiently phosphorylated by Sphk activity ($K_{m}:67.08\;{\mu}M,\;V_{max}\;:1507.5\;pmol/min/mg$). New product $C_{17}-S1P$ was separated from S1P in reversed-phase HPLC. In optimized conditions, 300 nM of phorbol 12-myristate 13-acetate (PMA) increased Sphk activity approximately twice while $20\;{\mu}M$ of N,N-dimethylsphingosine (DMS) reduced 70% of Sphk activity in F9-12 cells lysate. In conclusion, we established non-radioactive but convenient Sphk assay system by using HPLC and F9-12 cells.

• Archives of Pharmacal Research
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• v.19 no.6
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• pp.529-534
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• 1996

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of ketorolac in human serum using a new extraction method with a good recovery. Human serum samples (1.0 ml) spiked with known concentrations of ketorolac tromethamine and 10${\mu}g$ of ketoprofen as the internal standard (IS) were acidified with 200${\mu}l$ of 1 N HCl and extracted with 7 ml of n-hexane-ether (7:3 v/v). Extracts were centrifuged and organic layer was back-extracted with 400${\mu}l$ of 0.1% tromethamine solution. Twenty .mu.l of centrifuged aqueous layer was injected onto a reversed-phase octyl column and eluted with a mixture of acetonitrile, water, methanol, and triethylamine [35:55:10:0.1 (v/v), pH 3.0] at a flow rate of 1.0 ml/min. Ultraviolet detection of ketorolac and IS was carried out at 300 nm. The calibration curve obtained using peak area ratios showed a good linearity (in concentration range 10-150 ng/ml $r^2$=O.9944; in range 50-2000 ng/ml, r$^{2}$=0.9998). The mean intra-day accuracy and precision for this HPLC method were found to be 3.6 and 3.7%, respectively. The mean inter-day accuracy and precision were found to be 4.0 and 3.7%, respectively, in the concentration range 50-2000 ng/ml. The recovery of ketorolac from serum was 92.0 $({\pm}5.7)$ % at the concentration of 100 ng/ml. This method proved to be readily applicable to the assay of ketorolac in human serum.

• KSBB Journal
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• v.25 no.5
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• pp.449-452
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• 2010

L-Thyroxine possessing a chiral center, the naturally occurring thyroid hormone has been used for the treatment of thyroid dysfunctions and marketed as levothyroxine (L-thyroxine) sodium salt. In this study, after extraction of levothyroxine tablet as a pre-treatment process, direct enantiomer separation of thyroxine on crown ether derived chiral columns for determination of optical purity was performed using reversed mobile phase with acid additive. The chromatographic method developed in this study was applied in the determination of optical purity of several current domestic and foreign commercialized levothyroxine tablets. Optical purity values of these commercialized L-thyroxine sodium tablets except one were higher than 99 percents.

• The Korean Journal of Pain
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• v.20 no.2
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• pp.106-110
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• 2007

Background: A phosphodiesterase 5 inhibitor, sildenafil, has been effective against nociception. Several lines of evidence have demonstrated the role of the GABAergic pathway in the modulation of nociception. The impact of the GABA receptors on sildenafil was studied using the formalin test at the spinal level. Methods: Male SD rats were prepared for intrathecal catheterization. The formalin test was induced by subcutaneous injection of formalin solution. The change in the activity of sildenafil was examined after pre-treatment with GABA receptor antagonists ($GABA_A$ receptor antagonist, bicuculline; $GABA_B$ receptor antagonist, saclofen). Results: Intrathecal sildenafil dose-dependently attenuated the flinching observed during phase 1 and 2 in the formalin test. The antinociceptive effect of sildenafil was reversed by the $GABA_B$ receptor antagonist (saclofen) but not by the $GABA_A$ receptor antagonist (bicuculline) in both phases. Conclusions: Intrathecal sildenafil suppressed acute pain and the facilitated pain state. The antinociception of sildenafil is mediated via the $GABA_B$ receptor, but not the $GABA_A$ receptor, at the spinal level.

• Analytical Science and Technology
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• v.17 no.4
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• pp.289-294
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• 2004

A high performance liquid chromatographic method was designed for the quantitative analysis of oxymatrine in Sophora Radix. The separation of oxymatrine was performed by reversed-phase chromatography with a $C_{18}$ column and a buffered aqueous solution containing acetonitrile, and monitored by UV absorption at 215 nm. Extraction of oxymatrine in Sophora Radix was carried out using various solvents and extraction methods. The optimum extraction efficiency for the crushed Sophora Radix was achieved by reflux at $80^{\circ}C$ in 50% ethanol for five hours. Most extraction methods used to complicate pretreatments. In this study, sublimation was employed for a extraction method without going through complicate pretreatments. Sublimation was carried out under high vacuum ($1{\times}10^{-3}$ torr) and at high temperature ($200^{\circ}C$). Extraction efficiency using Sublimation was found to be inferior to other extraction methods.

• Korean Journal of Food Science and Technology
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• v.38 no.1
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• pp.5-9
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• 2006

Method for separation and quantification of methylxanthins, catechins, and theaflavins in Korean commercial teas (green, oolong, and black teas) was developed using reversed phase high-performance liquid chromatography (HPLC). After extraction with hot water, tea compounds were separated on Inertsil ODS-3v $(5\;{\mu}m)$ column, eluted with gradient of 7% acetonitrile and 93% of 20 mM phosphate buffer mixture for 7 min. Column effluent was monitored at 270 nm. This technique was effective for analyses of m methylxanthins, catechins, and theaflavins in teas and biological samples. In green and oolong teas, two kinds of methylxanthins and 7 of catechins were identified, whereas 4 theaflavins were only identified in black tea. Among seven catechins in green and oolong teas, EGCG showed highest amount, whereas ECG was highest in black tea. (theaflavins were found only in black teas) In all teas, theobromine content was lower than that of caffeine.

• Food Science and Biotechnology
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• v.15 no.3
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• pp.392-398
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• 2006

Four flavonoids 1-4 and one phytosterol 5 were isolated from methanol extract of Taekwangkong, one of the soybean cultivars, and the structures of these compounds were fully characterized by physical and spectral analysis. The content of compounds 1-4 as determined by $C_{18}$ reversed phase HPLC (high-performance liquid chromatography) coupled with diode-array detector were 12.1, 624.6, 18.0, and $219.6\;{\mu}g/g$, respectively, and the total phenolic content of this cultivar was measured as 3.7 mg gallic acid equivalent per g dry material (GAB/g). Also, compound 1 showed strong radical scavenging activity in the 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay ($IC_{50}\;=\;47.6\;{\mu}M$), five-fold higher than seen in the 1,1-diphenyl-2-picrylliydrazyl (DPPH) assay. These results lead to the conclusion that soybean not only has many phytoestrogens but also has potent antioxidant activity.

• Nutritional Sciences
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• v.9 no.4
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• pp.323-329
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• 2006

To more accurately estimate vitamin A and vitamin E intake of Koreans, food composition databases of the tocopherol and carotenoid contents of Korean foods are needed. In this study, the tocopherol $(\alpha-,\;\gamma-,\;and\;\delta-)$ and carotenoid ($\alpha-carotene,\;\beta-carotene,\;\beta-ctyptoxanthin$, lutein, and zeaxanthin) contents of 12 Korean cooked combination foods commonly consumed by children in Kwangju, Republic of Korea, were determined using reversed-phase HPLC. All samples were obtained from 3 different households in Kwangju during summer, 2005. All cooked foods in this study had detectable quantities of $\alpha-tocopherol,\;\gamma-tocopherol$ except for shoegogimugook, and $\delta-tocopherol$ except for myulchibokkeum Doejigogibokkeum had the highest $\alpha-tocopherol$ content (0.64 mg/l00 g edible portion), and $\gamma-tocopherol\;and\;\delta-tocopherol$ contents of gimbab were the highest among the foods (1.01 and 0.26 mg/l00 g edible portion). $\beta-carotene$ was found in all food samples. Gimbab had the highest contents of $\alpha-carotene,\;\beta-carotene$, and lutein among Korean cooked combination foods (158.3, 266.6, and $375.4{\mu}g/100g$ edible portion). Miyukgook contained only $\beta-carotene$ ($2.5{\mu}g/100g$ edible portion). Considerable sample-to-sample variability in tocopherol and carotenoid compositions were observed in several Korean cooked combination foods included in this study. Some of these carotenoids $(\alpha-carotene,\;\beta-carotene,\;and\;\beta-cryptoxanthin)$ are vitamin A precursors. The findings of this study may be valuable for use in Korean databases as well as nutrient consumption research for vitamin A and vitamin E.

• Journal of the Korean Society of Propulsion Engineers
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• v.21 no.1
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• pp.72-83
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• 2017

A thermo-fluid dynamic analysis was performed through the numerical simulation of a missile canister. Calculation was made in a fixed analytical volume and fully evaporated water was used as a coolant. To analyze the interaction among the hot gas, coolant, and mixture flow, Realizable $k-{\varepsilon}$ turbulence and VOF(Volume Of Fluid) model were chosen and parametric study was performed with the change of coolant flow rate. It could be found that the pressure on the canister top nonlinearly increased with the increase of coolant flow rate. Temperature and coolant distribution were closely related to the flow behavior in canister. Temperature on the canister bottom indicated a decrease being proportional to coolant flow rate in early times but after a specific time, the temperature increased with the tendency being reversed. In addition, the early part of temperature showed a fluctuating phenomenon because of the overall circulatory flow of mixture gas.