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Analysis of the Marker Compounds in Sagunja-tang by LC-ESI-MS (LC-ESI-MS에 의한 사군자탕의 지표성분 분석)

  • Seo, Chang-Seob;Shin, Hyeun-Kyoo
    • Korean Journal of Pharmacognosy
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    • v.50 no.1
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    • pp.65-71
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    • 2019
  • One of the oriental medicine prescriptions, Sagunja-tang consists of four herbal medicines (Ginseng Radix, Poria Sclerotium, Atractylodis Rhizoma Alba, and Glycyrrhiziae Radix et Rhizoma) and has been used as a medicine to enhance tonify the function of spleen and stomach in Korea. In this study, we conducted simultaneous analysis of the 9 marker components, liquiritin apioside, liquiritin, ginsenoside Rg1, liquiritigenin, ginsenoside Rb1, glycyrrhizin, atractylenolide III, atractylenolide II, and atractylenolide I in Sagunja-tang using a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). Marker compounds were separated on a Waters Acquity UPLC BEH $C_{18}$ analytical column ($2.1{\times}100mm$, 1.7 mm) and the column was maintained at $45^{\circ}C$. The mobile phase consists of 0.1% (v/v) aqueous formic acid and acetonitrile with gradient condition. The LC-MS analysis was performed using a Waters ACQUITY TQD LC-MS/MS system with multiple reaction monitoring (MRM) method in the positive and negative modes. The calibration curves of the nine marker components showed good linearity with coefficient of determination ${\geq}0.9984$ within tested range. The limits of detection and limits of quantification values were 0.27-2.42 ng/mL and 0.81-7.27 ng/mL, respectively. The concentrations of tested 9 analytes in the lyophilized Sagunja-tang sample using the established LC-ESI-MS/MS MRM method were detected up to 16.593 mg/g. These results can be useful as a basic data for the quality control of an oriental medicine prescriptions.

Purification and Characterization of Antioxidant Peptides from Lotus Nelumbo nucifera Seed Protein (연자육(Lotus Nelumbo nucifera Seed) 단백질로부터 항산화 펩타이드 분리 정제 및 특성)

  • Chathuri K. Marasinghe;Hyun-Woo Kim;Won-Kyo Jung;Jae-Young Je
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.56 no.1
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    • pp.21-27
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    • 2023
  • Lotus Nelumbo nucifera seed protein (LSP) was isolated by alkaline solubilization after removing fat and phenolics by hexane and ethanol treatment. Antioxidant peptides from LSP were produced with Alcalase® and pepsin and hydroxyl radical scavenging activities were determined. LSP-Alcalase® hydrolysates showed higher hydroxyl radical scavenging activity than LSP-pepsin hydrolysates. To purify antioxidant peptides, LSP-Alcalase® hydrolysates were subjected to high performance liquid chromatography (HPLC) separation on the C18 column and the active fraction was further purified using a SuperdexTM peptide 10/300 GL column. Finally, the active fraction (F8-2) was evaluated for antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical scavenging, and oxygen radical absorbance capacity (ORAC) assays. The EC50 values of the F8-2 were 105.81±0.02 ㎍/mL for DPPH and 32.26±0.02 ㎍/mL for hydroxyl radical and the F8-2 exhibited 7.22 μM trolox equivalent (TE)/100 ㎍ F8-2. Glutathione (GSH), which is a positive control, showed EC50 values of 19.87±0.01 ㎍/mL for DPPH and 15.95±0.03 ㎍/mL for hydroxyl radical and an ORAC value of 14.17±0.03 μM TE/100 ㎍ GSH. Finally, sixteen peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Ile-Tyr and Leu-Tyr showed higher antioxidant scores.

Establishment of a Method for the Analysis of Diarrhetic Shellfish Poisoning by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS를 이용한 설사성패류독소의 분석조건 확립)

  • Lee, Ka-Jeong;Suzuki, Toshiyuki;Kim, Poong-Ho;Oh, Eun-Gyoung;Song, Ki-Cheol;Kim, Ji-Hoe
    • Korean Journal of Food Science and Technology
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    • v.41 no.4
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    • pp.458-463
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    • 2009
  • To establish and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and accurate quantitation of diarrhetic shellfish poisoning (DSP) toxins, we compared the results from different mobile phases and columns used for their analysis and collision energies for MS/MS experiments. Clear peaks of okadaic acid (OA) and dinophysistoxin-1 (DTX1) were obtained by using a mobile phase comprising aqueous acetonitrile containing 2 mM ammonium formate and 50 mM formic acid. The collision energies were optimized to facilitate the most sensitive detection for each toxin, namely, OA, DTX1, pectenotoxin-2 (PTX2), or yessotoxin (YTX). Further, the maximum ion response was obtained at a collision energy of 48 V for OA and DTX1. We compared the analytical performance of $C_8$ and $C_{18}$ columns. A wide range of toxins namely, OA, DTX1, PTX2, and YTX, except DTX3, were detected by both the columns. Although DTX3 was only detected by the $C_8$ column, we found that the $C_{18}$ column was also suitable for the quantitation of OA and DTX1 the toxins responsible for inducing diarrhea. The limit of detection of OA and DTX1 by the established LC-MS/MS conditions was 1 ng/g, and the limit of quantitation of the toxins under the same conditions was 3 ng/g. The process efficiencies were 91-118% for oysters (Crassostrea gigas) and 96-117% for mussels (Mytilus galloprovincialis) further, we observed no significant effect of matrix during the ionization process in LC-MS/MS. The comparison between mouse bioassay (MBA) and LC-MS/MS yielded varying results because low concentrations of OA and DTX1 were detected by LC-MS/MS in some shellfish samples, which provided positive results on MBA for DSP. The analysis time required by MBA for DSP analysis can be reduced by LC-MS/MS.

The Correlation between Environmental Factors and Phytoplankton Communities in Spring and Summer Stratified Water-column at Jinhae Bay, Korea (진해만에서 춘계와 하계 성층기간 동안 환경요인과 식물플랑크톤 군집구조의 관계)

  • Son, Moonho;Hyun, Bong-Gil;Kim, Dong Sun;Choi, Hyun Woo;Kim, Young Ok;Baek, Seung Ho
    • Korean Journal of Environmental Biology
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    • v.30 no.3
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    • pp.219-230
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    • 2012
  • We surveyed 23 sites of Jinhae Bay in spring and summer 2010 in order to study the correlation between the variation of environmental factors, including salinity, temperature, and nutrients and the characteristics of phytoplankton community structures in summer stratification. Phytoplankton biomass was high in the surface water in summer; however, it was very low in the bottom water. The results showed a negative correlation between chlorophyll a (chl. a) and nutrients (silicate, nitrate, nitrite, ammonium, and phosphate) or nutrients ratio in summer; however, there was mostly a positive correlation between chl. a and these nutrients in spring. This inconsistent correlation between spring and summer was attributed to the phytoplankton community, because a diverse phytoplankton community has different nutrient uptake abilities. In addition, the results of CCA (canonical correspondence analysis) showed a negative correlation between phosphate and dominant species, including Chaetoceros spp., Skeletonema costatum-like spp., and Pseudo-nitzschia delicatissima in summer, but a strong positive correlation between DIN (dissolved inorganic nitrogen) and the dominant species, including Cryptomonas spp. and Pseudo-nitzschia multistriata in spring. Consistently, the dominant algal species in summer showed a relatively smaller size cells compared with those in spring, suggested that it may have related with the low nutrient levels at surface layer due to strong stratified water column of summer.

Identification of a Protein Kinase using a FITC-labelled Synthetic Peptide in Streptomyces griseus IFO 13350 (형광 Peptide를 이용한 Streptomyces griseus IFO 13350의 인산화 단백질 동정)

  • 허진행;정용훈;김종희;신수경;현창구;홍순광
    • Microbiology and Biotechnology Letters
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    • v.30 no.3
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    • pp.235-240
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    • 2002
  • Streptomycetes is a group of Gram-positive soil bacteria that growas a branching vegetative mycelium leading to the formation of spores, and display a physiological differenti-ation related to the synthesis of many secondary metabolites including antibiotics. Their complex life cycle and multicellular differentiation require various levels of regulation and types of signal transduction systems including eukaryotic-type serine/threonine protein kinases and prokaryotic-type histidine/aspartic acid protein kinases. Akt kinase that was found in cells is a sorine/threonine kinase controlling signal pathway for multi-tude of important cellular events. The activation or inactivation of Akt kinase in the cell is one of the critical regulatory points to deliver cell proliferation, differentiation, survival or apoptosis signal. To find the regula-tory protein homologous to Akt in Streptomyces, the fluorescien-labeled synthetic peptide (FITC-TRRSR-TESIT) was designed from the consensus sequence of target proteins for Akt kinase. From the difference of the mobility between the nonphosphorylated and phosphorylated synthetic peptides on Agarose gel electro-phoresis, the Akt-phosphorylating activity was monitored. The cell-free extract prepared from Streptomyces griseus IFO 13350 and the Akt homologous protein was purified by ammonium sulfate fractionation and many steps of column chromatographies such as, DEAE-Sepharose, Mono Q, Resource Phenyl-Soporose and Gel permeation column chromatographies. As a result, the protein phosphorylating the fluorescien-labeled Akt substrate was identified and it's molecular weight was estimated as 39 kDa on SDS-PAGE.

Seismic Behaviour of Exterior Joints in Post-Tensioned Flat Plate Systems (포스트 텐션 플랫 플레이트 외부 접합부의 내진 거동)

  • Han, Sang-Whan;Kee, Seong-Hoon;Kang, Tomas H.K.;Cho, Jong;Lee, Li-Hyung
    • Journal of the Korea Concrete Institute
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    • v.18 no.5 s.95
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    • pp.595-602
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    • 2006
  • An experimental study was conducted to investigate seismic behaviour of post-tensioned(PT) exterior slab-column connections used for the purpose to resist gravity loads only. For these, 2/3-scale, two PT post-tensioned exterior connections with two different tendon arrangement patterns and one conventional reinforced concrete(RC) exterior connection was tested under quasi-static, uni-directional reversed cyclic loading. During the lateral testing, gravity forces transferred to the column were kept constant to closely simulate a moment to shear ratio of a real building. One of the objectives of this study was to assess the necessity and/or the quantity of bottom bonded reinforcement needed to resist moment reversal which would occur under significant inelastic deformations of the adjacent lateral force resisting systems. The ACI 318 and 352 provisions for structural integrity were applied to provide the bottom reinforcement passing through the column for the specimens. Prior test results were also collected to conduct comparative studies for some design parameters such as the tendon arrangement pattern, the effect of post-tensioning forces and the use of bottom bonded reinforcement. Consequently, the impact of tendon arrangement on the seismic performance of the PT connection, that is lateral drift capacity and ductility, dissipated energy and failure mechanism, was considerable. Moreover, test results showed that the amount of bottom reinforcement specified by ACI 352. 1R-89 was sufficient for resisting positive moments arising from moment reversal under reversed cyclic loads. Shear strength of the tested specimens was more accurately predicted by the shear strength equation(ACI 318) considering the average compressive stress over the concrete($f_{pc}$) due to post-tensioning forces than that without considering $f_{pc}$.

Biochemical Study on Soft-rotted Sweetpotatoes (연부(軟腐)고구마의 생화학적(生化學的) 연구(硏究))

  • Chun, Jae-Kun
    • Applied Biological Chemistry
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    • v.10
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    • pp.47-61
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    • 1968
  • Sweetpotato infected by Soft-rot, Rhizopus nigricans, has been investigated for its biochemical changes. The results of the present experiment are summarized as follows: 1. From the Soft-rotted tuber, nineteen Ehrlich's positive substances and three fluorescent compounds which had not been found in a healthy tuber were detected with thin layer chromatography. 2, Ipomeamarone and umbelliferone, each of which are predominating substances among the Ehrlich's positive and fluorescent compounds, were isolated by silica gel column chromatography and studied for their identities. 3. Time course biosynthesis of the metabolites was studied and discussed for their possible biosynthetic pathways. 4. Among the metabolites, ipomeamarone and ipomeamarone-like substances have enzyme: inhibitive action against ${\alpha}$-amylase and probably other enzymes; the inhibition type was determined as the uncompetitive one. 5. Ipomeamarone obtained from soft-rotted sweetpotato was proved to have an uncoupling action as 2, 4-dinitrophenol.

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The Frequency and Distribution of Unexpected Red Cell Antibodies and Analysis of Antigen Exposure

  • Yang, Sung Sik;Yang, Hoi Joo;Park, Hyun Jun;Kwon, Seog Woon
    • Korean Journal of Clinical Laboratory Science
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    • v.45 no.4
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    • pp.164-169
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    • 2013
  • Red cell alloantibodies other than naturally occurring anti-A or anti-B are called unexpected red cell antibodies, and can be detected by performing an antibody screening. The frequency and distribution of unexpected antibody identified in Asan Medical Center were analyzed. We investigated a total of 135,238 cases of antibody screening test in AMC for 3 years from 2010 to 2012. Using column agglutination techniques, antibody identification tests were performed for the cases with positive antibody screening. Among 135,238 cases, 854 (0.6%) cases showed positive results of antibody screening test. In the order of frequency, 284 (33.3%) anti-Rh, 89 (10.4%) anti-MNS, 62 (7.3%) anti-Lewis, 34 (4.0%) anti-Kidd, 10 (1.2%) anti-Duffy, and 9 (1.1%) anti-P were identified. Multiple antibodies were detected in 199 (23.3%) cases. Among 381 subjects investigated for transfusion history, 299 (78.5%) had history of transfusion while 82 (21.5%) had unknown history. Thus the incidence of unexpected antibody was higher in the group with history of transfusion than the group without (p<0.001). Also, among 435 subjects investigated for the history of pregnancy, 46 (10.6%) had no history while 389 (89.4%) had history of pregnancy, showing higher incidence of unexpected antibody in the group with history of pregnancy than the group without pregnancy (p<0.001). Evaluated amounts and frequency of antigen exposure due to transfusion and pregnancy is suggested to increase the frequency of identification of unexpected antibody.

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An Antifungal Antibiotic Purified from Bacillus megaterium KL39, a Biocontrol Agent of Red-Pepper Phytophthora-Blight Disease

  • JUNG HEE KYOUNG;KIM SANG-DAL
    • Journal of Microbiology and Biotechnology
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    • v.15 no.5
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    • pp.1001-1010
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    • 2005
  • Bacillus megaterium KL39, an antibiotic-producing plant growth promoting rhizobacterium (PGPR), was selected from soil. The antifungal antibiotic, denoted KL39, was purified from culture filtrate by column chromatography using Dion HP-20, Silica gel, Sephadex LH-20, and prep-HPLC. Thin layer chromatography, employing the solvent system of ethanol:ammonia:water=8:1:1, showed the $R_{f}$. value of 0.32. The antibiotic KL39 showed a negative reaction with ninhydrin solution, positive with iodine vapor, and also positive with Ehrlich reagent. It was soluble in methanol, ethanol, butanol, and acetonitrile, but insoluble in chloroform, toluene, hexane, ethyl ether, or acetone. Its UV spectrum had the maximum absorption at 208 nm. Amino acid composition, FAB-mass, $^{1}H-NMR,\;^{13}C-NMR$, and atomic analyses showed that the antibiotic KL39 (MW=1,071) has a structure very similar to iturin E. The antibiotic KL39 has a broad antifungal spectrum against a variety of plant pathogenic fungi including Rhizoctonia solani, Pyricularia oryzae, Monilinia froeticola, Botrytis cinenea, Altenaria kikuchiana, Fusarium oxysporum, and F. solani. An MIC value of $10\;{\mu}g/ml$ was determined for Phytophthora capsici. Macromolecular incorporation studies with P. capsici using radioactive [$^{3}H-adenine$] as the precursor, indicated that the antibiotic KL39 strongly inhibits the DNA biosynthesis of the fungal cell. Microscopic observation of the antifungal action showed abnormal hyphal swelling of P. capsici. The purified antibiotic KL39 was very effective for the biocontrol of in vivo Phytophthora-blight disease of pepper.

Seasonal Changes of Zooplankton Distribution with Environmental Factors in Lake Jinyang (진양호 환경요인과 동물플랑크톤 군집 동태)

  • Yoon, Jong-Su;Jeong, Hyun-Gi;Kwon, Young-Ho;Shin, Chan-Ki;Hwang, Dong-Jin
    • Journal of environmental and Sanitary engineering
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    • v.23 no.4
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    • pp.45-54
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    • 2008
  • Our study indicates the zooplankton abundance with characteristics of water column and the vertical distribution in Lake Jinyang, South Korea. Seasonal changes of zooplankton community are determined by environmental parameters like water temperature, pH, dissolved oxygen, suspended solids and chlorophyll a. In lake Jinyang, this study showed that the zooplankton abundance in transition zone(St.1, St.2) was higher density than in lacustrine zone(St.3). Rotifers were dominant zooplankton and among them, Polyarthra spp., Keratella spp. and Nauplli(Copepoda) were common. But Cladoceran showed the low density. During survey period, zooplankton abundance with vertical distribution in surface layer(epilimnion) was higher than in bottom layer(hypolimninon). Zooplankton densities in Surface and middle layer showed positive relationship with water temperature and the densities in bottom layer(hypolimnion) showed positive relationship with chlorophyll a. Our assumption in spite of the short term study are supported by the facts that increase of temperature driven by climate change more maintains the thermocline duration by the summer temperature stratification. Thus the results suggest that the climate changes are an important source of changing zooplankton community feeding phytoplankton. So the zooplankton should be monitoring by the ecological management of Lake Jinyang to cope with climate changes like flood plain or drought.