• Title/Summary/Keyword: reversed-phase HPLC

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Purification and Characterization of the Siderophore from Bacillus licheniformis K11, a Multi-functional Plant Growth Promoting Rhizobacterium. (다기능 PGPR균주 Bacillus licheniformis K11이 생산하는 항진균성 Siderophore의 정제와 특성)

  • Woo, Sang-Min;Woo, Jae-Uk;Kim, Sang-Dal
    • Microbiology and Biotechnology Letters
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    • v.35 no.2
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    • pp.128-134
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    • 2007
  • Previously, we isolated plant growth promoting rhizobacterium (PGPR) Bacillus licheniformis K11 which could produce auxin, cellulase and siderophore. The siderophore of B. licheniformis K11 $(siderophore_{K11})$ was determined to be a catechol type siderophore which is produced generally by Bacillus spp. B. licheniformis K11 could produce the siderophore most highly after 96 h of incubation under nutrient broth at $20^{\circ}C$ with initial pH 9.0. For the production of the $siderophore_{K11}$, trehalose and $NH_4Cl$ were the best carbon and nitrogen sources in Davis minimal medium, respectively. The $siderophore_{K11}$ was Produced in M9 medium (pH 9.0) after 4 days at $20^{\circ}C$, and purified from culture broth of B. licheniformis K11 by using Amberlite XAD-2, Sephadex LH-20 column chromatography, and reversed-phase HPLC. The $siderophore_{K11}$ had the biocontrol activity against spore germination of P. capsici and F. oxysporum on potato dextrose agar (PDA). The results indicate that the $siderophore_{K11}$ is an antifungal mechanism of B. licheniformis K11 against phytopathogenic fungi.

Synthesis of Cocoa Butter Alternative from Coconut Oil Fraction and Palm Oil Fractions by Lipase-Catalyzed Interesterification (Coconut Oil 분별유와 Palm 분별유로부터 효소적 Interesterification에 의한 코코아 버터 대체유지 합성)

  • Bae, Sang-Kyun;Lee, Kyung-Su;Lee, Ki-Teak
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.39 no.10
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    • pp.1487-1494
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    • 2010
  • Structured lipid (SL) for cocoa butter alternative was synthesized by interesterification of coconut oil fraction and palm stearin (6:4 and 8:2, by weight) in a shaking water bath at $60^{\circ}C$ and 180 rpm. It was performed for various reaction times (1, 2, 3, and 6 hr). The reaction was catalyzed by sn-1,3 specific Lipozyme TLIM (immobilized lipase from Thermomyces lanuginosus). SL-solid part was obtained from acetone fractionation at $0^{\circ}C$. SL-solid part was blended with other palm oils and fractions for desirable property of cocoa butter alternative (SL-solid part : palm middle fraction : palm stearin solid : palm oil, 70.4:18.4:2.9:8.3, by weight). In reversed-phase HPLC analysis, triacylglycerol species of cocoa butter alternative had partition number of 40 (10.77%), 42 (13.06%), 44~46 (17.38%) and 48 (51.88%). Major fatty acids of cocoa butter alternative were lauric acid (16.5%), myristic acid (12.28%), palmitic acid (46.03%), and linoleic acid (14.75%). Solid fat content (SFC) and polymorphic form (${\beta}'$ form) of cocoa butter alternative prepared were similar to those of commercial cocoa butter replacer (CBR).

Comparative Analysis of Catechins and Antioxidant Capacity in Various Grades of Organic Green Teas Grown in Boseong, Korea (보성산 유기농 녹차의 품질에 따른 카테킨 함량과 항산화능 비교 분석)

  • Park, Kyung-Ryun;Lee, Sang-Gil;Nam, Tae-Gyu;Kim, Young-Jun;Kim, Young-Rok;Kim, Dae-Ok
    • Korean Journal of Food Science and Technology
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    • v.41 no.1
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    • pp.82-86
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    • 2009
  • The objective of this study was to evaluate the effect of various solvents on extraction of bioactive phenolics and to analyze the antioxidant capacity and contents of individual catechins in various grades of green teas organically grown in Boseong, Korea. The organic green teas, based on their harvest seasons, were categorized into five grades such as Woo-Jeon, Se-Jak, Jung-Jak, Dae-Jak, and coarse tea. Solvents used to extract phenolics from these teas included water at $23^{\circ}C$ and $70^{\circ}C$ as well as 80% (v/v) aqueous methanol and ethanol. In general, aqueous organic solvents of methanol and ethanol led to higher extraction yields of phenolics than water at $23^{\circ}C$ and $70^{\circ}C$. Total phenolics and antioxidant capacity of the teas extracted with the aqueous organic solvents were approximately 1.5 to 3.2 and 1.8 to 3.8 times higher than those with water at $23^{\circ}C$ and $70^{\circ}C$, respectively. Coarse tea, the lowest grade of green tea, showed approximately 30-60% lower total phenolics and antioxidant capacity compared with the higher grade ones. Reversed-phase HPLC analysis was performed quantitatively to identify individual catechins, gallic acid, and caffeine in teas extracted with 80% (v/v) aqueous methanol. Based on their dry weights, the organic green teas contained about 1.7 to 2.9% of caffeine. Content (mg/g dry weight) of tea catechins decreased in the following order: Woo-Jeon (155.4) > Se-Jak (147.7) > Jung-Jak (143.2) > coarse tea (135.1) > Dae-Jak (130.5). (-)-Epigallocatechin gallate was the most abundant among the catechins analyzed. The highest grade of green tea, Woo-Jeon, had the highest amount of (-)-epigallocatechin gallate at 77.4 mg/g dry weight. Overall, the higher grade of organic green teas tended to have the higher level of antioxidant capacity and catechins.

Screening of Domoic Acid, a Marine Neurotoxin, in Korean Shellfishes (국내산 패류의 신경독소 domoic acid 검색)

  • Koh, Eun-Mi;Kwon, Hoon-Jeong
    • Korean Journal of Food Science and Technology
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    • v.34 no.6
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    • pp.1130-1133
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    • 2002
  • Domoic acid, and amnesic shellfish poison, is a neurotoxin frequently found in shellfishes. Guidance level for the consumable shellfish has been established as $20\;{\mu}g$ domoic acid/g by Health and Welfare Canada and U.S. FDA. Domoic acid is produced by pennate diatom, a Nitzschia pungens f. multiseries ingested by the shellfish. Content of domoic acid in shellfish samples collected along the Korean shoreline from May to December of 1999 was analyzed. The collection included 1 Gastropoda (Murex shell) and 11 Bivalvias (oyster, little neck clam, orient hard clam, venus clam, surf clam, ark shell, hard-shelled mussel, pen shell, jack-knife clam, pink butterfly shell, and granulated ark shell). Samples were homogenized, extracted with 50% methanol, filtered, and analyzed by reversed-phase liquid chromatography at 242 nm with mobile phase consisting of 10% acetonitrile and 0.1% trifluoroacetic acid. Recovery of the HPLC analysis was 95.80% (${\pm}1.09$). All tested samples showed no domoic acid at the detection limit of 50 ng/g.

Analysis of coenzyme Q10 in human plasma by high performance liquid chromatography (고성능액체크로마토그라피를 이용한 혈장 내 코엔자임 큐텐 분석)

  • Park, Yong-Sun;Park, Sang-Boem;Song, Sean-Mi;Kim, Yong-Woo;Lee, Kyoung-Ryul
    • Analytical Science and Technology
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    • v.22 no.6
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    • pp.514-518
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    • 2009
  • Coenzyme $Q_{10}$($CoQ_{10}$), a vitamin E-like substance, represents a components of the complex antioxidant system of the human organism. $CoQ_{10}$ levels in human plasma were determined by high performance liquid chromatography (HPLC) with UV detection. It was dissociated from lipoproteins by methanol and extracted into n-hexane with liquid-liquid extraction procedure, after centrifugation, the supernatant was dried under nitrogen gas stream. The residue was dissolved in the absolute ethanol. Determination of $CoQ_{10}$ was performed on a $C_{18}$ reversed-phase analytical column with ultraviolet detection at 275 nm and the mobile phase containing 15% (v/v) ethanol in methanol at a flow rate of 1.7 mL/min. The low limit of quantitation was 0.02 mg/L (S/N=10), the linearity between the concentration and peak height is from 0.1 to 2.0 mg/L. Twenty-four randomly selected plasma samples from apparently healthy, 27 to 44 year old individuals (males and females) were analyzed for total $CoQ_{10}$. The average level in these subjects was $0.62{\pm}0.13mg/L$ with the range of 0.41-0.98 mg/L. This method has a specific and a sufficient limit of quantitation (LOQ) for analysis of $CoQ_{10}$ in human plasma in both a clinical study and research at laboratories.

Isolation and Characterization of Elastase Inhibitor from Areca catechu (빈랑으로부터 Elastase 저해물질의 분리 및 특성조사)

  • 조중제;이건국;조병기;최정도
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.26 no.1
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    • pp.163-186
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    • 2000
  • We have previously screened 150 medicinal plants on the inhibition of elastase and found a significant inhibitory effects of the extracts of Areca catechu L. on the aging and inflammation against the skin tissues. To isolate and identify the compounds having biological activity, we was further purified by each of the solvent fractions, silica gel column chromatography, preparative TLC and reversed-Phase HPLC. Peak in HPLC, which coincided with the inhibitory activity against elastase, was identified as Phenolic substance using various colorimetric methods, UV, and IR. $IC_{50}$/ values of phenolic substance purified from Areca catechu were 26.9 $\mu\textrm{g}$/$m\ell$ for porcine pancreatic elastase (PPE) and 60.8 $\mu\textrm{g}$/$m\ell$ for human neutrophil elastase (HNE). This Phenolic substance showed more potent activity than those of reference compounds, oleanolic acid (76.5 $\mu\textrm{g}$/$m\ell$ for PPE, 219.2 $\mu\textrm{g}$/$m\ell$ for HNE) and ursolic acid (31.0 $\mu\textrm{g}$/$m\ell$ for PPE, 118.6 $\mu\textrm{g}$/$m\ell$ for HNE). According to the Lineweaver-Burk Plots, the inhibition against both PPE and HNE by this phenolic substance was competitive with substrate. Phenolic substance from Areca catechu exhibited high free radical scavenging effect ($SC_{50}$/ : 6 $\mu\textrm{g}$/$m\ell$) and inhibited effectively hyaluronidase activity ($IC_{50}$/: 210 $\mu\textrm{g}$/$m\ell$). These results suggest that the Phenolic substance Purified from Areca catechu showed anti-aging effect by protecting connective tissue proteins.

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Simultaneous determinations of anthracycline antibiotics by high performance liquid chromatography coupled with radial-flow electrochemical cell (고성능 액체 크로마토그래피/방사흐름 전기화학전지를 이용한 안트라사이클린계 항생제의 동시 정량)

  • Cho, Yonghee;Hahn, Younghee
    • Analytical Science and Technology
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    • v.20 no.4
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    • pp.308-314
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    • 2007
  • The analytical method of HPLC with the radial-flow electrochemical cell (RFEC) has been developed to determine doxorubicin, epirubicin, nogalamycin, daunorubicin and idarubicin simultaneously by employing a reversed-phase chromatography. Anthracyclines were detected at -0.74 V vs. a Ag/AgCl (0.01 M NaCl) reference electrode, a potential of diffusion current plateau in the mobile phase. At a $V_f$ of 1.0 mL/min doxorubicin, epirubicin, daunorubicin and idarubicin appeared at a retention time ($t_r$) of 6.4 min, 7.4 min, 12.7 min and 18.4 min, respectively, while at a $V_f$ of 0.6 mL/min, doxorubicin, epirubicin, nogalamycin, daunorubicin and idarubicin appeared at a $t_r$ of 9.9 min, 11.5 min, 13.5 min, 19.6 min and 28.7 min, respectively. The linearity between each anthracycline injected ($2.40{\times}10^{-7}M{\sim}1.42{\times}10^{-5}M$) and peak area (charge) was excellent with the square of the correlation coefficient ($R^2$) higher than 0.999. The detection limits were $1.0{\times}10^{-8}M{\sim}1.5{\times}10^{-7}M$ for the five anthracyclines. Within-day precision for the five anthracyclines were in reasonable relative standard deviations less than 3 % ($1.00{\times}10^{-6}M{\sim}1.42{\times}10^{-5}M$) except the lower concentrations less than $0.7{\mu}M$. Solid phase extractions of $1.00{\times}10^{-5}M$ epirubicin, $0.48{\times}10^{-5}M$ nogalamycin and $1.52{\times}10^{-5}M$ daunorubicin from human serum with a $C_{18}$ cartridge resulted in 97 %, 100 % and 90 % of recoveries, respectively.

Purification and Isolation for Antihypertensive Peptides from Beef Heart and Spleen (쇠고기 부산물로부터 혈압 상승 억제 펩타이드 분리 및 정제)

  • Jang, S. H.;Jang, A.;Kim, K. J.;Cheon, Y. H.;Min, J. S.;Lee, S. O.;Lee, M.
    • Journal of Animal Science and Technology
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    • v.45 no.2
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    • pp.319-326
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    • 2003
  • Angiotensin-I converting enzyme(ACE)inhibitor was isolated from beef by-products. The beef by- product hydrolysates prepared with various proteases were tested for the inhibitory effects against ACE. The proteases used were proteinase A from bakers yeast, protease type ⅩIII fungal and thermolysin. The maximum inhibitory effect was observed after hydrolysis for 12hrs(beef heart) and 24hrs(beef spleen), respectively. After gel filtration, IC50 value was 0.37mg/ml in beef heart and 1.84mg/ml in beef spleen. After RP-HPLC, the IC50 value of peak 1, peak 2, peak 3 and peak-4 were 0.28mg/ml, 0.26mg/ml, 0.25mg/ml and 0.35mg/ml, respectively. In the results of amino acid composition of peak 1, peak 2, peak 3 and peak 4, it was observed that peak 1 was consisted mainly of glycine and methionine, peak 2 was proline, cystine and methionine, peak 3 was proline and peak 4 was alanine, methionine and leucine. In conclusion, beef heart hydrolysate treated with thermolysin+ proteinase A was shown to have the highest inhibitory effect for 12hrs incubation at 37$^{\circ}C$.

A Study on Contents of Vitamin K1 in Local Agricultural Products (지역농산물의 비타민 K1 함량 조사)

  • Lee, A Reum;Kim, Joo Hee;Park, Jae-Ho;Kim, Youngho;Hong, Eui Yon;Kim, Haeng-Ran;Choi, Youngmin;Lee, Junsoo;Eom, Hyun-Ju
    • The Korean Journal of Food And Nutrition
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    • v.29 no.3
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    • pp.301-306
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    • 2016
  • Vitamin K (phylloquinone) is an essential cofactor in the synthesis of active blood-clotting factors II, VII, IX and X. Deficiency of vitamin K leads to inadequate activity of these factors, resulting in bleeding. In this study, we investigated vitamin $K_1$ content of agricultural products that are widely and specifically grown in Korea including 9 leaves and vegetables, 16 fruits, and 11 cereals and specialty crops. Vitamin $K_1$ analysis of the agro-samples was by a validated, modified, reversed phase-HPLC method with fluorescence detection after post-column derivatization. The vitamin $K_1$ content ranged from 1.83 to $682.73{\mu}g$/100 g in leaves and vegetables, 0.17 to $28.22{\mu}g$/100 g in fruits, and ND to $279{\mu}g$/100 g in cereals and specialty crops. Among the 36 samples, high content of vitamin $K_1$ were found in Gugija (Lycium chinense Miller) leaves (average $682.73{\mu}g$/100 g) and Hansan ramie leaves (average $423.12{\mu}g$/100 g); however, mushroom, amaranth and Chinese artichoke showed no detectable levels. The results of ourstudy provide reliable vitamin $K_1$ content of Korean grown agricultural products that expand nutritional information and food composition database.

Enzymatic Interesterification and Melting Characteristic for Asymmetric 1,2-Distearoyl-3-Oleoyl-rac-Glycerol Triacylglycerol Enriched Product (효소적 반응을 이용한 비대칭형 1,2-Distearoyl-3-Oleoyl-rac-Glycerol 혼합물의 생성 및 융점 특성)

  • Kim, Jin Young;Lee, Ki Teak
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.43 no.1
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    • pp.93-101
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    • 2014
  • Asymmetric 1,2-distearoyl-3-oleoyl-rac-glycerol (SSO) triacylglycerol (TAG) is used as a cocoa butter replacer (CBR). In this study, it was produced by lipase-catalyzed interesterification of fully hydrogenated soybean oil (FHSBO) and oleic ethyl ester (OEE) in a batch type reactor at $75^{\circ}C$, 250 rpm. Different molar ratios (FHSBO : OEE=1:1, 1:2 and 1:3, w/w) and various reaction times (1, 2, 3, 4, and 5 hr) were also tested. The optimized condition for SSO was a FHSBO : OEE molar ratio of =1:1 at reaction times of 2, 3, 4, and 5 hr. Enzymatic synthesis generated SSO/SOS, as well as the other TAGs (e.g., PSO/POS, SOO/OSO, SSS), ethyl esters, monoacylglycerol (MAG), and diacylglycerol (DAG). After scale-up, fractionation by solvent (methanol and acetone) fractionation and column chromatography was applied. To reduce ethyl esters, high-melting TAGs (e.g., SSS), and SOO/OSO in reactants, solvent fractionation was applied. Using a silica gel column (sample : silica gel=2:1, wt%), MAG and DAG were removed at $25^{\circ}C$. The major fatty acid composition of the final products (with a high SSO/SOS content) was palmitic acid (C16:0, 10.9~12.9 area%), stearic acid (C18:0, 52.2~54.9 area%), and oleic acid (C18:1, 34.2~35.5 area%). In reversed-phase HPLC analysis, the major TAG species of the final product (FHSBO : OEE=1:1, 2 hr) were SSO/SOS (82.31 area%) and PSO/POS (14.51 area%). Based on the $[SS]^+$ : $[SO]^+$ ratio obtained by RP-HPLC/APCI-MS, the final product had a higher SSO (AAB type TAG) content than cocoa butter (CB). The solid fat index (SFI) of CB and the final product obtained were similar with a narrow melting point range around ~32 to $35^{\circ}C$.