• Title/Summary/Keyword: thin layer chromatography (TLC)

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Development of Doxorubicin overproducing Streptomyces Strain using Protoplast Regeneration (방선균 원형질체 재생에 의한 독소루비신 고생산성 균주개발)

  • 박희섭;박현주;김용훈;임상민;김동일;류욱상;김상린;김응수
    • KSBB Journal
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    • v.18 no.4
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    • pp.289-293
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    • 2003
  • Doxorubicin is an anthracycline-family polyketide compound with a very potent anti-cancer activity, typically produced by Streptomyces peucetius. In order to increase doxurubicin productivity, a semi-industrial doxorubicin-producing Streptomyces strain named BR-Dox was cultured in a R2YE liquid medium containing CaCO$_3$, and then converted to a cell wall-free protoplast using lysozyme treatment method, followed by PEG-mediated cell wall regeneration. Among several protoplast-regenerated Streptomyces BR-Dox strains, two independent isolates named BR-Dox4 and BR-Dox6 were visually selected using thin layer chromatography (TLC) based on the pigment overproducing phenotype. Comparing with Streptomyces BR-Dox parental strain, two protoplast-regenerated strains, BR-Dox4 and BR-Dox6 exhibited 25.2% and 12.2% higher doxorubicin productivity analyzed by high pressure liquid chromatography (HPLC), respectively. This result suggests that a protoplast-regeneration of an antibiotics-producing Streptomyces strain should be a promising strain development approach for antibiotics overproduction in Streptomyces species.

Isolation and Identification of Antimicrobial Compound from Plantain (Plantago asiatica L.) (질경이로부터 항균성 화합물의 분리 및 동정)

  • 김건희;김순임;한영실
    • Korean journal of food and cookery science
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    • v.15 no.4
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    • pp.410-417
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    • 1999
  • Antimicrobial activity of Plantain(Plantago asiatica L.) was investigated. Methanol extract of dried Plantain was fractionated to hexane, chloroform, ethylacetate, butanol and aqueous fraction. Ethylacetate fraction among these fractions showed the highest inhibitory effect on the microorganisms such as B. subtilis, E. coli, and V. parahaemolyticus at 500 $\mu\textrm{g}$/disc. Ethylacetate fraction was further fractionated into 8 fractions by silica gel column and thin layer chromatography(TLC). The results showed that ethylacetate fractions No. 2 and 3 had the highest anti-microbial activity. They were mixed again, re-separated, and seven fractions were obtained. Among them, No.4 and 6 fraction had the highest inhibitory effect on the microorganisms, which were then separated into four fractions. In the 3rd fractionation, No.4 fraction was identified as hexadecanoic acid by HPLC, $^1$H-NMR and GC-MS.

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Antimicrobial Substance against Escherichia coli O157:H7 Produced by Lactobacillus amylovorus ME1

  • Jung, Byung-Moon;Woo, Suk-Gyu;Chung, Kun-Sub
    • Food Science and Biotechnology
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    • v.17 no.3
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    • pp.679-682
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    • 2008
  • A lactic acid bacterium producing an antimicrobial substance against Escherichia coli O157:H7 was isolated from raw milk and identified as Lactobacillus amylovorus ME-1. In addition to E. coli O157 :H7, the antimicrobial substance also inhibited the growth of Bacillus cereus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyrogenes, and Yersinia enterocolitica. The antimicrobial substance was stable at pH 2-12 and $121^{\circ}C$ for 15 min and insensitive to proteinase K, protease, amylase, and catalase. Purification of the antimicrobial substance was conducted through methanol and acetonitrile/ethylacetate extraction, ultrafiltration with a 500 Da cutoff, thin layer chromatography (TLC) with silicagel 60, and high performance liquid chromatography (HPLC) with a $C_{18}$ reverse phase column. The ${\lambda}_{max}$ of the purified antimicrobial substance was determined as 192 nm by ultra violet (UV) scanning, while the molecular weight was estimated as 453 Da based on the mass spectrum. Accordingly, the current results suggest that the antimicrobial substance from the L. amylovorus ME-1 was not a bacteriocin, but rather a new non-proteinaceous substance distinct from acidophilin, acidolin, diacetyl, and reuterin.

Optimization of the 32P-postlabeling Assay for Detecting Benzo(a)pyrene-induced DNA Adduct Formation in Zacco platypus

  • Lee, Jin Wuk;Lee, Sung Kyu
    • Journal of Environmental Health Sciences
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    • v.40 no.1
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    • pp.55-62
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    • 2014
  • Objectives: $^{32}P$-postlabeling assay is the most sensitive method of detecting DNA adduct formation. However, it is limited by a low sample throughput and use of radioisotopes (RI). In this study, we modified it to minimize these limitations and applied it to Z. platypus exposed to Benzo(a)pyrene (BaP) in order to investigate DNA adduct formation (effect biomarker for pollutants) in Z. platypus for assessing risk of waterborne BaP exposure. Methods: DNA hydrolysis was performed only with Micrococcal nuclease (MNase), RI reduction test was performed and the overlapping steps between thin layer chromatography (TLC) and radioisotope high-performance liquid chromatography (RI-HPLC) were omitted. The application of a modified method to Z. platypus exposed to BaP was performed. Results: The results revealed that the amount of RIs used can be reduced roughly 10-fold. Because the analysis time was shortened by 8.5 hours, the sample throughput per hour was increased compared with the previous method. The results of applying modified $^{32}P$-postlabeling assay to Z. platypus, DNA adduct formation in Z. platypus showed dose-dependency with the BaP concentration. Only BPDE-dGMP was detected as a DNA adduct. Conclusion: These results demonstrate that the modified $^{32}P$-postlabeling assay is a suitable method for detecting DNA adduct formation in Z. platypus exposed to waterborne BaP and will be useful in risk assessment of carcinogenic effect in aquatic environment due to BaP.

Study of Selenium Compound in Favorite Korean Foodstuffs (한국 식품중의 Se 화합물의 함량에 관한 연구)

  • Chun, Sea-Yeol
    • Korean Journal of Food Science and Technology
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    • v.5 no.1
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    • pp.55-64
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    • 1973
  • Garlic and traditional fovarite foodstuffs of plant origin have been analyed for selenium and sulfur containing amino acid content selenium compound were assayed using a $^{77m}Se$ neutron activation analysis, cystine and methionine determination by paper and thin-layer chromatography. The results obtained indicate that the content of selenium and sulfur containing amino acid are highest in garlic. The results also show that the selenium is a more abundant in particular speies such as garlics produced in Tan Yang and Wei Sung. The effect of Korean garlic and favorite typically found in the Korean diet has also been studied. Some vegetables known to contain a relatively high level of sulfur and selenium (garlic and onion) do loss significent quantities of selenium as a result of cooking.

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Isolation, Identification and Determination of Antioxidant in Ginger (Zingiber officinale) Rhizome

  • Cho, Kang-Jin;Kim, Jin-Weon;Choi, In-Lok;Kim, Jung-Bong;Hwang, Young-Soo
    • Journal of Applied Biological Chemistry
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    • v.44 no.1
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    • pp.12-15
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    • 2001
  • The antioxidative compounds and antioxidant contents of ginger (Zingiber officinale) rhizomes were determined. Substances reextracted using ethyl acetate from crude methanol extract of fresh ginger rhizome were separated through thin layer chromatography. Ten phenolic antioxidative bands were visualized through color reactions using ferric chloride-potassium ferricyanide and 1,1-diphenyl-2-picrylbydrazyl (DPPH). The antioxidative compounds were purified through preparative TLC and high performance liquid chromatography (HPLC), among which, five antioxidants were identified as 4-, 6-, 8-. and 10-gingerols and 6-shogaol on the basis of their molecular weights determined through LC-MS. As shown in experiments using DPPH free radicals, 6-Gingerol and PT4-HP8 (unknown) were revealed to be more efficient than BHT (butylated hydroxy toluene). Contents of gingerols were determined through reverse phase HPLC. Total gingerol contents (sum of 6-,8-, and 10-gingerols) in rhizomes of different ginger varieties varied significantly. The HG55 (collected at Wanju district in Korea) and the HG52 (imported from Brazil) showed the highest gingerol contents.

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Production of Indole-3-Acetic Acid by the Cyanobacterium Arthrospira platensis Strain MMG-9

  • Ahmed, Mehboob;Stal, Lucas J.;Hasnain, Shahida
    • Journal of Microbiology and Biotechnology
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    • v.20 no.9
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    • pp.1259-1265
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    • 2010
  • The filamentous cyanobacterium Arthrospira platensis strain MMG-9 was isolated from a rice field. The ability of this strain to synthesize the bioactive compound indole-3-acetic acid (IAA) was demonstrated. IAA was extracted from the culture of A. platensis strain MMG-9 and its identity was confirmed by thin-layer chromatography (TLC) as well as by high-performance liquid chromatography (HPLC). The IAA precursor L-tryptophan was required for IAA biosynthesis. Released IAA increased with the increase of the initial concentration of L-tryptophan in the medium and with the incubation time. A. platensis strain MMG-9 accumulated more IAA than it released into the medium. The bioactivity of the secreted IAA was shown by its effect on the formation of roots by Pisum sativum. There was a significant positive effect of the supernatant of cultures of A. platensis strain MMG-9 on the number of lateral roots of P. sativum, whereas a negative effect on root length was observed.

Structural Characterization of a Flavonoid Compound Scavenging Superoxide Anion Radical Isolated from Capsella bursa-pastoris

  • Kweon, Mee-Hyang;Kwak, Jae-Hyock;Ra, Kyung-Soo;Sung, Ha-Chin;Yang, Han-Chul
    • BMB Reports
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    • v.29 no.5
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    • pp.423-428
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    • 1996
  • A superoxide anion radical scavenger isolated from Capsella bursa-pastoris was characterized by infrared (IR) spectroscopy, sugar analysis, ultraviolet (UV) spectroscopy, $^{1}H$ and $^{13}C$ nuclear magnetic resonance (NMR) spectroscopies, and fast atom bombardment (FAB) mass analysis. The compound was assumed to be a flavonoid-O-glycoside from IR spectrum and UV absorption maxima. When the sugar composition of the compound was examined by thin layer chromatography (TLC) and gas chromatography (GC) of the acid hydrolysate, only glucose was detected. According to the results of UV spectrotroscopy by using shift reagents, the compound was supposed to be luteolin (5,7,3',4'-tetrahydroxy flavone) or chrysoeriol (5,7,4'-trihydroxy-3'-methoxy flavone) with glucose. Based on $^{1}H$- and $^{13}C-NMR$ spectroscopies, the compound was deduced as 7,4'-dihydroxy-5,3'-dimethoxy-${\alpha}$-6-c-glucosyl-${\beta}$-2"-o-glucosyl flavone. In FAB mass analysis the compound was finally characterized as 7,4'-dihydroxy-5,3'-dimethoxy-${\alpha}$-6-c-glucosyl-${\beta}$-2"-o-glucosyl flavone ($C_{29}H_{34}O_{16}$, M.W.=638).

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Solid-Phase Extraction of Curcuminoid from Turmeric Using Physical Process Method (물리적 가공법을 이용한 강황으로부터 Curcuminoid의 고체상추출)

  • Lee, Kwang-Jin;Yang, Hye-Jin;Jeong, Sang-Won;Ma, Jin-Yeul
    • Korean Journal of Pharmacognosy
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    • v.43 no.3
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    • pp.250-256
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    • 2012
  • In order to extract the curcuminoid such as curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) in turmeric (Curcuma longa), solvent extraction methods (dipping and ultrasonic extraction method) and solid-phase extraction (SPE) were used. RP-HPLC (reverse-phase high-performance liquid chromatography) and TLC (thin-layer chromatography) were used for identification and analysis the three curcuminoid. From the experimental results, it is evident that the percentage of curcuminoid extracted from turmeric by ultrasonic extraction method was higher than dipping method. The percentage of curcumin extracted from turmeric by pure methanol was higher than any aqueous methanolic composition. Moreover, the total peak area of three curcuminoid was above 92% in RP-HPLC using solid-phase extraction. These results will form a database for investigating the constituents of natural products and the resources of pharmaceutical, nutrition, and cosmetic products.

Quantitative Analysis of Kirenol in Siegesbeckia glabrescens and S. pubescens by HPLC-UV (HPLC-UV에 의한 진득찰과 털진득찰의 Kirenol 정량분석)

  • Nugroho, Agung;Lee, Kyung-Tae;Park, Hee-Juhn
    • Korean Journal of Pharmacognosy
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    • v.43 no.4
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    • pp.286-290
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    • 2012
  • Many diterpenoids from Siegesbeckia species (Compositae) and their anti-inflammatory actions have been examined. In this research, high-performance liquid chromatography-ultraviolet spectrophotometer (HPLC-UV) method was used to compare the quantitative level of kirenol (ent-pimarane-type diterpenoid) in the aerial parts of Korean S. glabrescens and S. pubescens and the Chinese Siegesbeckiae Herba. Fingerprints of the two HPLC chromatograms of Korean S. glabrescens and S. pubescens were similar, but considerably different from Chinese Siegesbeckiae Herba. The content of kirenol in S. pubescens ($16.51{\pm}0.10$ mg/ml dry weight as mean${\pm}$RSD) was higher than S. glabrescens ($13.48{\pm}0.12$ mg/g dry weight). These values were considerably higher than the Chinese Siegesbeckiae Herba ($1.55{\pm}0.74$ mg/g dry weight). Thin layer chromatography (TLC) analysis demonstrated the containing of kirenol in the three plant materials, but the presence of siegeskaurolic acid (entkaurane-type diterpenoid) only in the Chinese Siegesbeckiae Herba.