• Title/Summary/Keyword: HPLC Chromatography

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Screening and Purification of an Anti-Prostate Cancer Compound, Deoxypodophyllotoxin, from Anthriscus sylvestris Hoffm (전호(Anthriscus sylvestris Hoffm)로부터 전립선 암세포 저해물질인 deoxypodophyllotoxin 의 탐색 및 분리)

  • Cho, Hyo-Jin;Yu, Sun-Nyoung;Kim, Kwang-Youn;Sohn, Jae-Hak;Oh, Hyun-Cheol;Ahn, Soon-Cheol
    • Journal of Life Science
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    • v.19 no.1
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    • pp.9-14
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    • 2009
  • The prostate cancer is the critical health problem, increasing of its related death in worldwide. Unfortunately present surgery and chemotherapeutic choices seem to be impossible in curing or controlling prostate cancer, because metastasis occasionally advances even after these potentially curative therapies. Therefore, there is immediate need to alternative chemoprevention and chemotherapeutic agents. Over one hundred species of dried medicinal herbs were tested for proliferation inhibitory effects on prostate cancer cell line, PC-3. One of them, Anthriscus sylvestris was selected because of potent anti-proliferation effect. The dried root of A. sylvestris was extracted with 100% methanol for 2-3 days and its extract was fractionated by using ethyl acetate. And ethyl acetate layer was subjected to column chromatographies on silica gel, reverse phase-18 (RP-18) and Sephadex LH-20, in turn. Finally, the pure compound was obtained by crystallization in methanol at $4^{\circ}C$ for overnight and identified as deoxypodophyllotoxin by NMR spedorscopic and physico-chemical analyses. In addition, it was confirmed that deoxypodophyllotoxin clearly inhibits the proliferation of PC-3 cells in a dose and time-dependent manner.

Isolation of Anticarcinogenic Isoflavone-conjugated Glycoproteins from a Submerged Liquid Culture of Agaricus blazei Mycelia by the Autolysis Process (신령버섯균사체 액체배양물의 자가분해에 의한 항암성 isoflavone-conjugated glycoprotein 분리)

  • Kim, So Young;Kim, Young Suk;Jang, Joung Soon;Kim, Boh Hyun;Rakib, Abdur Md.;Kim, Gon Sup;Kim, Jeong Ok;Ha, Yeong Lae
    • Journal of Life Science
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    • v.24 no.12
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    • pp.1316-1324
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    • 2014
  • Most beta-glucans obtained from various fruit bodies of mushrooms and mushroom mycelial cultures have high-molecular weight glycoproteins, conjugated with beta-glucans. We report that isoflavone-conjugated glycolproteins (designated as gluvone) were isolated and exhibited stronger anticarcinogenic activities. Agaricus blazei mycelia (ABM) was cultured in a liquid medium containing soybean flakes for 14 days. The liquid culture was autolyzed by incubating at $53^{\circ}C$ (pH 5.5) for 3 h. A crude glycoprotein (CGP) fraction with a cytotoxic effect on a mouse ascite cancer cell line (S-180) and a human breast cancer cell line (MCF-7) was isolated from the autolyzed ABM cultures by 80% ethanol treatment. Gluvone was isolated from the CGP with Sephadex G-75 column chromatography. It exhibited a stronger anticancer effect than CGP against the S-180 cell-induced female ICR mouse ascites carcinogenesis. Gluvone with 9,400 daltons was identified as a glycoprotein conjugated with isoflavone. According to HPLC and GC analysis, in conjunction with $^1H$-NMR spectral analysis, it contained 60% carbohydrates (glucose, fructose, and ribose), 31% protein, and 2% isoflavone (daidzein and genistein), which is a novel material. These results indicate that a strong anticarcinogenic gluvone was isolated from the autolyzed product of a submerged liquid culture of ABM, suggesting that autolysis could be a useful tool to produce antitumor agents.

Analysis of Florfenicol in Meats (식육 중 항생제 florfenicol의 분석)

  • Kuk, Ju-Hee;Song, Young-Me;Bae, Min-Seok;Go, Myoung-Jin;Yoo, Myung-Sang;An, Eun-Suk;Park, Eun-Heui;Go, Myoung-Jin;Kang, Kil-Jin
    • Journal of Food Hygiene and Safety
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    • v.23 no.4
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    • pp.319-323
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    • 2008
  • Analytical method for determination of florfenicol was developed for estimate veterinary drug residue of unestablished MRLs in meat. The method was validated in correspondence with the CODEX guideline for florfenicol residue in meat. The samples mixed with sodium sulfate were extracted with ethyl acetate. After clean-up, the residue was dissolved in mobile phase and analyzed using high-performance liquid chromatography with fluorescence detector. The calibration curve showed good linearity($r^2=0.9997$) within the concentration range of $0.05{\sim}1.0\;mg/kg$. The limit of detection(LOD) and limit of quantification(LOQ) were validated at 0.012 and 0.039 mg/kg, respectively. The recoveries in fortified meat ranged from 85.6 to 95.6%($1.1{\sim}5.3%$ RSD) at the 0.05 to 0.4 spiking levels. We monitored 150 samples of meats that were purchased in Korea(Seoul, Busan, Daegu, Daejeon and Gwangju). Among tested samples, florfenicol was detected in 1 of pig at the level of 0.040 mg/kg, and below LOQ in 1 of cattle, 2 of pig and 2 of chicken. The residues of florfenicol in the tested samples were within the MRLs.

Synergistic Inhibition of IgY, Auricularia auricula, and Lactic Acid Bacteria from Kimchi and Tarak on Helicobacter pylori (Helicobacter pylori 의 생육억제에 대한 유산균, 난황항체 및 목이버섯의 상승효과)

  • Yoo, Hye-Lim;Lee, Young-Duck;Han, Bok-Kyung;Choi, Hyuk-Joon;Park, Jong-Hyun
    • The Korean Journal of Food And Nutrition
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    • v.26 no.1
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    • pp.35-43
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    • 2013
  • The substances of lactic acid bacteria (LAB) isolated feom Kimchi and Tarak, L. mesenteriodes LAB kw5, and S. thermophilus LAB KW15 were investigated for growth effect of Helicobacter pylori with IgY and Auricularia auricula. Inhibition of H. pylori was confirmed at LAB KW5 and KW15 supernatants. Interestingly, anti-H. pylori substance in LAB KW5 and KW15 supernatants were sensitive to lipase, but insensitive to protein hydrolase and carbohydrate hydrolase. The inhibition zone toward H. pylori was not shown with the lipase-treated supernatants. Therefore, there seemed to be lipid-like substances in the cultures. By the analyses with gas chromatography, undecanoic acid ($C_{11:0}$), palmitic acid ($C_{16:0}$), stearic acid ($C_{18:0}$), and oleic acid ($C_{18:1}$) were detected at the culture substances from L. mesenteroides LAB KW5 and S. thermophilus LAB KW15, and more eicosadienoic acid ($C_{20:2}$) from L. mesenteroides LAB KW5. Anti-H. pylori substances of LAB with IgY and A. auricula extract were analyzed for inhibition effect of H. pylori. The inhibition increased more by the range from 57% to 86% by the mixture. The substances with IgY and A. auricula extract showed more effective inhibition of H. pylori than single or double trials.

Characteristics and purification of proteoglycan from Phellinus igniarius (Phellinus igniarius로부터 분리한 단백다당류의 분리 및 특성)

  • Kim, Seon-Hee;Jung, In-Chang;Kwon, Yong-Il;Kim, So-Yeun;Lee, Jong-Suk;Lee, Hang-Woo;Lee, Jae-Sung
    • Applied Biological Chemistry
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    • v.43 no.1
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    • pp.57-62
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    • 2000
  • The proteoglycan, intracellular and extracellular, extracted from the liquid culture of Phellinus igniarius were purified and characterized. The mycelial productivity was proved to be better in shaking culture compared to standing culture. The productivity of intracellular proteoglycan of Phellinus igniarius appeared to be similar in two culturing methods. The standing culture of Phellinus igniarius produced 6 times as much extracellular proteoglycan compared to shaking culture. The proteoglycan were purified to a single peak by ion exchange chromatography(DEAE-cellulose) followed by gel filtration(Sepharose 2B). PIEPDG contained 79.0% total sugar and 7.2% protein. PIEPAG contained 56.7% total sugar and 40.8% protein. PIIPDG contained 64.8% total sugar and 17.4% protein. PIIPAG contained 56.9% total sugar and 41.5%n protein. The molecular weights of all the fractions were estimated to be above 100,000, from 134KDa of PIEPDG to 560 KDa of PIEPAG. The results of sugar analysis by HPLC showed that PIEPDG contains glucose only. The sugar part of PIIPDG and PIIPAG were consisted of glucose and inositol. The PIEPAG contained three kinds of monosaccharides, glucose, fructose and inositol.

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Antioxidant and α-Glucosidase Inhibitory Activities of the Extracts of Aster koraiensis Leaves (국내산 벌개미취 잎 추출물의 α-glucosidase 억제능 및 항산화 활성 평가)

  • Lee, Tae Gu;Hyun, Soo Wang;Lee, Ik Soo;Park, Bong Kyun;Kim, Jin Sook;Kim, Chan Sik
    • Korean Journal of Medicinal Crop Science
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    • v.26 no.5
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    • pp.382-390
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    • 2018
  • Background: The plant Aster koraiensis has long been used as an ingredient in folk medicine. It has been reported that Aster koraiensis extract (AKE) prevents the progression of diabetes-induced retinopathy and nephropathy. However, although these beneficial effects of AKE on diabetes complications have been identified, the antidiabetic effects of AKE have not yet been completely investigated and quantified. In the present study, the glucose-lowering and antioxidant effects of aqueous and ethanolic AKEs were evaluated. Methods and Results: The glucose-lowering effects of aqueous and ethanolic (30%-, 50%-, and 80%-ethanol) AKEs were investigated via ${\alpha}$-glucosidase inhibitory assays. The mode of inhibition by AKEs on ${\alpha}$-glucosidase was identified through kinetic analysis. The total antioxidant capacity of each of the 4 AKEs was evaluated by assessing their conversion rate of $Cu^{2+}$ to $Cu^+$. The content of chlorogenic acid and 3,5-di-O-caffeoylquinic acid, the bioactive compounds in AKE, in each extract were analyzed by high performance liquid chromatography (HPLC). The AKEs showed potent ${\alpha}$-glucosidase inhibitory activity with mixed inhibition mode, and significant antioxidant capacity. Conclusions: These results of this study suggested that the AKEs tested had ${\alpha}$-glucosidase inhibitory and antioxidant effects. Among the extracts, the 80% ethanol extract showed the most significant ${\alpha}$-glucosidase inhibitory activity, with a half maximal inhibitory concentration ($IC_{50}$ value) of $1.65{\pm}0.36mg/m{\ell}$ and a half maximal effective concentration ($EC_{50}$ value) for its antioxidant activity of $0.42{\pm}0.10mg/m{\ell}$. It can therefore be used as a source of therapeutic agents to treat diabetes patients.

Comparison of Anti-oxidative and Anti-inflammatory Effect of Atractylodes Interspecific Hybrid Cultivar Roots (삽주 육성품종 뿌리의 항산화 및 항염증 효과 비교)

  • Jeong, Hyeon Soo;Jeong, Jin Tae;Lee, Jeong Hoon;Park, Chun Geon;Choi, Je Hun;Jang, Gwi Yeong;Kim, Jang Wook;Chang, Jae Ki;Kim, Dong Hwi;Lee, Seung Eun
    • Korean Journal of Medicinal Crop Science
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    • v.26 no.5
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    • pp.391-400
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    • 2018
  • Background: Atractylodes radix is a well-known medicinal crop having many physiological effects. This study was conducted to select useful Atractylodes japonica ${\times}$ Atractylodes macrocephala (AJM) cultivars by comparing anti-oxidative and anti-inflammatory efficacies. Methods and Results: Seven extracts from AJM cultivars were used to treat lipopolysacchride (LPS)-treated BV2 cells, and the effects on cell viability and inhibition on reactive oxygen species (ROS) and nitric oxide (NO) production were analyzed. In vitro scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and peroxynitrite ($NOO^-$) radicals were also investigated. Contents of total phenol, atractylenolide I, and atractylenolide III in the AJM extracts were measured using high performance liquid chromatography (HPLC) or spectrophotometry. The experiments show that none of the seven extracts was cytotoxic above 89.2% at $20-250{\mu}g/m{\ell}$. Extracts of Gowon, Dawon, Sangchul, and Huchul inhibited ROS generation in a dose-dependent manner, and Sangchul extract showed the highest inhibition on ROS production. All the AJM extracts showed effective inhibitory activity after on NO release in the LPS-treated BV2 cells, and Sangchul extract showed the highest activity. Sangchul extract had the most potent scavenging activities for $NOO^-$ and had some DPPH radical scavenging effect. Sangchul extract also had the highest content at total phenol and atractylenolide I content. Atractylenolide III was not detected in the AJM extracts. Conclusions: The results suggested that Sangchul was the most useful anti-oxidative and anti-inflammatory resource among the AJM cultivars.

The Study on the Analysis Method of Tetrodotoxin in Puffer Fish (복어 중 테트로도톡신 분석법에 관한 연구)

  • Kang, Young-Woon;Lee, Yoon-Suk;Park, Sung-Kug;Seo, Jung-Heok;Kim, Mee-Hye
    • Journal of Food Hygiene and Safety
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    • v.27 no.1
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    • pp.37-41
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    • 2012
  • The current standard for testing tetrodotoxin (TTX) in foodstuffs is the mouse bioassay (MBA) in Korea as in many other countries. However, this test suffers from potential ethical concerns over the use of live animals. In addition, the mouse bioassay does not test for a specific toxin thus a sample resulting in mouse incapacitation would need further confirmatory testing to determine the exact source toxin (e.g., TTX, STX, brevotoxin, etc.). Furthermore, though the time of death is proportional to toxicity in this assay, the dynamic range for this proportional relationship is small thus many samples must be diluted and new mice be injected to yield a result that falls within the quantitative dynamic range. Therefore, in recent years, there have been many efforts in this field to develop alternative assays. High performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) has been emerged as one of the most promising options. A LC-MS-MS method involves solid-phase extraction (SPE) and followed by analysis using an electrospray in the positive ionization mode and multiple reactions monitoring (MRM). To adopt LC-MS-MS method as alternative standard for testing TTX, we performed a validation study for the quantification of TTX in puffer fish. This LC-MS-MS method showed good sensitivity as limits of detection (LOD) of $0.03{\sim}0.08{\mu}g/g$ and limits of quantification (LOQ) of $0.10{\sim}0.25{\mu}g/g$. The linearity ($r^2$) of tetrodotoxin were 0.9986~0.9997, the recovery were 80.9~103.0% and the relative standard deviations (RSD) were 4.3~13.0%. The correlation coefficient between the mouse bioassay and LC/MS/MS method was higher than 0.95.

Selection of Biogenic Amine-reducing Microorganisms from a Traditional Korean-style Fermented Food, $Cheonggukjang$ (전통 발효 청국장으로부터 biogenic amine 저생성 미생물의 선발)

  • Choi, Jae-Young;Hong, Sung-Wook;Chung, Kun-Sub
    • Korean Journal of Food Science and Technology
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    • v.44 no.2
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    • pp.196-201
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    • 2012
  • Microorganisms, having the lower decarboxylase activity, among the isolated strains from $cheonggukjang$ and rice-straw in this study were selected by using biogenic amine (BA) media. The selected strains were identified as $Bacillus$ $subtilis$ HH12, $B.$ $subtilis$ HR254, and $Paenibacillus$ $barcinonensis$ KR97, by using 16S rRNA analysis. PCR analysis showed that the histidine decarboxylase ($hdc$) gene was absent in the HH12, HR254, and KR97 strains. However, PCR analysis showed that the tyrosine decarboxylase ($tdc$) gene was present in the HH12, HR254, and KR97 strains. Quantitative analysis of the selected strains by using high-performance liquid chromatography showed that histamine was absent in the HH12, HR254, and KR97 strains. However, these 3 strains showed tyramine concentrations of 6.09, 3.68, and 6.30 mg/L, respectively. These strains produced lower concentrations of amines (approximately 7.9, 0, and 9.3% amines in the HH12, HR254, and KR97 strains, respectively) than the $B.$ $subtilis$ MC138 strain, which showed the higher protease activity.

Immuno-stimulating Activities of Mannose-rich Polysaccharides Isolated from Korean Black Raspberry Wine (복분자주에서 분리한 Mannan 다당의 면역증진 활성)

  • Lee, Ho
    • Korean Journal of Food Science and Technology
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    • v.44 no.3
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    • pp.343-349
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    • 2012
  • Polysaccharides isolated from Korean black raspberry wine were examined for their chemical properties and immuno-modulating activities. The molecular mass of RB-1b-I, the main polysaccharide in black raspberry wine, was estimated as 180 kDa and it contained a significant proportion of mannose (76.8%) and 4 different minor component sugars such as galactose (15.8%), arabinose (3.8%), glucose (2.6%) and rhamnose (1.2%). This indicated that RB-1b-I was mainly present as a mannan, which had originated from the cell walls of fermenting yeasts. On the other hand, RB-1b-I induced high levels of macrophage activation as well as mitogenicity regarding murine splenocytes in vitro. The intravenous administration of RB-1b-I significantly augmented NK cytotoxicity against YAC-1 tumor cells. RB-1b-I also showed potent anti-complementary activity in a dose-dependent manner via both alternative and classical pathways. Results indicated that Korean black raspberry wine contains peculiar polysaccharides which provide beneficial immuno-stimulating activities for human health.