Kiwi pectinesterase 저해제의 정제 및 특성

고은경,김명화,허원녕 목포대학교 자연자원개발연구소 1999 자연자원연구 Vol.2 No.-

Kiwi pectinesterase inhibitor(PEI)를 분리하기 위하여 CNBr-sepharose 4B에 orange pectinesterase(PE)를 공유결합으로 결합시켜 affinity chromatography용 수지를 제조하였다. 제조된 수지를 사용하여 PEI를 강력하게 선택적으로 결합시켜 pH 9.5의 높은 이온 강도를 갖는 용액으로 단일한 활성 peak로 용출·분리하였다. 분리된 kiwi PEI의 분자량은 12.5% SDS-PAGE 전기영동상에서 약 16.6 KDa 정도의 밀접한 이중 band로 나타났다. 분리된 PEI를 -25,℃ 5℃ 및 실온에서 30일 동안 보관·저장하였을 때 냉장, 냉동상태에서는 저해활성이 2주정도 경과시 25% 정도 감소하고 그 이후에는 큰 변화 없이 유지되었으나, 실온에서는 저해활성이 급격히 감소하여 저장 4주 정도에 거의 활성을 상실하였다. 이들의 활성특성을 조사해 본 결과 최적저해활성 pH는 7.5이고 최적저해반응 온도는 20℃였고, 70℃까지는 내열성을 지녔다. 또한 0.2M NaCl의 기질농도에서 최적저해 활성을 나타내었으며, kiwi PEI의 PE에 대한 저해작용은 noncompetitive inhibition인 것으로 나타났다. The resin for affinity chromatography was prepared to separate pectinesterase inhibitor(PEI) from ripened kiwi fruit(Actinidia chinensis) by CNBr-activated Sepharose 4B being covalently bound by orange petinesterse(PE). The affinity resin strongly and selectively bound PEI, which could be eluted in high yield as a single, homogeneous and sharp peak by pH 9.5 without loss of inhibitory activity. The separated PEI had maintained almost inhibitory activity at -25℃ and 5℃ during 30 days but lost it at room temperature in 4 weeks. The PEI possessed a molecular weight of 16.6 KDa, as estimated by 12.5% SDS-PAGE. The optimum pH, the optimum temperature, the thermostable range and NaCl concentration of substrate for PEI inhibitory activity appeared to be 7.5, 20℃, below 70℃ and 0.2 M respectively. The kind of inhibition on tomato pectinesterase was found to be noncompetitive, using citrus pectin as substrate.

한국산 보통토마토(Lycopersicon esculentum)와 방울토마토(L. esculentum var. cerasiforme) 펙틴에스테라제 정제 및 특성

허원녕,고은경 목포대학교 자연자원개발연구소 1998 자연자원연구 Vol.1 No.-

토마토 PE의 정제는 보통토마토 및 방울토마토로부터 PE를 추출하여 DEAE-sephadex와 heparin sepharose로 chromatography를 실시하여 정제하였다. 음이온 교환수지에서 비흡착된 양이온성의 PE들은 친화성수지인 heparin sepharose에 의하여 10 mM∼300 mM NaCl linear gradient하였을 때 보통토마토 PE와 방울토마토 PE 모두 150mM NaCl에서 흡착·용출되었다. 방울 토마토 PE보다 보통토마토 PE의 비활성 및 정제도가 컸으며, 전기영동상에 나타난 분자량은 방울토마토의 경우 약 30.5 KDa, 보통토마토 27.8 KDa 정도로 나타났다. 두 종류 토마토 PE의 최적 반응온도는 모두 60℃이고, 이로부터 구한 활성화 에너지값은 각각 보통토마토 PE가 , 방울토마토 PE가 로 나타내었으며, 45℃까지 열 안전성을 지녔다. 또 한 두 종류의 PE 모두 pH 9.0에서 최적반응을 나타내었고, pH 4.0∼10.0까지의 범위내에서 대체로 안정성을 지녔으며, 0.1M NaCl의 기질 농도에서 최적 활성을 나타내었다. Pectinesterases(PE) (EC. 3.1.1.11) of normal tomato and cherry tomato were extracted by using 1.0M NaCl form the precipitation of tomato pericarp pulp which was mixed with distilled water(1:1) and homogenized. Tomato PEs were purified by means of ammonium sulfate fractionation(30∼75%), ion chromatography on DEAE-Sephadex and affinity chromatography on Heparin- Sepharose. Normal tomato PE thus obtained had higher value of purification fold and specific activity than cherry tomato, and was confirmed to be homogeneous state electrophoretically. The molecular weights of normal tomato and cherry tomato were 27.8 and 30.5 KDa respectively. The two kinds of tomato PE had the optimum pH of 9.0, the optimum temperature of 60℃ with activation energy of 4,344 (normal tomato) and , stability up to 45℃, pH stability of the range of pH 4.0∼10.0 and optimum activity with pectin substrate of 0.1M NaCl.

과일류에서 Pectinesterase Inhibitor의 추출

허원녕,김명화,고은경 목포대학교 자연자원개발연구소 1999 자연자원연구 Vol.2 No.-

PEI의 추출재료를 선발, 추출방법, 불투명과즙의 저장에 미치는 영향을 조사한 결과는 다음과 같다. 1. PEI의 추출은 kiwi가 가장 우수하였고 이는 완충액이나 증류수 모두에서 효과적이었다. 2. Kiwi PEI의 황산암모늄 염석 분획은 0∼0.8 포화도에서 효과적이었다. 3. PEI의 활성이 가장 큰 kiwi PEI를 오렌지 주스에 첨가한 결과는 대조구에 비하여 혼탁도 유지에 효과적이었다. Pectinesterase(EC. 3.1.1.11, PE) inhibitors(PEI) were extracted by using pure water and high salt solution from several citrus fruits and kiwi fruit. Kiwi fruit PEI had the highest activity among the experimented fruits. They were effectively fractionated at 0∼0.8 ammonium sulfate saturation degree and kiwi PEI addition to Valencia orange juice was effective on cloud maintenance during its storage.

Protective effect of ethanol extract collected from Styela clava tunics against CCl4-induced hepatotoxicity in ICR mice

Eun-Kyoung Koh,Jun Go,Ji Eun Kim,Sung Hwa Song,Ji Eun Sung,Chan Kyu Park,Hyun Ah Lee,Young Lim,Dae Youn Hwang 한국실험동물학회 2015 한국실험동물학회 학술발표대회 논문집 Vol.2015 No.8

Protective Effect of Aqueous Extracts of Styela Clava Tunic Against Apoptosis of HepG2 Cells Induced by Hydrogen Peroxide

Eun Kyoung Koh(고은경),Young Ju Lee(이영주),Ji Eun Kim(김지은),Moon Hwa Kwak(곽문화),Jun Go(고준),Hong Joo Son(손홍주),Hee Seob Lee(이희섭),Young Jin Jung(정영진),Dae Youn Hwang(황대연) 한국생명과학회 2014 생명과학회지 Vol.24 No.6

미더덕껍질(Styela clava tunic, SCT)은 항염증 복합체, 창상필름, 골재생 유도 등을 포함한 다양한 의학적인 치료영역에 이용 되고 있다. 본 연구에서는 미더덕껍질 열수추출물(aqueous extract of Styela clava tunic, AE-SCT)의 H₂O₂에 의해 유발된 세포사멸의 보호 효과를 알아보기 위하여 세포 활성도의 변화에 관련된 요인을 측정하였다. 그 결과, AE-SCT는 3.3 mg/g의 플라보노이드와 32.3 mg/g의 페놀화합물을 포함하고 있었으며, HepG2 세포주에 독성을 유발하지 않았다. 또한, H₂O₂ 처리 후 AE-SCT를 처리하는 실험에서 AE-SCT는 H2O2에 의해 유발된 세포사멸을 개선하는 효과를 나타내지 않았다. 그러나, H₂O₂ 처리전에 AE-SCT를 사전 처리하는 예방효과 실험에서, 세포생존율은 H₂O₂만 처리한 그룹에 비하여 AE-SCT를 처리한 그룹에서 유의적으로 증가하였으며, 특히 AE-SCT를 50 μg/ml 처리한 농도에서 가장 높았다. 또한, FACS분석과 DAPI 염색에서도 사멸 세포의 수는 H₂O₂만 처리한 그룹에 비하여 AE-SCT를 처리한 그룹에서 유의적으로 감소하였다. 더불어, H₂O₂의 처리에 의해 유도된 Bax/Bcl-2 발현비율은 AE-SCT처리에 의해 농도의존적으로 감소되었다. 이러한 연구 결과는 AE-SCT가 H₂O₂에 의해 유발된 세포사멸을 예방하는 우수한 효과를 가지고 있음을 제시하고 있어 향후 다양한 항산화 제품 개발을 위한 기초자료로 사용될 수 있을 것으로 사료된다. Styela Clava tunic (SCT) has found some applications in many areas of medical treatment including as an anti-inflammatory compound, a wound healing film, in guided bone regeneration, and as a food additive. The protective effect of SCT aqueous extract (AE-SCT) on cell death induced by H₂O₂ treatment was investigated by measuring the changes in cell viability in HepG2 cells after AE-SCT treatment. High concentrations of antioxidant compounds including flavonoids (3.3 mg/g) and phenolics (32.3 mg/g) were detected in AE-SCT but no significant cytotoxicity was observed in HepG2 cells treated with AE-SCT. The viability of HepG2 cells was also not changed by treatment with different concentrations of AE-SCT after H₂O₂ treatment. However, cell viability was significantly increased in cells treated with three different concentrations of AE-SCT before H₂O₂ treatment. The greatest increase in cell viability was observed in the group treated with 50 μg/ml AE-SCT, when compared with vehicle-treated group. FACS and DAPI staining analysis indicated that the decrease in number of dead cells was dependent on the concentration of AE-SCT. Alterations in the Bax/Bcl-2 ratio after H₂O₂ treatment were significantly restored by treatment with different concentrations of AE-SCT. These results indicate that AE-SCT, which contains high levels of antioxidants, may protect cells against death induced by H₂O₂ treatment.

Neuroprotective Effects of a Butanol Fraction of Rosa hybrida Petals in a Middle Cerebral Artery Occlusion Model

( Go Eun Yang ),( Dong Sun Park ),( Sun Hee Lee ),( Dae Kwon Bae ),( Yun Hui Yang ),( Jang Been Kyung ),( Da Jeong Kim ),( Ehn Kyoung Choi ),( Jin Tae Hong ),( Heon Sang Jeong ),( Hee Jung Kim ),( Su 한국응용약물학회 2013 Biomolecules & Therapeutics(구 응용약물학회지) Vol.21 No.6

The neuroprotective effects of a butanol fraction of white rose petal extract (WRPE-BF) were investigated in a middle cerebral artery occlusion (MCAO) model. Seven week-old male rats were orally administered WRPE-BF for 2 weeks and subjected to MCAO for 2 h, followed by reperfusion. Twenty-four h later, MCAO-induced behavioral dysfunctions were markedly improved in a dose-dependent manner by pretreatment with WRPE-BF. Moreover, higher dose of WRPE-BF not only decreased infarction area but also effectively reduced astrogliosis. The expression of inducible nitric oxide synthase, cyclooxygenase-2, and glial fi brillary acidic protein in MCAO model were markedly inhibited by WRPE-BF treatment. Notably, WRPE-BF decreased nitric oxide and malondialdehyde levels in the striatum and subventricular zone of stroke-challenged brains. These data suggested that WRPE-BF may exert its neuroprotective effects via anti-oxidative and anti-infl ammatory activities against ischemia-reperfusion brain injury and could be a good candidate as a therapeutic target for ischemic stroke.

Hepatoprotective Effect of Gallotannin-enriched Extract Isolated from Gall on Hydrogen Peroxide-induced Cytotoxicity in HepG2 Cells

Go, Jun,Kim, Ji Eun,Koh, Eun Kyoung,Song, Sung Hwa,Kang, Hyun Gu,Lee, Young Hee,Kim, Han Do,Hong, Jin Tae,Hwang, Dae Youn Medknow PublicationsMedia Pvt Ltd 2017 Pharmacognosy magazine Vol.13 No.50

<P><B>Background:</B></P><P>Gall (Galla Rhois [GR]) is known to have antibacterial, anti-inflammatory, antimetastatic, and anti-invasion activities and exert hepatoprotective effects. However, the hepatoprotective effects of gallotannin-enriched GR (GEGR) and their mechanisms have not yet been investigated.</P><P><B>Objective:</B></P><P>The potential protective effect of GEGR against hepatotoxicity induced by hydrogen peroxide (H<SUB>2</SUB>O<SUB>2</SUB>) was investigated.</P><P><B>Materials and Methods:</B></P><P>Changes in cell viability, apoptosis protein expression, and reactive oxygen species (ROS) generation were determined in HepG2 cells that were pretreated with four different concentrations of GEGR (6.25–50 μg/ml) for 24 h before H<SUB>2</SUB>O<SUB>2</SUB> exposure.</P><P><B>Results:</B></P><P>GEGR consisted of gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) and showed remarkable 2,2-diphenyl-1-picrylhydrazyl scavenging activity (inhibitory concentration 50% = 0.212 μg/ml). The lethal dose 50% and effective dose 50% values for the response of HepG2 cells to GEGR were determined to be 178 and 6.85 μg/ml, respectively. Significant reductions in the immunofluorescence intensity indicating apoptosis were also detected in the nuclei of HepG2 cells stained with 4’,6-diamidino-2-phenylindole and Annexin V after GEGR treatment. The Bax/Bcl-2 ratio and active caspase-3 level were higher in H<SUB>2</SUB>O<SUB>2</SUB> + vehicle-treated cells. However, these levels gradually decreased to those of the No-treated group in the GEGR pretreated group even though little or no decrease was observed in response to low GEGR concentrations. Furthermore, the GEGR pretreated group showed a reduced level of 2’,7’-dichlorofluorescein diacetate stained cells, indicating ROS generation relative to the H<SUB>2</SUB>O<SUB>2</SUB> + vehicle-treated group.</P><P><B>Conclusion:</B></P><P>The results of this study provide strong evidence that GEGR can prevent cell death induced by H<SUB>2</SUB>O<SUB>2</SUB> in HepG2 cells through the induction of antioxidant conditions.</P><P><B>SUMMARY</B></P><P><P>The gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) are the main constituents of water extracts of GR</P><P>GEGR was more potent in DPPH scavenging, and gallotannin contributes to this extract activity</P><P>GEGR significantly reduced the increase of apoptosis, Bax/Bcl-2 ratio, and active caspase-3 level after H2O2 treatment</P><P>GEGR pretreatment showed protection against H<SUB>2</SUB>O<SUB>2</SUB>-induced ROS production in DCFH-DA staining analysis.</P></P> >[FIG OMISSION]</BR><P><B>Abbreviations used:</B> COX: Cyclooxygenase; DAPI: 4’,6-diamidino-2-phenylindole; DMSO: Dimethyl sulfoxide; DPPH: 2,2-diphenyl-1-picrylhydrazyl; GEGR: Gallotannin-enriched Galla Rhois; GR: Galla Rhois; HPLC: High-performance liquid chromatography; H<SUB>2</SUB>O<SUB>2</SUB>: Hydrogen peroxide; MMP: Metallopeptidase; MTT: 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ROS: Reactive oxygen species; UV-Vis: Ultraviolet-visible.</P>

Protective Effect of Aqueous Extracts of Styela Clava Tunic Against Apoptosis of HepG2 Cells Induced by Hydrogen Peroxide

Eun Kyoung Koh,Young Ju Lee,Ji Eun Kim,Moon Hwa Kwak,Jun Go,Hong Joo Son,Hee Seob Lee,Young Jin Jung,Dae Youn Hwang 한국실험동물학회 2014 한국실험동물학회 학술발표대회 논문집 Vol.2014 No.8

Evolutionary dynamic of HA gene of swine H1N2 influenza virus in Korea from 2009 to 2022

Go-Eun Shin,Eun-Yong Lee,Kyoung-Ki Lee,Ilseob Lee,Bok-Kyung Ku,Hye-Young Jeoung 한국예방수의학회 2023 예방수의학회지 Vol.47 No.4

Pigs are considered a “mixing vessel” that can produce new influenza strains through genetic reassortments, which threaten public health and cause economic losses worldwide. We performed surveillance of swine influenza virus (SIV) using 114,140 nasal swabs from 11,414 Korean farms from 2009 to 2022, and characterized their genetic evolution at each farm level. A total of 169 farms out of the 11,414 (1.48%) were SIV-positive. The positivity rate for the H1N2 subtype, which is most dominant in the pig population, was 37.87% (64/169). Through hemagglutinin (HA) gene analysis, 64 H1N2-positive farms were classified into Eurasian avian-like (46/64, 71.88%), triple-reassortant (14/64, 21.88%), pdm09 (3/64, 4.69%), and classical swine (1/64, 1.56%) groups. The estimated evolutionary rate of HA in H1N2 from 2009 to 2022 in Korea is 1.5309 × 10-3/site/year (95% HPD intervals from 1.0003 × 10-3 to 2.1735 × 10-3) with an estimated mean growth rate of 0.0114. Estimates of the relative genetic diversity of clades over time suggested that the HA of H1N2 exhibited an increase in population size. The results of this study showed that the Eurasian avian-like-HA of the H1N2 subtype was dominant in the pig population. The continued evolution of the HA of H1N2, which is critical for cell entry, might lead to genetic diversity and the loss of vaccine cross-protection. These results indicate that continus surveillance is imperative for monitoring the evolution of the swine influenza virus.

The evolutionary dynamic of porcine reproductive and respiratory syndrome virus type 2 in Korea

Kyoung-Ki Lee(Kyoung-Ki Lee),Hye-Young Jeoung(Hye-Young Jeoung),Go-Eun Shin(Go-Eun Shin),Ji-Young Park(Ji-Young Park),Bok-kyung Ku(Bok-kyung Ku) 한국예방수의학회 2022 한국예방수의학회 학술대회자료집 Vol.2022 No.-