Ginsenoside Rg₁의 NMR 데이터 동정

이대영(Dae-Young Lee),조진경(Jin-Gyeong Cho),이민경(Min-Kyung Lee),이재웅(Jae-Woong Lee),박희정(Hee-Jeong Park),이윤형(Youn-Hyung Lee),양덕춘(Deok-Chun Yang),백남인(Nam-In Baek) 고려인삼학회 2008 Journal of Ginseng Research Vol.32 No.4

수삼으로부터 용매추출, 용매분획 및 silica gel column chromatography를 반복하여 ginsenoside Rg₁을 분리하였다. Ginsenoside Rg₁의 결정특성, 녹는점, 비선광도, IR 데이터, FAB/MS 데이터, TLC에서의 Rf 값, HPLC에서의 r.t. 및 NMR 데이터를 표준화한 조건으로 측정하여 문헌 값과 비교 고찰하였다. 특히 ginsenoside Rg₁의 ¹H- 및 <SUP>13</SUP>C-NMR 데이터를 HSQC 및 HMBC와 같은 2D-NMR 실험을 통하여 정확하게 동정하였다. The fresh ginseng roots were extracted in aqueous methanol (MeOH), and the obtained extracts were partitioned using ethyl acetate (EtOA), n-butanol (n-BuOH), and water, successively. The repeated silica gel column chromatography for n-BuOH fraction afforded a purified ginsenoside Rg₁ The physico-chemical, spectroscopic and chromatographic data of ginsenoside Rg₁ such as crystallization characteristics, melting point, specific rotation, infrared spectrometry (IR) data, fast atom bombardment/mass spectrometry (FAB/MS) data, nuclear magnetic resonance (NMR) data, retention factor (Rf) in thin layer chromatography (TLC) experiment, and retention time (r.t.) in HPLC analysis, were measured and compared with those reported in literatures. Especially, the previous literatures reported different data for ginsenoside Rg₁ in the ¹H- and <SUP>13</SUP>C-NMR experiments. This paper gives the exactly assigned NMR data through 2D-NMR experiments, such as ¹H-¹H correlation spectroscopy (COSY), hetero nuclear single quantum correlation (HSQC), and hetero nuclear multiple bond connectivity (HMBC).

Cytochrome P - 450 유도물질 처리한 흰쥐 간의 마이크로좀 Aryl Hydrocarbon Hydroxylase 활성도와 혈액의 Polyamines 농도와의 관계

박기현,이윤형 ( Ki Hyun Park,Youn Hyung Lee ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.3

The relationship between polyamines in blood and aryl hydrocarbon hydroxylase (AHHase, EC, 1.14.14.1) activities in hepatic microsomes was observed when rats (Sprague - Dawley, male, 150 160 g) were treated intraperitoneally with phenobarbital (Pb) or 3 - methylcholanthrene (3 - MC) as potent inducers of cytochrome P - 450 (P - 450) isoenzymes. In the case of Pb - treatment for an induction of P - 450 - b, P - 450 contents of hepatic microsomes were significantly enhanced and total polyamine levels in blood had no change After administration of 3 - MC as a carcinogen and an inducer of P - 450 - c, the major polyamines measured in blood were putrescine and spermidine and total polyamine level was increased over 2 - fold. In particular, putrescine concentration was enhanced drastically when compared with control value (10 - fold). These results indicate that only the putrescine was accumulated abnormally because ornithine decarboxylase and polyamine oxidase as enzymes of polyamine synthesis or degradation were stimulated by metabolites of 3 - MC treated. On the other hand, hepatic microsomal AHHase activities by the induction of P - 450 - c were increased over 13 - fold. In this respect, a relationship is suggested between AHHase activities enhanced from induction of P - 450 - c and synthesis or degradation of polyamines.

Cytochrome P-450 유도물질을 처리한 흰쥐 간의 마이크로좀 Aryl Hydrocarbon Hydroxylase 활성도와 혈액의 Polyamines 농도와의 관계

박기현,이윤형,Park, Ki-Hyun,Lee, Youn-Hyung 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.3

Cytochrome P-450 (P-450) 유도물질인 phenobarbital (Pb)과 3-methylcholanthrene (3-MC)을 흰쥐 (S.D., male, 150-160 g)에 처리한 후 간 마이 크로좀의 P-450 유도에 따른 Aryl Hydrocarbon Hydroxylase (AHHase) 활성도와 혈 액중의 polyamines 분포 및 농도와의 관계를 검토하였다. P-450-b 유도물질인 Pb 처리는 P-450 함량을 상당히 증가시키면서 대조구의 polyamines 분포 (putrescine, spermidine, spermine)와는 달리 spermidine, spermine의 분포를 나타내지만 총 polyamines 농도는 차이가 없었다. P-450-c 유도물질이며 강력한 발암물질인 3-MC 처리는 putrescine, spermidine의 분포로서 특히 putrescine은 대조구의 10배로, 그리고 총 polyamines 농도는 2배 증가되었으며 AHHase 활성도는 12배 이상 증가폭을 보였다. 이와 같은 현상은 putrescine 생합성에 관여하는 ornithine decarboxylase가 3-MC의 대사산물에 의해 그 활성이 촉진되어 비정상적으로 putrescine이 축적되며 polyamine oxidase가 spermine의 분해를 증가시킨 것으로 P-450-c의 유도에 따른 AHHase 활성도 증가와 polyamines의 생합성 또는 분해와는 밀접한 관계가 있는 것으로 사료된다. The relationship between polyamines in blood and aryl hydrocarbon hydroxylase (AHHase, EC, 1.14.14.1) activities in hepatic microsomes was observed when rats (Sprague - Dawley, male, 150-160 g) were treated intraperitoneally with phenobarbital (Pb) or 3-methylcholanthrene (3-MC) as potent inducers of cytochrome P-450 (P-450) isoenzymes. In the case of Pb-treatment for an induction of P-450-b, P-450 contents of hepatic microsomes were significantly enhanced and total polyamine levels in blood had no change After administration of 3-MC as a carcinogen and an inducer of P-450-c, the major polyamines measured in blood were putrescine and spermidine and total polyamine level was increased over 2-fold. In particular, putrescine concentration was enhanced drastically when compared with control value (10-fold). These results indicate that only the putrescine was accumulated abnormally because ornithine decarboxylase and polyamine oxidase as enzymes of polyamine synthesis or degradation were stimulated by metabolites of 3-MC treated. On the other hand, hepatic microsomal AHHase activities by the induction of P-450-c were increased over 13-fold. In this respect, a relationship is suggested between AHHase activities enhanced from induction of P-450-c and synthesis or degradation of polyamines.

Differential Inhibition of Liver Microsomal Cytochrome P-450-Dependent Monooxygenases by Hesperetin

문자영,이윤형,박기현,Mun, Ja-Yeong,Lee, Youn-Hyung,Park, Ki-Hyun 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.3

Cytochrome P-450-dependent monooxygenase (P-450-MO)는 스테로이드 등의 대사에 도 관여하지만 이물질 특히 환경오염물질 등의 생체내 활성화 대사를 촉진한다. 또한 P-450은 여러 동위 효소로 구성되어 있으며 각각의 기질 특이성을 나타낸다. 따라서 동위 효소 유도 물질로 phenobarbital (PB)과 3-methylcholanthrene (MC)을 흰쥐 (SP-D, male, 150-160g)에 각각 처리하여 간의 microsomes (mis)에 존재하는 P-450을 스펙트럼 차이로 부터 그리고 단일항체와 $^{35}S$-rabbit antimouse IgG를 사용한 방사면역학적인 방법에 의해 P-450-b (P-450) type과 P-450-c (P-448) type이 주된 동위 효소로 각각의 mis에 존재하는 것을 확인하였다. 이와 같은 mis에 hesperetin (2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one)을 첨가한 스펙트럼 특성은 동일한 modified type II (Peak, 415 nm; trough, 385 nm)를 나타내므로서 hesperetin이 MO에 대한 억제효과가 있음을 알았다. hesperetin의 benzo(a)pyrene hydroxylase (BPH), 7-ethoxycoumarin O-deethylase (ECDE), aniline hydroxylase (AH)에 대한 inhibitory potency ($I_{50}$)는 PB-mis 또는 MC-mis, 즉, P-450의 동위 효소에 따라 다소 차이는 있으나 측정된 MO 들의 활성도에 상관 없이 비교적 강력한 것으로 나타났다. 한편 MO에 대한 hesperetin의 inhibition type과 inhibition constant ($K_i$)를 측정한 결과 PB-mis-BPH에 대하여 noncompetitive inhibitor로 작용했으며 이때의 $K_i$ 값은 $5.42\;{\mu}M$ 이었다. 그리고 MC-mis-BPH에는 mixed inhibitor로 $K_i$ 값은 $14.5\;{\mu}M$로 전자보다 높았다. 또한 MC-mis-ECDE에는 hesperetin이 mixed inhibition을 나타냈으며 $K_i$ 값은 $1.67\;{\mu}M$로 competitive inhibition을 나타내는 PB-mis-ECDE의 $18\;{\mu}M$보다 상당히 낮았을 뿐 아니라 BPH의 값들보다도 훨씬 낮은 경향이었다. PB-mis-와 MC-mis-AH에는 hesperetin이 같은 mixed inhibitor로 작용했으며 $K_i$ 값은 각각 20.5, $34\;{\mu}M$로 높은 수치를 보였다. 따라서 hesperetin의 MO에 대한 선택적이며 차별적인 억제효과는 측정되는 mis에 따라 그리고 MO에 따라 상당한 차이를 보이고 있으며 특히 MC-microsomal P-450-dependent ECDE에 가장 강력한 억제효과를 나타냈다. The effect of hesperetin (2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-One) on the inhibition of the liver microsomal monooxygenases was investigated from the male Sprague Dawley rats (150-160 g) pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC) in vitro. The major form of PB- or MC-induced P-450 isozyme was identified from characteristic CO-difference spectra (450 nm and 448 nm) and from results by using the specific monoclonal antibodies of P-450 isozymes and the secondary antibody ($^{35}S$-rabbit antimouse IgG), namely the major form of PB-microsomal P-450 was P-450-b type and that of MC-microsomal-450 was P-450-c (P-448) type. A very similar spectral changes of the modified type II (peak: 415 nm, trough: 385 nm) were caused from the PB- and MC-aerobic liver micro somes added with hesperetin, respectively. From the result of this spectral changes and of the high inhibitory potency ($I_{50}=17-75\;{\mu}M$), we found that this compound is an effective inhibitor towards of the oxidative reactions of microsomal monooxygenases tested, namely benzo(a)pyrene hydroxylase (BPH), 7-ethoxycoumarin o-deethylase (ECDE) and aniline hydroxylase (AH). Hesperetin exerted a mode of noncompetitive inhibition on the BPH of PB-microsomes and of mixed inhibition on the same enzyme of MC-microsomes with inhibition constant ($K_i$) of 5.42 and $14.5\;{\mu}M$, respectively. On the other hand, the hesperetin inhibited competitively the ECDE of PB-microsomes and its $K_i$ value was $18\;{\mu}M$, whereas the ECDE of MC-microsomes was markedly inhibited as a mixed mode by this compound and the $K_i$ value was remarkably low ($1.67\;{\mu}M$). In the case of the AH of PB and MC-microsomes, the activities exhibited the same mode of mixed inhibition and the $K_i$ values in general were higher than those of BPH or ECDE. From this observations hesperetin clearly demonstrated that it is an inhibitor of the oxidative metabolism of exogenous substances catalyzed by the liver microsomal P-450-dependent monooxygenases. In particular, this compound indicated that it is a new potent inhibitor of ECDE from results of the high affinity for microsomes containing a great proportion of P-450-c in total P-450 content. We also found that the differences exist between the mechanisms of inhibition according to the form of P-450 isozyme present in microsomes of differently pretreated rats and/or according to the monooxygenase tested.

산철쭉(Rhododendron yedoense var. poukhanense) 꽃으로부터 Terpenoid의 분리.동정

홍윤희,송명종,한재택,장태오,이윤형,김성훈,김승애,박미현,백남인,Hong, Yoon-Hee,Song, Myoung-Chong,Han, Jae-Taek,Jang, Tae-O,Lee, Youn-Hyung,Kim, Sung-Hoon,Kim, Seung-Ae,Park, Mi-Hyun,Baek, Nam-Ln 한국응용생명화학회 2003 한국농화학회지 Vol.46 No.2

산철쭉 끝을 MeOH로 추출하고, 이를 EtOAc, n-BuOH과 $H_2O$로 분배 추출하였다. EtOAc추출물과 n-BuOH 추출물에 대하여 silica gel과 ODS column을 이용하여 chromatography하거나, 아세틸화 한 후 sillica gel column chromatography를 수행하여 2종의 triterfenoid와 3종의 diterpenoio를 분리하였다. 각 화합물은 $^1H-^lH\;COSY$, HMQC, HMBC와 같은 2D-NMR기법을 포함한 스펙트럼 데이터를 해석하여 $2{\alpha}\;3\{beta}-dihydroxylolean-12-ene (1), ursolic acid (2), grayanotoxin IV (3), grayanotoxin I (4) 및 grayanotoxin III (5) 등으로 동정하였다. Extracts were obtained from the flower of Rhododendro yedoense var. poukhanense. (7 kg) in 80% aqueous MeOH and successively fractionated with solvent of EtOAc, n-BuOH and $H_2O$, successively. Silica gel and ODS column chromatographies of the EtOAc and n-BuOH fractions were repeatedly carried out by using the various solvent systems to give five terpenoids. Chemical structures of the isolated terpenoids were determined as $2{\alpha},3{\beta}-dihydroxylolean-12-ene$ (1), ursolic acid (2), grayanotoxin IV (3), grayanotoxin I (4) and grayanotoxin III (5) based on the interpretation of several spectral data including 2D-NMR such as $^1H-^1H\;COSY$, HMQC and HMBC.

스파티필름(Spathiphyllum cannifolium) 지상부로부터 지질화합물의 분리

이대영 ( Dae Young Lee ),박지해 ( Ji Hae Park ),유종수 ( Jong Su Yoo ),송명종 ( Myoung Chong Song ),백남인 ( Nam In Baek ),이윤형 ( Youn Hyung Lee ) 한국응용생명화학회 2008 Applied Biological Chemistry (Appl Biol Chem) Vol.51 No.1

산복사나무(Prunus davidiana) 열매로부터 Triterpenoid의 분리 및 동정

이민지 ( Min Jee Lee ),김지혜 ( Ji Hye Kim ),차병주 ( Byeong Ju Cha ),서경화 ( Kyeong Hwa Seo ),백남인 ( Nam In Baek ),이윤형 ( Youn Hyung Lee ) 한국응용생명화학회 2016 Journal of Applied Biological Chemistry (J. Appl. Vol.59 No.2

산복사나무(Prunus davidiana) 열매를 실온에서 80% MeOH수용액으로 추출하고 이 추출물을 EtOAc 분획, n-BuOH 분획, 물 분획으로 나누었다. EtOAc 분획에 대하여 SiO2과 ODScolumn chromatography를 반복 실시하여 3종의 화합물을 분리 및 정제하였다. NMR, MS, 및 IR data 등을 해석하여, ursolicacid (1), corosolic acid (2), 그리고 α-amyrin (3)로 구조동정하였다. The fruits of Prunus davidiana were extracted with 80 % aqueous methanol at room temperature. The concentrated extract was partitioned as ethyl acetate (EtOAc), n-butyl alcohol, and water fractions. From the EtOAc fraction, three triterpenoids were isolated through the repeated silica gel (SiO2) and octadecyl SiO2 column chromatographies. Based on physico-chemical and spectroscopic data including nuclear magnetic resonance, mass spectrometry, and infrared, the compounds were identified to be ursolic acid (1), corosolic acid (2), and α-amyrin (3).

산복사 나무 열매로부터 플라보노이드의 분리 및 구조 동정

이민지(Min-Ji Lee),김지혜(Ji-Hye Kim),서경화(Kyung-Hwa Seo),차병주(Byeong-Ju Cha),백남인(Nam-In Baek),이윤형(Youn-Hyung Lee) 한국원예학회 2015 한국원예학회 학술발표요지 Vol.2015 No.5

쪽(Polygonum tinctortium) 세포의 형질전환 및 쪽 세포에서 Sodium Butyrate가 Green Fluorescent Protein 발현에 미치는 영향

박성길,정충식,이종진,이윤형,정인식,Park, Sung-Kil,Chung, Choong-Sik,Lee, Jong-Jin,Lee, Youn-Hyung,Chung, In-Sik 한국응용생명화학회 2001 Applied Biological Chemistry (Appl Biol Chem) Vol.44 No.4

쪽(Polygonum tinctortium) 세포에서 외래 단백질 발현을 검토하기 위하여 green fluorescent protein(GFP)가 내재하는 pCAMBIA1302를 형질 전환시켰으며 Western blot 분석에 의해 GFP의 발현을 확인하였다. Sodium butyrate가 GFP생성에 미치는 효과를 검토한 결과, 10 mM에서 세포성장이 지연되었으며, 15 mM 이상에서는 정지되었다. 세포 내 GFP 생성량은 세포 접종 후 3일째 5 mM sodium butyrate를 첨가하였을 때가 0일째 처리에 비해 120% 증가하였다. 또한 접종후 3일 후 5 mM의 sodium butyrate를 처리한 경우가 10 mM의 경우보다 GFP의 수율이 50% 증가하였다. 본 실험을 통하여 세포 접종 후 3일째, 5 mM의 농도로 처리한 sodium butyrate가 외래 단백질의 발현을 효과적으로 증가시키는 결과를 확인하였다. To examine the expression of foreign protein in Polygonum tinctorium cells, plasmid pCAMBIA1302 encoding Green Fluorescent Protein(GFP) was used to transform the cells and the expression was confirmed using Western blot analysis. When the effect of sodium buryrate on the formation of GFP was examined, cell growth was retarded at the addition of 10 mM and was stalled at more than 15 mM. The amount of GFP production was increased by 15% when 5 mM of sodium butyrate was added at three-days after inoculation as compared to at 0-day. Moreover, when sodium butyrate was added at three-days after inoculation, the amount of GFP was increased by 50% at the addition of 5 mM of sodium butyrate as compared to 10 mM.

생화학,분자생물학 : DEB 처리에 의해 유도된 벼 돌연변이 집단으로부터 도열병 감수성 돌연변이 분리

김혜경 ( Hye Kyung Kim ),이상규 ( Sang Kyu Lee ),한무호 ( Mu Ho Han ),전용희 ( Yong Hee Jeon ),이기환 ( Gi Hwan Yi ),이윤형 ( Youn Hyung Lee ),부성희 ( Seong Hee Bhoo ),한태룡 ( Tae Ryong Hahn ),전종성 ( Jong Seong Jeon ) 한국응용생명화학회 2005 Applied Biological Chemistry (Appl Biol Chem) Vol.48 No.4