Photodynamic apoptosis and antioxidant activities of Brassica napus extracts in U937 and SK-HEP-1 cells

김근기,이민우,박재은,이준영,홍창오,이상몽,김용균,김근기 한국응용생명화학회 2017 Applied Biological Chemistry (Appl Biol Chem) Vol.60 No.4

Brassica napus is the most common feedstock for biodiesel production, and its cultivation area has been rapidly increased. Thus, B. napus residues left in the field after harvest are valuable resources. However, there have been few studies on biologically active substances from B. napus plant. The objective of this study is to evaluate cytotoxicity/photodynamic activity and antioxidant activity of B. napus plant extracts. B. napus plants were sequentially extracted with organic solvents (hexane, chloroform, ethanol, and water) and then screened for antioxidant activity and cytotoxicity against leukemia U937 and human liver cancer SK-HEP-1 cells. Among the solvent extracts, the cytotoxicity was the highest when cells treated with chloroform extract and irradiated. Degree of apoptosis substantially increased in both cell types in concentrationdependent manner, and non-irradiated cells showed similar results as the control cells. For the highest concentrations (100 lg/ml), toxicity effect in U937 and SK-HEP-1 cells was 94.62 ± 0.15% and 74.16 ± 1.54%, respectively. We observed the number of cells significantly decreased, and vesicles were floating in B. napus chloroform extract (BNCE) and light condition. BNCE induced DNA laddering pattern (between 300 and 1000 bp) and caspase-3/7 activation in both U937 and SK-HEP-1 cells. Total apoptotic U937 and SK-HEP-1 cells following BNCE 100 lg/ ml and light treatment were significantly increased (92.62 ± 2.07% and 59.71 ± 4.38%, respectively) compared with control. Our results showed that U937 cells were more sensitive than SK-HEP-1 cells. For the antioxidant activity, B. napus ethanol extract was the highest (IC50 = 0.52 mg/ml).

포스터 초록 ( 농약 및 환경 ) : 시설 House 토양에서 분리한 길항균의 항균 Spectrum 과 그 특성

김근기,박기훈,강규영 한국응용생명화학회(구 한국농화학회) 1996 한국응용생명화학회 학술발표회 Vol.1996 No.2

초피첨가 전통장류의 항균 및 항암활성

김근기,박현철,손홍주,김용균,이상몽,최인수,최영환,신택순,Kim, Keun-Ki,Park, Hyean-Cheal,Son, Hong-Joo,Kim, Yong-Gyun,Lee, Sang-Mong,Choi, In-Soo,Choi, Young-Whan,Shin, Teak-Soon 한국생명과학회 2007 생명과학회지 Vol.17 No.8

초피나무(Z. piperitum) 열매는 다양한 생리활성을 갖고 있으며, 과피 용매추출물은 세균에 대한 강한 항균활성을 갖고 있어, 간장과 된장제조에 초피를 첨가하여 생리활성효과를 조사하였다. 초피를 간장제조에 1%, 2%, 4% 첨가하여 제조하고, 간장추출물을 식중독균(Sta. aureus, Sal. typhimurium, V. parahemolyticus, E. coli 0157:H7)에 대한 항균활성을 조사했다. 초피를 2% 첨가한 간장은 E. coli 0157:H7과 V. parahemolyticuS 의 생육을 각각 70%와 50% 억제했으며, Sal. typhimurium 과 Sta. aureus 에 대한 항균효과는 농도와 간장제조시기에 따라 40% ${\sim}$ 60% 의 생육을 억제했다. 초피된장의 용매추출물에서는 약한 항균활성을 확인할 수 있었다. 초피간장의 추출물을 백혈병세포 U-937에 처리하여 caspase-3 활성으로 항암활성을 조사했다. 그 결과 초피의 량을 1%, 2%, 4% 첨가한 간장의 추출물은 초피를 침가하지 않은 간장보다 caspase-3 활성이 각각 4배, 12배, 15배 활성이 증가하는 것이 확인되어졌다. 이는 초피간장의 추출물이 apoptosis를 유도하는 것을 의미하며, 된장의 용매추출물은 caspase-3 활성에 아무런 영향이 없었다. 초피간장 추출물을 처리한 세포의 죽음이 necrosis인지 apoptosis인지 알아보기 위하여 세포내의 DNA fragmentation을 확인한 결과, 초피간장 추출물을 처리한 세포는 DNA 단편화가 형성되어졌다. 초피틀 1% 첨가한 것에서는 fragmentation을 확인하기 힘들었고, 2%와 4%초피를 첨가한 간장추출물은 DNA fragmentation을 강하게 일으켰다. 초피를 첨가한 간장추출물이 caspase를 활성화시키고, DNA fragmentation을 일으키는 apoptosis 기작에 의해 암세포를 죽음으로 유도하는 초피간장의 항암효과를 입증했다. The fruits of Zanthoxylum piperitum are known as having various physiology vitality, and the abstraction ingredient of the pericarp is also known as having strong antibiotic activities against various bacteria. Therefore, this study was carried out to estimate the effect of physiology vitality when the abstraction ingredient of Z. piperitum was added in soy sauce(Chopi-kanjang) and soybean paste(Chopi-doenjang). For the antibiotic activity against the pathogens of sitotoxism such as Staphylococcus aureus, Salmonella typhimurium, Vibrio parahemolyticus, Escherichia coli 0157:H7, the extracts of the Chopi-kanjang was added 1%, 2%, 4% pericarp of Z. piperitum in the manufacturing process of soy source. According to the results, the growth of E. coli 0157:H7 and V. parahemolyticus were respectively inhibited as 70% and 50% by the Chopi-kanjang added 2% of the ingredient. For the antibiotic effects of the aforementioned Chopi-kanjang against Sal. typhimurium and Sta. aureus, the growth of those pathogens was also inhibited between 40% and 60% according to the manufacturing period of Chopi-kanjang. It was confirmed that the antibiotic activity using the mixture of the abstraction ingredient and Chopi-doenjang was lower than those of Chopi-kanjang. In order to estimate the anticancer activity using by caspase-3 activity, the mixture of the abstraction ingredient of the pericarp of Z. piperitum and Chopi-kanjang was treated to leukemia cells. According to the results, the activities of caspase-3 using the mixture added 1%, 2% and 4% of the abstraction ingredient were respectively increased as much as 4, 12, 15 times comparing with the control which was treated with the soy source only. It could be that the mixture of the abstraction ingredient of the pericarp of Z. piperitum and soy source induced apoptosis, and the mixture of the abstraction ingredient and soybean paste had no effect on the activity of caspase-3. In order to find out the death of the aforementioned cells caused by necrosis or apoptosis, DNA fragmentation in the cell was examined. U-937 cells showed apoptotic DNA fragmentation in the incubation with Chopi-kanjang extract.

Burkhoderia sp. AK-17에 의한 잎들깨 재배의 생물학적 조절

김근기,김용균,손홍주,최영환,강규영 Plant molecular biology and biotechnology research 2005 Plant molecular biology and biotechnology research Vol.2005 No.

There are various crop diseases in green houses that are caused by the cultural environments, especially high temperature and moisture. To solve the forementioned problems, farmers are overusing agricultural chemicals, causing other damages by the chemical residue. In this study, antagonistic bacteria as biological control agents were isolated to produce the environmentally-friendly crops for use in green houses. Eighteen species of antagonistic bacteria were totally isolated from the soil and plants in the Perilla fields, and AK-17 showed the highest activity among the isolates. According to the results of anti-fungal spectrum against several pathogens by AK-17, the antagonism effect of the isolates was remarkable against grey mold rot by Botrytis cinerea, sclerotinia rot by Sclerotnia sclerotiorum, and stem rot by Rhizoctonia solini. To evaluate the biological control effects of the isolates against the major diseases of Perilla, studies were carried out to evaluate the preventive and the curative effects of the diseases throughout the pot experiments. According to the forementioned experiments, the preventive and the curative effects by the isolates against sclerotinia rot were respectively showed as 55% and 92%. For the grey mold rot, those were 40% and 78%, respectively. As to the evaluation of the growth-promoting effect by AK-17, the length and the biomass of the tested plants were increased to 120% and to 164%, respectively. For the leaf numbers and area were respectively increased to 120% and 220%. Furthermore, AK-17 was identified as Burkhoderia sp. according to the results of physiological properties and genetic methods. 시설원예단지의 재배환경은 고온다습으로 많은 병해가 발생하며, 이를 방제하기위해 과다한 농약을 사용하므로 농약 잔류성에 대한 피해가 심각한 우려를 낳고 있다. 미생물을 이용한 생물학적 방제법 개발로 청정 시설원예작물을 생산할 목적으로 길항균을 분리하였다. 잎들깨 재배토양과 식물체로부터 길항세균 18종을 분리하였으며 분리한 길항세균 중에 AK-17이 가장 활성이 뛰어나 이를 이용하여 주요 식물병원균에 대한 항균스펙트럼을 조사하였다. 그 결과 잿빛곰팡이병원균의 Botrytis cinerea와 균핵병원균의 Sclerotinia sclerotiorum 및 줄기썩음병원균인 Rhizoctonia solant에 대한 항균효과가 뛰어났다. 잎들깨의 주요 병에 대한 생물학적 방제실험은 병발생 억제효과와 병방제효과를 pot 실험으로 실시했다. 그 결과 균핵병은 55%의 병발생 억제효과와 92%의 방제효과가 있었고, 잿빛곰팡이병은 40%의 병발생 억제효과와 78%의 방제효과를 확인할 수 있었다. AK-17의 식물 생육촉진효과는 신장이 120%, 생체중이 164% 증가되었으며, 엽수와 엽면적은 각각 120%와 220%의 증가효과를 보였다. 그리고 AK-17을 생리·생화학적방법과 유전학적 방법으로 동정한 결과, Burkhoderia sp.로 확인되었다.

Isolation, identification, and apoptosis activity of the photosensitizer methyl pheophorbide A from Perilla frutescens leaves

김근기,Ha Jun Young,Yi Gibum,Bae Hwan Hee,Go Young Sam,김유진,이광민,홍창오 한국응용생명화학회 2022 Applied Biological Chemistry (Appl Biol Chem) Vol.65 No.4

Photodynamic therapy (PDT) is a promising cancer treatment with fewer side effects, and it eliminates tumors in target tissues with reactive oxygen species produced by photosensitizers (PS) and light. In this study, we isolated methyl pheophorbide A, which induces photodynamic cell death in the U937 and SK-HEP-1 cells, from perilla leaves. Its potential as a material for the development of a new PS was also evaluated. The methyl pheophorbide A is a dark green porphyrin compound isolated from methanol extract of perilla leaves. Apoptosis occurred was methyl pheophorbide A treated and irradiated with light, and there was no significant change where light not treated. In both U937 and SK-HEP-1 cells, apoptotic body, vesicle formation, and DNA ladder were confirmed in the light-irradiated. The caspase-3/7 activity an important factor in apoptosis, was 101.50 ± 14.24% when treated with 0.25 μg/ml methyl pheophorbide A in U937, and 91.32 ± 16.23% when treated with 1.00 μg/ml in SK-HEP-1 cells. The apoptotic phenomenon appeared more strongly when the methyl pheophorbide A concentration was increased with irradiating light. This study demonstrates the isolation, identification, and phototoxic activity of methyl pheophorbide A in perilla leaves. We expect that this study will be useful in the search for PS candidates using natural products.

Stevia rebaudiana Bertoni의 Steviol 생합성 효소 ent-Kaurenoic Acid 13-Hydroxylase의 특성

김근기,Hitoshi Shibata ( Keun Ki Kim ) 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.6

Chloroplasts isolated from Stevia rebaudiana Bertoni leaves contained an enzyme activity which catalyzed hydroxylation of ent-kaurenoic acid (ent-kaur-16-en-19-oic acid; ent-KA) to steviol (ent-13-hydroxy kaur-16-en-19-oic acid), the diterpenoid carboxylic alcohol which is the aglycone of sweet stevioside-related glycosides. [^(14)C]-methylated ent-KA was used to localize ent-KA hydroxylase. [^(14)C]-methyl-KA was most actively was transformed into methyl-steviol in chloroplast. The enzymatic activity was found in stroma fraction but not in thylakoid membrane in Stevia rebaudiana Bertoni. However, ent-KA 13-hydroxylase activity was not detected in strotna fraction of either Spinacia deracea and Solidago altissima. The reaction products using [^(14)C]-methyl-KA were purified and identified on TLC autoradiogram. The hydroxylation of ent-KA from stromal protein to form steviol required NADPH and oxygen. FAD and riboflavin stimulated the enzyme activity 1.5-and 1.7-fold, respectively. It also turned out that the activity of this enzyme using methyl-KA as a substrate was 16.7% that of ent-KA. The purified ent-KA 13-hydroxylase did not act on t-cinnamic acid, 4-hydroxyphenyl acetic acid, choline and resorcinol, known as monooxygenase and hydroxylase substrates.

Aspergillus terreus로부터 항진균성 물질의 분리 및 구조분석

김근기,박기훈,문석식,강규영 ( Keun Ki kim,Ki Hun Park,Suk sik Moon,Kyu Young Kang ) 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.6

In the course of search antagonistic fungi from soil in green house, four kind of fungi (AF1, AF2, AF3, AF4) were isolated, which have activities aganist Phytophthora capsici, Botrytis cinera, Rhizoctomia solani, Pythium ultimum and Fusarium oxysporum. The AF2 was identified according to the morphological description of Aspergillus terreus. This antagonistic fungus inhibiting various plant pathogens was effective to reduce disease incidence of cucumber seedlings caused by mixed inoculum of Rhizoctonia solani, Pythium ultimum and Fusarium oxysporum. Antifungal compound I was isolated and purified by fresh chromatography from A. terreus. The ¹H and ^(13)C assignment of compound I was achieved from two-dimensional ¹H-¹H COSY, HMQC, HMBC with the add of homonuclear and heteronuclear double resonance experiment. The compound I was identified butyrolactone I (α-oxo-β-(p-hydroxyphenyl)-γ-(p-hydroxy-m-3,3-dimethyl-allylbenzyl)-γ-methoxy-carbonyl-γ-butyrolactone, C_(24)H_(24)O_7, M.W.=424).

Aspergillus terreus로부터 항진균성 물질의 분리 및 구조분석

김근기,박기훈,문석식,강규영,Kim, Keun-Ki,Park, Ki-Hun,Moon, Suk-Sik,Kang, Kyu-Young 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.44 No.4

In the course of search antagonistic fungi from soil in green house, four kind of fungi (AF1, AF2, AF3, AF4) were isolated, which have activities against Phytophthora capsici, Botrytis cinera, Rhizoctonia solani, Pythium ultimum and Fusarium oxysporum. The AF2 was identified according to the morphological description of Aspergillus terreus. This antagonistic fungus inhibiting various plant pathogens was effective to reduce disease incidence of cucumber seedlings caused by mixed inoculum of Rhizoctonia solani, Pythium ultimum and Fusarium oxysporum. Antifungal compound I was isolated and purified by fresh chromatography from A. terreus. The $^1H$ and $^{13}C$ assignment of compound I was achieved from two-dimensional $^1H-^1H\;COSY$, HMQC, HMBC with the add of homonuclear and heteronuclear double resonance experiment. The compound I was identified butyrolactone I (${\alpha}$-oxo-${\beta}$-(p-hydroxyphenyl)-${\gamma}$-(p-hydroxy-m-3,3-dimethyl-allylbenzyl)-${\gamma}$-methoxycarbonyl-${\gamma}$-butyrolactone, $C_{24}H_{24}O_7$, M.W.=424). 시설내 토양으로부터 식물병원균에 활성을 갖는 길항균을 탐색하는 과정에서 주요 식물 병원균인 Phytophthora capsici, Botrytis cinerea, Rhizoctonia solani, Pythium ultimum 및 Fusarium oxysporum에 활성을 갖는 4종류의 길항균(AF1, AE2, AE3, AF4)을 분리하였다. 이들 중 가장 높은 활성을 나타낸 AE2균을 동정한 결과, 생육한 균은 짙은 오렌지색과 황갈색을 띄었으며, 현미경의 morphology는 column형의 conidial head와 foot cell로 부터 곧게 뻗은 분생자와 2중층의 포자낭병을 형성했다. 그리고 MY20 agar 배지에서 투명한 둥근세포의 형성등이 Aspergillus terreus의 형태와 일치 하였다. Pot에서 병원균과 A. terreus를 동시에 처리하여 오이의 생육을 조사한 결과, 토양중량당 A. terreus의 균체를 1% 처리에 40%, 5% 처리에 57%, 10% 처리에는 75% 병발생율을 억제시켰다. 그리고 A. terreus의 배양여액으로부터 activity-guided fractionation을 실시하여 항진균성 물질인 화합물 I 을 얻었다. $^1H$, $^{13}C\;NMR$, DEPT, $^1H-^1H\;COSY$, HMQC, HMBC 및 질량스텍트럼을 분석한 결과 화합물 I 은 butyrolactone I (${\alpha}$-oxo-${\beta}$-(p-hydroxyphenyl)-${\gamma}$-(p-hydroxy-m-3,3-dimethyl-allylbenzyl)-${\gamma}$-methoxycarbonyl-${\gamma}$-butyrolactone, $C_{24}H_{24}O_7$, M.W. = 424)로 동정되었다.

포스터 발표논문 초록 ( 토양 / 농약 / 환경 분야 Poster 초록 ) : Aspergillus terreus가 분비하는 항균물질의 활성과 구조결정

김근기,박기훈,문석식,강규영 한국응용생명화학회(구 한국농화학회) 1997 한국응용생명화학회 학술발표회 Vol.1997 No.1

Physiological and Biochemical Roles of the Accumulation of Sweet Glycosides in Stevia rebaudiana Bertoni

김근기 한국응용생명화학회(구 한국농화학회) 1997 한국응용생명화학회 학술발표회 Vol.1997 No.1