들깨잎 Polyphenol oxidase의 특성에 관한 연구

김안근,박수선,장영수,Kim, An-Keun,Park, Soo-Sun,Chang, Young-Soo 한국생약학회 1996 생약학회지 Vol.27 No.4

Effects of hydrogen peroxide$(H_2O_2)$ on polyphenol oxidase (PPO) in Perillae Folium were investigated. The inactivation of this enzyme was dependent on $H_2O_2$ concentration. and the initial lag period was not shown. Preincubation of Perillae Folium PPO with $H_2O_2$ in the absence of a substrate resulted in rapid loss of enzymatic activity. The inactivation of PPO by $H_2O_2$ dependents temperature and pH. OH radical scavengers such as mannitol and sodium formate did not protect the enzyme against inactivation by $H_2O_2$. Substrate analogue such as phenylalanine protected the enzyme against inactivation by $H_2O_2$. and copper chelator such as sodium azide also protected the enzyme.

고초균에서 폴리페놀로 유도된 DNA 손상에 대한 폴리페놀산화효소의 억제효과

김안근(An Keun Kim),김유경(Yoo Kyung Kim),강영순(Young Sook Kang) 대한약학회 2005 약학회지 Vol.49 No.4

Antimutagenic activity of the enzymatic browning reaction products (EBRPs) was investigated by using the spore rec-assay with Bacillus subtilis strains H17 (rec+) and M45 (rec-). The EBRPs tested were prepared from the reactions of five different kinds of polyphenols with polyphenol oxidase isolated from the leaves Perilla frutescens. In the spore rec-assay, most of the polyphenolic compounds tested showed positive, whereas only their tested compound showed negative respectively. In addition of polyphenol oxidase inhibitors such as cysteine, glutathione and ascorbic acid to the reaction mixtures consisted with the polyphenol oxidase and polyphenols, the mutagenic effects were increased in the spore rec- assay. These results show that the activity of polyphenol oxidase may play an important role in the reduction of mutagenicity of polyphenols.

붉은 서나물 잎 (Erechitites hieracifolia Raf.)에서의 Polyphenoloxidase 활성측정 및 항산화효오 특성분석

김안근(Kim An Keun),이상은(Lee Sang Eun),김국환(Kim Kuk Hwan),권영이(Kwon Young Ee) 대한약학회 2002 약학회지 Vol.46 No.4

Polyphenoloxidase activity (PPO) in the leaves of Erechitites hieracifolia was estimated by Warburg's manometric method. The emzyme was most reactive toward chlorogenic acid followed by caffeic acid. Biethyldithiocarbamate and potassium cyanide were shown powerful inhibition rate to the polyphenoloxidase from the leaves of Erechitites hieracifolia . We confirme d antioxidant activity of the leave s ofErechitites hieracifolia by DPPH (1,1- diphenyl -2-picrylhydrazyl) radical scavenging method. Electrophorectic isoenzyme banding patterns of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were observed by native PAGE. The correlation of PPO and antioxidant enzymes is not investigated yet. That is need to further study.

아스코르빈산과 티올류 및 유기산이 폴리페놀 산화효오 활성에 미치는 영향

김안근(An Keun Kim),김유경(Yoo Kyung Kim) 大韓藥學會 2001 약학회지 Vol.45 No.4

The effects of ascorbic acid, thiols such as cysteine, n-acetyl-l- cyteine, glutathione, thiourea, 2-mercaptoethanol and dithiotreithol and organic acids such as magic acid, citric acid, glycolic acid, taurine and kojic acid on polyphenol oxidase (PPO) activity were studied in order to establish if it reacts with oxidized product an[Vor directly inhibits the enzyme. To investigate the mechanism, the quantiscation of t-butylcatechol and 4-methylcatechol (phenolic compounds) as substrates, their oxidized product and sul-phydryl colorless additional compounds were determined by high performance liquid chromatograph (HPLC) method. Chromatographic results indicate that ascorbic acid, organic acids and lower level of cgsteine reduced oxidized product of substrates back to their respective positions oleo-diphenols. On the other hand, other thiols and high level of cysteine reacted with oxidative product of o-diphenols and then produced sulphydryl colorless compounds. Cysteine apperars to have two types of mechanism of actions in the formation of oxidative products of substrates depending on its concentration; ascorbic acid-type and other thiols-types. The effect of ascorbic acid with thiols on polyphenol oxidase was determined by same method. Chromatographic results indicate that ascorbic acid was more reactive with oxidized product of substrates than thiols.

들깨잎 polyphenol oxidase의 세포내 분포 및 특성

김안근(An Keun Kim),김유경(Yoo Kyung Kim) 대한약학회 1999 약학회지 Vol.43 No.6

Polyphenol oxidase (PPO) activity in 200Xg (cell wall), 4,000Xg (plastid), 100,000Xg (mitochondrial) and soluble fractions of the perilla leaves was monitored in the upper, middle and lower sections of the plant. In the course of plant growth, PPO activities in plastid and mitochondrial fractions were decreased, while those in cell wall fraction were maintained. During growing process, specific activities and PPO activities of each fraction were decreased, while total phenol content were decreased in middle (middle) and then increased in later stage (lower). Cell wall, plastid, mitochondrial (pellet) and soluble fraction had slightly different pH optima and substrate specificities. Isoenzyme patterns were identical in two bands for PPO activity in different subsellular fractions. Their molecular weight were 37KD and 48KD respectively.

산화적 스트레스 및 항산화제가 항산화효소 활성에 미치는 영향

김안근(An Keun Kim),김지현(Ji Hyun Kim) 한국응용약물학회 2001 Biomolecules & Therapeutics(구 응용약물학회지) Vol.9 No.4

N/A The effect of oxidative stress on the alterations of different antioxidant enzyme activities was investigated in human skin melanoma cell line (SK-MEL-2). Oxidative stress was induced by the exposure to hydrogen peroxide (H_2O_2). SK-MEL-2 cells were treated with antioxidants such as vitamin E and selenomethionine in combination with H_2O_2. SK-MEL-2 cells were exposed to various concentrations of H_2O_2 and measured the time course of changes in cell viability and antioxidant enzyme activities for 24 hr. Oxidative stress was induced by the exposure to 2.5mM hydrogen peroxide (H_2O_2) resulted in declining significantly for 24 hr. GPX and CAT activities peaked at 3 hr and returned to control levels by 24 hr. On the contrary, SOD activity was inactive before 6 hr but recovered at 24 hr. In case vitamin E (Vit E) and selenomethionine (SeMet) were used at nontoxic concentrations (25μM Vit E/500μM Se-Met) to oxidative stress was induced by the exposure to hydrogen peroxide (H_2O_2) led to a 3- and 5-fold increase on the viability comparing to control and caused an increase in GPX activity respectively.

효소적 갈변 생성물의 DNA 손상에 대한 효과

김안근(An Keun Kim),이지은(Ji Eun Lee) 한국생약학회 2000 생약학회지 Vol.31 No.2

Antimutagenicity profiles of the enzymatic browning reaction products(EBRP) were investigated. The rec-assay with Bacillus subtilis strains H17(rec^+) and M45(rec^-) was carried out using their spores. The biological activities were evaluated for seven different enzymatic browning reaction products, which resulted from the reactions of seven polyphenols with polyphenol oxidase isolated from Ginkgo biloba leaves. In the spore rec^- assay, most of the polyphenolic compounds tested were positive, whereas their enzymatic browning reaction products were tested negative. The mutagenicity of enzymic browning mixtures of the polyphenols and the enzymes obtained from Ginkgo biloba leaves showed negative results in the mutagenicity test using Bacillus subtilis strains H17(rec^+) and M45(rec^-). In the case where polyphenol oxidase inhibitors were added in the enzymatic reaction mixtures with polyphenols, the polyphenols showed mutagenic effect in the spore rec^- assay. This suggests that the activity of polyphenol oxidase is decreased.

Genistein이 햄스터 난소세포의 항산화효소활성과 발현에 미치는 영향

김민혜,김안근,Kim, Min-Hye,Kim, An-Keun 대한약학회 2007 약학회지 Vol.51 No.1

Reactive oxygen species (ROS) are produced in the metabolic process of oxygen in cells. The superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in cells systemize the antioxidant enzymes to control the oxidative stress. Genistein is one of the isoflavonoids, and its role in controlling cellular oxidative stress is presently the active issue at question. In this study; we analyzed genistein-induced survival rates of the CHO-K1 cells, activities of antioxidant enzymes, ROS levels, and expression levels of antioxidant enzyme genes in order to investigate the effect of genistein on cellular ROS production and antioxidative systems in CHO-K1 cells. As results, the survival rate of cells was decreased as the dose of genistein increases (12.5${\sim}$200 ${\mu}$M). Genistein increased cellular ROS levels, while it reduced total SOD activities and the expression of CuZnSOD. In conclusion, we suggest that genistein may induce oxidative stress via down-regulation of SOD.

햄스터 난소세포에서 Daidzein과 Genistein에 의해 유도된 산화적 스트레스에 대한 Vitamin C의 효과

김민혜,김안근,Kim, Min-Hye,Kim, An-Keun 대한약학회 2007 약학회지 Vol.51 No.4

The oxidative stress causes many diseases like cancer, aging, cardiovascular disease, degenerative neurological disorders (Parkinson’s disease, and Alzheimer's disease) by damage of cell membrane, protein deformation, and damage of DNA due to the oxidation of lipid of cell membrane, protein of tissue or enzyme, carbohydrate, and DNA. It is caused by the reactive oxygen species (ROS) that is produced in the metabolic process of oxygen in cell. The superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in cell systemize the antioxidative enzymes to control the oxidative stress. In this research, it is measured that the survival rate of cell by the typical isoflavonoid of daidzein or genistein, activity of antioxidative enzyme, and ROS level, in order to study the effect of isoflavonoid over the ROS production in cell and antioxidative system. As the similar action of the isoflavonoid with the estrogen is examined, women are encouraged to get bean. In view of this trend, it is very important to find out a combination medicine that lowers the oxidative stress caused by the daidzein in the ovarian cell. In the combined treatment of the typical antioxidant of vitamin C to oxidative stress which induced by daidzein recover the control level particularly lowering the ROS in cell by 30%. However, it made no effect in the combined treatment with genistein. Therefore, the research took the combination effect of daidzein with vitamin C in order to check it effect over the antioxidative system. In conclusion, it was disclosed that the oxidative stress caused by daidzein is related to the lowering activity of SOD, and the specific combination effect of daidzein with vitamin C is related to the recovery of SOD activity.

들깨잎 폴리페놀 산화효소의 pH 및 온도에 의한 영향

김유경,김안근,Kim, Yoo-Kyung,Kim, An-Keun 대한약학회 2004 약학회지 Vol.48 No.6

Polyphenol oxidase-catalyzed oxidation of substrates (t-butylcatechol, 4-methylcatechol, chlorogenic acid, caffeic acid and pyrocatechol) were performed in the Ph range 4~8. Co ncentrations of substrate's major oxidation products were monitored by high performance liquid chromatograph. The nature and amounts of products formed were highly pH dependent. They also were ifluenced by kinds of substrates. Major oxidation product of 4-methylcatechol appeared the maxium value at pH 5, them of chlorogenic acid, caffeic acid and pyrocatechol at pH 6.0 and that of t-butylcatechol at pH 5~7. Time-dependent PPO activity was determined at $4^{\circ}C\;and\;30^{\circ}C$. PPO extracted by phosphate buffer containing triton X-114 (t-PPO) was more stable than PPO by phosphate buffer (b-PPO). The result of electrophoresis, at first PPO was showed only a band at 48 kd. After 1~3 days a partial degrade band was appeared in b-PPO and three partial degrade bands in t-PPO. No activity band was appeared in PPOs at $30^{\circ}C$ and b-PPO at $4^{\circ}C$ after 4 days. And a band (37 kDa) in t-PPO was remained finally and disappered. PPO from Perillae leaves has two activity bands at 48 and 37 kDa in previous paper. It was supposed that PPO in the leaves of Perilla frutescens was a protein having one molecular weight as 48 kDa. And 37 kDa protein, relatively proteolysis-resistant, was a proteolyzed form of a major form.