초임계이산화탄소를 이용한 탈카페인녹차 열수추출물의 포유동물 세포주를 이용한 염색체이상시험

구윤창,이현순,박병규,김은진,이선주,김경헌,김영석,정영신,이광원,Koo, Yun-Chang,Lee, Hyun-Sun,Park, Byung-Gyu,Kim, Eun-Jin,Lee, Sun-Joo,Kim, Kyoung-Hoen,Kim, Young-Suk,Chung, Young-Shin,Lee, Kwang-Won 한국환경성돌연변이발암원학회 2006 한국환경성돌연변이·발암원학회지 Vol.26 No.4

There are $10{\sim}30%$ polyphenol and $2{\sim}4%$ caffeine in green tea. Caffeine is a kind of alkaloid containing nitrogen which cause stimulation, impatience, headache, insomnia, low birth weight infant. Because of these negative effect, decaffeined beverage came out and decaffeined coffee already have a big market since 1970s. Having proving the physiologic functions of green tea, high consumption of coffee is shifting to green tea. Because of the carcinogenic effect of the organic solvents, decaffeine processing with supercritical carbon dioxide has industrialized and have an advantage in environment-friendly and minimized flavor loss. Decaffeined green tea using supercritical carbon dioxide is considered to be safe but there are not enough study. We investigated the chromosome aberration test with mammalian cell line, CHL. When the cells were treated with 5000, 2000, 1000 ${\mu}g/ml$ and compared with the negative controls, there were no significant(P>0.05) increased chromosome aberration. Same results was observed when adding S9 mixture or not. As a result, water extract of decaffeined green tea using supercritical carbon dioxide does not induce chromosome aberration.

Inhibitory Effect of Yellow Myrobalan (Terminalia chebula) Extract on Fibrosis Induced by Carbon Tetrachloride in Rat Liver

구윤창,홍충의,남미현,김지훈,양성용,원남희,박태선,이광원 한국식품과학회 2013 Food Science and Biotechnology Vol.22 No.3

Ethyl acetate layer of methanol extract (TCE)of yellow myrobalan (Terminalia chebula) contains 2.4%of chebulic acid, a standard compound in TCE, which have a strong anti-oxidative effect and was reported in our previous study. Thirty-one male SD rats were randomly divided into 5 groups (normal control, CCl4 control, TCE control, CCl4+high dose TCE, and low dose TCE). Liver fibrosis was induced by i.p. injection of CCl4 and TCE were orally administrated. TCE decreased the up-regulated malondealdehyde value and increased the down-regulated ratio of GSH/GSSG content and activities of GRd, GPx,and GST compared to the CCl4 control group. Decreased dysfunction and inflammatory reaction in liver was confirmed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, relative liver weight and infiltration of neutrophils. Also, TCE inhibited phenotype change of hepatic stellate cells (HSC) by reducing α-smooth muscle actin (α-SMA) gene expression and protein production. Inhibition of collagen 1A1 gene expression and protein production were also confirmed. From these results, TCE may be a useful material for preventing liver fibrosis.

초임계이산화탄소를 이용한 탈카페인녹차 열수추출물의 포유동물 세포주를 이용한 염색체이상시험

구윤창(Yun-chang Koo),이현순(Hyun-sun Lee),박병규(Byung-gyu Park),김은진(Eun-jin Kim),이선주(Sun-joo Lee),김경헌(Kyoung Hoen Kim),김영석(Young Suk Kim),김광옥(Kim Kwang ok),정영신(Young-shin Chung),이광원(Kwang-won Lee) 한국환경성돌연변이발암원학회 2006 한국환경성돌연변이·발암원학회지 Vol.26 No.3

There are 10~30% polyphenol and 2~4% caffeine in green tea. Caffeine is a kind of alkaloid containing nitrogen which cause stimulation, impatience, headache, insomnia, low birth weight infant. Because of these negative effect, decaffeined beverage came out and decaffeined coffee already have a big market since 1970s. Having proving the physiologic functions of green tea, high consumption of coffee is shifting to green tea. Because of the carcinogenic effect of the organic solvents, decaffeine processing with supercritical carbon dioxide has industrialized and have an advantage in environment-friendly and minimized flavor loss. Decaffeined green tea using supercritical carbon dioxide is considered to be safe but there are not enough study. We investigated the chromosome aberration test with mammalian cell line, CHL. When the cells were treated with 5000, 2000, 1000 ㎍/㎖ and compared with the negative controls, there were no significant (P > 0.05) increased chromosome aberration. Same results was observed when adding S9 mixture or not. As a result, water extract of decaffeined green tea using supercritical carbon dioxide does not induce chromosome aberration.

HPLC/DAD를 이용한 림술푸론, 에타메트설푸론메틸, 트리베누론메칠, 클로리무론에칠 4종 성분의 잔류농약 분석법 개발

구윤창(Yun-Chang Koo),양성용(Sung-Yong Yang),왕증(Zeng Wang),안은미(Eun-Mi An),허경(Kyoung Heo),김형국(Hyengkook Kim),신한승(Han-Seung Shin),이진원(Jin-Won Lee),이광원(Kwang-Won Lee) 한국식품영양과학회 2010 한국식품영양과학회지 Vol.39 No.8

Sulfonylurea계 제초제인 rimsulfuron, ethametsulfuronmethyl, tribenuron-methyl, chlorimuron-ethyl의 네 가지물질의 잔류농약 분석을 위해 HPLC를 이용한 분석방법을 개발하였다. ODS 칼럼(250 ㎜×4.6 ㎜, 5 ㎛ diameter particle size)를 사용하였으며 칼럼오븐은 35oC로 유지하고 용매 A(20 mM KH₂PO₄, pH 2.5)와 용매 B(acetonitrile)를 45:55의 비율로 1 mL/min의 유속으로 흘려주었다. 시료 주입량은 20 μL였으며 분석조건 하에서 rimsulfuron, ethametsulfuron-methyl, tribenuron-methyl, chlorimuron-ethyl의 retention time은 각각 5.92, 6.54, 9.28, 14.35분이었고 정량한계는 각각 0.02, 0.01, 0.001, 0.004 ㎎/㎏이었다. 회수율 실험은 백미, 사과, 콩에 4종의 농약 표준품을 각각 0.05, 0.1, 0.5㎎/㎏의 세 가지 농도로 spiking하여 수행하였으며 86.12∼116.26%의 회수율을 얻었고 표준오차는 모든 실험에서 10%이하였다. The method for residue analysis of four sulfonylurea pesticides, rimsulfuron, ethametsulfuron-methyl, tribenuron-methyl and chlorimuron-ethyl was examined and analyzed by HPLC with ODS column (250 ㎜×4.6 ㎜, 5 ㎛ diameter particle size) which was maintained at 35oC. Mobile phase consisted of solvent A (20 mM KH₂PO₄, pH 2.5) and solvent B (acetonitrile). Isocratic elution of the column with 45% solvent A and 55% solvent B at a flow rate of 1 mL/min resulted in retention times of 5.92, 6.54, 9.28, and 14.35 min for rimsulfuron, ethametsulfuron-methyl, tribenuron-methyl, and chlorimuron-ethyl, respectively. All injection volumes were 20 μL. The limit of quantitation was 0.02, 0.01, 0.001, and 0.004 ㎎/㎏ for rimsulfuron, ethametsulfuron-methyl, tribenuron-methyl, and chlorimuron-ethyl, respectively. Recovery rate test was performed with three farm products, rice, apple and soybean. Four sulfonylurea pesticides were spiked at concentrations of 0.05, 0.1 and 0.5 ㎎/㎏. The recovery rates were ranged from 86.12% to 116.26% and the standard deviations of all experiments were within 10%.

차전초(Plantago asiatica)로부터 분리된 Plantamajoside의단회와 14일 반복투여 독성시험 및 유전독성학적 안전성 평가

박병규,이현순,정성훈,구윤창,홍충의,이선주,이광원 한국독성학회 2007 Toxicological Research Vol.23 No.1

The isolated plantamajoside from Plantago asiatica that is often used as a marker compound in chemotaxonomic studies has various bioactivites such as the inhibitions of cyclic AMP phosphodi-esterase and 5-lipoxygenase, microbial growth and inflammation, and currently demands the generation of toxicity data. The purpose of this study was to examine the toxicities of the single and 14 days repeated dose toxicity in Sprague-Dawley rats orally administrated with plantamajoside at dose levels of 0, 500, 1000, and 2000 mg of dried material/kg body weight/day. The results showed that there was no difference in body weight change, food intake, water consumption, or relative organ weight among different dose groups. Also we observed no death and abnormal clinical signs were observed during the experimental period. Between the groups orally administered Plantago asiatica and the control group, there was no statistical significance in hematological test or serum biochemical values. There were no gross findings at final sacrifice. There was no evidence of histopathological alteration mediated by 14 days treatment with Plantago asiatica. These results suggest that no observed adverse effect level (NOAEL) of the oral application was considered to be more than 2000 mg/kg in rats under the conditions employed in this study. Another observation was performed to investigate the safety of Plantago asiatica in respect of genotoxicity. This substance was examined that Salmonella typhimurium reversion assay (Ames test) in strain TA98, TA100, TA1535. In the reverse mutation test, Plantago asiatica did not induce mutagenicity in Samonella typhimurium with and without metabolic activation. These results indicated that Plantago asiatica had no genotoxicity.

HPLC를 이용한 Isoproturon, Phenmedipham, Pyridate 및 Nitenpyram 4종 성분의 잔류농약 분석법 개발

양성용(Sung-Yong Yang),구윤창(Yun-Chang Koo),Zeng Wang,허경(Kyeong Heo),김형국(Hyeongkook Kim),안은미(Eun-Mi An),신한승(Han-Seung Shin),이진원(Jin-Won Lee),이광원(Kwang-Won Lee) 한국식품영양과학회 2010 한국식품영양과학회지 Vol.39 No.8

Bis-carbamate계(phenmedipham), urea계(isoproturon), thiocarbamate계(pyridate) 및 vinyllidenediamine계(nitenlyram) 4종의 농약을 HPLC를 이용하여 분석하는 방법을 개발하였다. 사용된 칼럼은 C-18(250 ㎜×4.6 ㎜, 5 ㎛ diameter particle size)이며 isoproturon, phenmedipham은 acetonitrile과 물을 50:50으로 섞어서 사용하였으며, pyridate는 acetonitrile과 물을 85:15로, nitenpyram은 phosphoric acid를 이용하여 pH 2.5로 맞춘 50 mM KH₂PO₄와 acetonitrile을 90:10으로 섞어 유속 1 mL/min의 isocratic 조건으로 분석하였다. 시료 주입량은 10 μL이며 retention time은 isoproturon 6.12분, phenmedipham 8.63분, pyridate 9.40분, nitenpyram 12.76분, 정량한계는 모두 0.05 ㎎/㎏이었다. 회수율 실험은 쌀, 사과 및 대두에 4종의 농약 표준품을 각각 0.05, 0.1 및 0.5 ㎎/㎏이 되도록 세 가지 농도로 spiking 하였으며, 회수율 및 분석오차는 70.18~118.08% 범위와 10% 이내를 만족하였다. A method for the determination of four pesticide compounds, urea (isoproturon), bis-carbamate (phenmedipham), thiocarbamate (pyridate) and vinyllidenediamine (nitenpyram) were examined and analyzed by HPLC with C-18 column (250 ㎜×4.6 ㎜, 5 ㎛ diameter particle size). Mobile phase consisted of deionized water, acetonitrile and 50 mM KH₂PO₄ (pH 2.5). Isoproturon and phenmedipham analytical condition was isocratic elution of the column with 50% solvent A (acetonitrile) and 50% solvent B (deionized water); pyridate was 85% solvent A (acetonitrile) and 15% solvent B (deionized water) at a flow rate of 1 mL/min; and nitenpyram analytical condition was 90% solvent A (50 mM KH₂PO₄, pH 2.5) and 10% solvent B (acetonitrile) at a flow rate of 1 mL/min. In results, retention times were 6.12, 8.63, 9.40 and 12.76 min for isoproturon, phenmedipham, pyridate and nitenpyram, respectively. All injection volumes were 10 μL and the limit of quantitation was 0.05 ㎎/㎏ for four pesticide compounds, respectively. Recovery rate test was performed with three farm products, rice, apple and soybean. Four pesticide compounds were spiked at concentrations of 0.05, 0.1 and 0.5 ㎎/㎏. The recovery rates were ranged from 70.18% to 118.08% and the standard deviations of all experiments were within 10%.

차전초 추출물을 투여한 랫드에서의 Fe-NTA 유발 산화스트레스에 대한 신장보호 효과

홍충의,이광원,홍승택,구윤창,양성용,이지영,이얀회이,하영민 한국식품위생안전성학회 2011 한국식품위생안전성학회지 Vol.26 No.2

Plantago asiatica L. (PA), which is widely distributed in Korea, Japan and China, has traditionally been used as a popular folk medicine for the treatment of liver diseases. A variety of activities of PA was reported, that is hepatoprotective, anti-inflammatory, anti-glycation and anti-oxidant effect. Ferric nitrilotriacetate (Fe-NTA) is a potent nephrotoxic agent and has been reported to induce renal proximal tubular necrosis. In the present study, pretreatment with PA extract (PAE) in Wistar rat followed by Fe-NTA i.p. treatment (13.5 mg Fe/kg body weight) was performed to detect the renal protective effect of PAE. Only Fe-NTA treated group showed increases in the level of serum blood urea nitrogen (BUN) and serum creatinine (Cr), and renal tissue malondialdehyde (MDA), product of lipid peroxidation. Moreover, the level of biomarkers indicate the antioxidants status, reduced glutathione (GSH), glutathione-S-transferase (GST) and glutathione reductase (GR) were decreased. However, PAE pre-treated group showed decreases in the levels of serum BUN, serum Cr and renal tissue MDA in concentration dependent manner and increases in the level of GSH, GST and GR. These results are significantly different (p < 0.05) to the other groups. Our data suggest that PAE may be used as an chemopreventive material against Fe-NTA-mediated renal oxidative stress.

스트렙토조토신 유발 당뇨 쥐의 산화스트레스에 대한 매생이 추출물의 신장 보호 효과

남미현(Mi-Hyun Nam),구윤창(Yun-Chang Koo),홍충의(Chung-Oui Hong),양성용(Sung-Yong Yang),김세욱(Se-Wook Kim),정혜림(Hye-Lim Jung),이화(Hwa Lee),김지연(Ji-Yeon Kim),한아람(Ah-Ram Han),손원락(Won-rak Son),표민철(Min-Cheol Pyo),이광원(Kwan 한국식품과학회 2014 한국식품과학회지 Vol.46 No.5

당뇨 모델 쥐에서 매생이 추출물과 그 지표물질인 pheophorbide A의 신장 보호 효과를 확인하였다. 5주령의 SD 랫드를 일주일간 순화시킨 뒤 40 ㎎/㎏ BW의 농도로 streptozotocin을 복강 투여하였으며, 한달 동안의 유도기간을 거쳐 모든 당뇨 유발군에서 혈당이 400-500 ㎎/dL가 된 것을 확인한 이후 9주 동안의 샘플 투여를 진행하였다. 신장 손상과 관련한 혈액 생화학적 지표인 BUN, creatinine에서는 당뇨 유발군 사이에 개선되는 경향을 보이진 않았다. 신장 조직에서 산화스트레스에 의해 유발되는 지질 과산화물을 측정 하였을 때 당뇨 유발군인 STZ군(0.19±0.04)은 정상 대조군인 Con군(0.05±0.00)에 비하여 3.5배 많은 MDA를 생성하였지만 매생이 추출물 4, 20 ㎎/㎏ BW투여한 CL, CH군은 MDA의 생성량이 0.05±0.02, 0.08±0.02, 지표물질인 pheophorbide A를 0.2 ㎎/㎏ BW을 투여한 PhA군은 0.12±0.01 nmol/㎎ protein로 샘플투여군에서 과산화물이 유의적으로 감소하는 것으로 보아(p<0.05) CFE와 PhA의 항산화 작용을 확인할 수 있었다. 항산화 바이오마커인 GSH와 GSSH 함량 및 GPx, GR, GST 활성을 측정한 결과 CFE와 PhA는 산화스트레스에 의한 신장 조직의 GSH 함량이 감소하는 것을 막아 당뇨로 인한 신장 손상에 보호 효과가 있는 것을 확인하였다(p<0.05). 또한 STZ에 유도된 당뇨 쥐의 신장조직엔 많은 양의 과산화물이 축적되어 있으며 이를 소거하기 위해 GPx의 활성이 높아졌는데, AC군과 PhA군에서 GPx 의 증가정도를 유의적으로 완화시켰다(p<0.05). GR 활성은 CL, CH, PhA군에서 Con군 수준까지 올라오는 것으로 보아 신장의 산화적 손상을 정상수준까지 회복 시키는 것으로 보였으며, GST 결과로 미루어 보아 CFE와 PhA는 신장 조직에서 일차적으로 과산화물을 포집하고 GSH를 이용하여 체내의 독성 물질을 분해시켜 STZ로 유도된 당뇨로부터 신장 보호 효과를 나타내었다. 또한, 조직내에서 SOD와 CAT의 활성 측정 결과, CFE와 PhA는 지질과산화물의 증가로 산화적 손상이 가속화 될 때, 항산화 효소계의 활성을 증가시키고 산화 스트레스에 의한 조직 손상을 완화 시켜주는 것으로 확인되었다. 신장 조직의 병리학적 관찰 결과 CL, CH 및 PhA군에선 STZ군에 비해 비이상적인 글리코겐의 축적이 유의적으로 저해 되는 것을 관찰할 수 있었다(p<0.05). 위와 같은 실험 결과를 통하여 매생이 추출물과 PhA가 당뇨병성 신장손상에 보호 효과가 있다는 것을 확인하였다. In this study, we evaluated the effect of Capsosiphon fulvescens extract (CFE) and its active compound, pheophorbide A (PhA), on diabetic kidney failure. Diabetes mellitus (DM) was induced by a single intraperitoneal injection of streptozotocin (STZ; 40 ㎎/㎏ body weight (BW)). After a week, the rats were orally administered CFE (4 and 20 ㎎/㎏ BW) or PhA (0.2 ㎎/㎏ BW) once a day for 9 weeks. After scarification, renal tissue samples were collected for biochemical and histochemical analyses. Our study showed that the treatment with CFE and PhA significantly decreased lipid peroxidation level and the activities of glutathione peroxidase and glutathione-S-transferase (p<0.05), but it increased glutathione level and the activities of glutathione reductase, superoxide dismutase, and catalase in the renal tissues (p<0.05). The CFE- and PhA-treated rats with DM showed improved histochemical appearance and decreased abnormal glycogen accumulation. Therefore, we suggest that PhA-containing CFE could exert renal protective effects against STZinduced oxidative stress.

가자(Terminalia chebula Retz.) 추출물의 간섬유화 억제활성

이현순(Hyun-Sun Lee),구윤창(Yun-Chang Koo),이광원(Kwang-Won Lee) 한국식품과학회 2009 한국식품과학회지 Vol.41 No.5

간성상세포(HSC)는 간섬유화와 간경변에 중요한 역할을 한다고 알려져 있다. 간손상에 의해 둥근 모양의 간성상세포는 활성화되어 세포외기질(ECM)을 생산하는 myofibroblast와 같은 모양으로 활성화 된다. 활성화된 간성상세포의 특징은 빠른 증식 속도와 collagen과 같은 세포외 기질의 생산이다. 활성화된 간성상세포의 제거방법은 apoptosis를 유도하는 것이다. 가자 추출물은 정상 간세포(rat primary hepatocyte), 간세포주(HepG2) 및 활성화된 간성상세포주인 T-HSC/Cl-6에 1,000 μg/mL의 농도까지 처리하여 세포독성을 확인하였다. 그 결과 hepatocyte나 HepG2에서는 최고 농도에서도 독성이 없었으나 T-HSC/Cl-6는 U-shape 모양으로 사멸하는 것을 확인 하였다. T-HSC/Cl-6의 사멸이 apoptosis에 의한 것인지를 Annexin-V/PI double staining을 통하여 확인한 결과 apoptosis에 의해 T-HSC/Cl-6의 사멸이 일어나는 것을 확인하였다. Activation of hepatic stellate cells (HSCs) is known to be responsible for hepatic fibrosis and cirrhosis. When round-shape quiescent HSCs go to activation by liver injury, production of extracellular matrix is increased, and its shape becomes myofibroblast-like shape. The activated HSCs are characterized by the high rate of proliferation and the increased production of extracellular matrix. One way of the regeneration of activated HSCs is an apoptosis induction followed by removing the activated myofibroblast-like cells. The effect of extract of Terminalia chebula Retz. (TCE) on cytotoxicity was evaluated using the rat primary hepatocyte, HepG2 and T-HSC/Cl-6 by incubating these cells with TCE up to the dose of 1,000 μg/mL. At the maximum dose of TCE, no cytotoxicity was found on primary hepatocyte and HepG2, but cytotoxic effect of TCE was found on activated HSCs, and T-HSC/Cl-6 in a U-shaped dose-response manner with the highest effect at 500 μg/mL of TCE. Finally, we confirmed the occurrence of apoptotic cell death by annexin-V/PI double staining. The population of annexin-V positive cells was increased in a dose dependent manner.

생강나무(Lindera obtusiloba Blume) 잎 70% 에탄올 추출물의 단회와 14일 반복투여 독성시험의 안전성 평가

홍충의(Chung-Oui Hong),서문영(Young Seomun),구윤창(Yun-Chang Koo),남미현(Mi-Hyun Nam),이현아(Hyun Ah Lee),김지훈(Ji-Hoon Kim),왕증(Wang-Zeng),양성용(Sung Yong Yang),이성희(Sung-Hee Lee),노수환(Su Hwan No),이광원(Kwang-Won Lee) 한국식품영양과학회 2009 한국식품영양과학회지 Vol.38 No.10

생강나무는 한국, 일본, 중국 등지에서 자생하며 열, 복통, 타박상, 출혈 등에 민간요법으로 사용되어온 식물이다. 이 논문의 목적은 오래전부터 민간요법으로 사용되어온 생강나무 잎의 안전성을 평가하고자 생강나무 잎 70% 에탄올 추출물을 암수 각 군당 5마리씩 5000(단회투여 독성시험), 0, 500, 1000, 2000(14일 반복투여 독성시험) ㎎/㎏의 용량으로 단회경구투여와 14일 반복경구투여를 실시하여 15일간의 사망률, 일반증상, 체중변화, 조직병리, 혈액학적 및 혈액 생화학적 검사를 실시하였다. 시험기간 중 사망동물은 암수 모든 시험군에서 관찰되지 않았으며, 일반증상, 체중변화, 조직병리, 혈액학적 및 혈액생화학적 검사에서 투여와 관련된 변화는 관찰되지 않았다. 이상의 결과로 보아 생강나무의 단회투여 독성은 5000 ㎎/㎏ 용량을 상회하며, 14일 반복투여의 최고용량인 2000 ㎎/㎏ 용량에서도 아무런 독성이 나타나지 않는 것으로 보아 최대무독성량은 이 농도를 상회하는 것으로 판단된다. 이로써 생강나무는 안전한 소재로서 식재료 또는 건강기능식품의 원료로서도 사용이 가능하다고 볼 수 있다. Lindera obtusiloba Blume (LO), which is widely distributed in Korea, Japan and China, has traditionally been used as a popular folk medicine for the treatment of fever, abdominal pain, bruise and extravation. The purpose of this study was to examine the toxicities of the single and 14-day repeated doses in Sprague-Dawley rats orally administrated with LO at doses of 0, 500, 1000, 2000 (14-day repeated toxicity test) and 5000 (single toxicity test) mg (dry weight)/㎏ of body weight/day. The results showed that there was no difference in body weight change, food intake, water consumption, or organ weight among different dose groups. Also we observed no death and abnormal clinical signs during the experimental period. Between the groups orally administered LO and the control group, there was no statistical significance in hematological test or serum biochemical values. There were no evidences of histopathological alteration as well as abnormal gross finding mediated by single and 14 days treatments with LO. These results suggest that no observed adverse effect level (NOAEL) of the oral application of LO was considered to be more than 2000 mg/kg in rats under the conditions employed in this study.