[학위논문]미나리(Oenanthe javanica DC.)로부터 산화적 스트레스에 의한 간손상 보호 물질의 분리 및 기작 연구 In vitro and In vivo Hepatoprotective Effects of Oenanthe javanica DC. against Oxidative Stress원문보기
본 실험은 화순에서 생육되는 불미나리(Oenanthe javanica DC.)를 이용하여 산화적 손상으로부터 세포를 보호할 수 있는 물질을 찾아내고, 그 활성을 확인하는데 초점을 두었다. 미나리를 대상으로 4가지 용매(Hot-, Cold-water, Ethanol, Methanol)를 사용하여 추출을 실시하였고, 그 중 ...
본 실험은 화순에서 생육되는 불미나리(Oenanthe javanica DC.)를 이용하여 산화적 손상으로부터 세포를 보호할 수 있는 물질을 찾아내고, 그 활성을 확인하는데 초점을 두었다. 미나리를 대상으로 4가지 용매(Hot-, Cold-water, Ethanol, Methanol)를 사용하여 추출을 실시하였고, 그 중 에탄올 추출물이 가장 많은 페놀성 화합물을 함유하고 있으며, 가장 높은 항산화 활성을 보유하고 있음을 확인하였다. OJE는 추출물의 극성에 따라 hexane, chloroform, ethyl acetate, water 순으로 분획한 후 DPPH radical 과 super oxide anion radical 소거실험을 실시한 결과, 가장 높은 활성을 보인 ethyl acetate (OJE-E) 층을 대상으로 thin layer chromatography를 실시하여 높은 항산화 활성을 나타낸 밴드를 확인하고 HPLC로 분리하였다. 미나리의 활성물질은 caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid] 로 확인되었으며, caffeic acid 는 항산화 활성을 가진 물질의 하나로 커피콩, 감자, 채소 등에도 들어있는 것으로 알려져 있다. Caffeic acid 의 산화적 스트레스에 대한 간 보호효과를 알아보기 위해 HepG2 세포를 이용하여 in vitro assay 를 수행하였다. 세포생존 (viability) 정도를 측정하기 위해 XTT assay를 수행한 결과 caffeic acid (30~50 ug/mL)를 처리한 세포는 활성산소의 하나인 H₂O₂ 처리에 의한 세포 독성을 유의적으로 억제 하였으며, ROS 농도가 현저히 저하되었다. 산화적 스트레스에 대한 항산화 효과를 알아보기 위하여, MDA, GSH 함량과 SOD, GPx, Catalase, GST 활성을 측정하였다. 무처리 대조군과 H₂O₂ 만 처리한 군 (HPO), caffeic acid 20 ug/mL 과 H₂O₂ 를 처리한 군 (CAL) 그리고 caffeic acid 50 ug/mL 과 H₂O₂ 를 처리한 군(CAH)으로 나누어 실험하였다. 지질 과산화물인 MDA는 H₂O₂ 만을 처리한 HPO 군에서는 다른 실험군 들에 비하여 유의하게 증가하였으며 대조군과 caffeic acid 를 처리한 후 H₂O₂를 처리한 군(CAL, CAH) 간에는 유의차가 없었다. GSH 함량과 SOD, GPx, Catalase, GST 활성은 H₂O₂ 만을 처리한 HPO 군에서는 유의하게 낮은 활성을 보였고, Caffeic acid를 전처리 한 군은 대조군과 유의한 차이가 없었다. 이와 같은 결과는 caffeic acid의 처리가 세포의 산화적 스트레스를 억제하고 있음을 시사한다. 위와 같은 결과를 토대로 동물 실험을 진행하기위하여, 24마리의 8주령 수컷 rat을 대상으로 in vivo assay 를 수행하였다. Caffeic acid (10 mg/kg, 40 mg/kg)를 2주 동안 경구 투여하였으며, 마지막 투여 세 시간 후 CCl₄ 를 경구 투여하여 간독성을 유발시켰다. Rat 에서 수집한 혈액 분석 결과, CCl₄ 만 투여한 Rat 에서는 간독성 지표인 GOT 와 GPT 수치가 증가하였고 CCl₄ 투여 전 caffeic acid 를 경구 투여한 rat 에서는 그 수치가 유의적으로 감소하였으며, 세포 괴사도 감소하였다. Rat 의 간과 신장을 균질화 한 후 MDA, GSH 함량과 SOD, GPx, catalase, GST 활성을 측정하여 본 결과, MDA 함량은 CCl₄ 만 처리한 군에서 유의적으로 증가하였으며, GSH 함량과 SOD, GPx, catalase, GST 활성은 유의적으로 낮게 나타났다. 그러나 CCl₄ 투여 전caffeic acid 를 경구 투여한 rat 에서는 이 같은 효과가 보이지 않았으며, 대조군과의 유의차가 없었다. 본 연구의 결과로부터, 불미나리로부터 분리된 caffeic acid는 산화적손상으로부터 세포를 보호하며, CCl₄ 투여로 인해 발생하는 간독성에 대한 억제효과를 갖고있음을 확인하였다. 따라서, 불미나리 에탄올 추출물 또는 이로부터 분리된caffeic acid는 간세포를 보호하는 천연 항산화제로 활용될 수 있을 것으로 기대된다.
본 실험은 화순에서 생육되는 불미나리(Oenanthe javanica DC.)를 이용하여 산화적 손상으로부터 세포를 보호할 수 있는 물질을 찾아내고, 그 활성을 확인하는데 초점을 두었다. 미나리를 대상으로 4가지 용매(Hot-, Cold-water, Ethanol, Methanol)를 사용하여 추출을 실시하였고, 그 중 에탄올 추출물이 가장 많은 페놀성 화합물을 함유하고 있으며, 가장 높은 항산화 활성을 보유하고 있음을 확인하였다. OJE는 추출물의 극성에 따라 hexane, chloroform, ethyl acetate, water 순으로 분획한 후 DPPH radical 과 super oxide anion radical 소거실험을 실시한 결과, 가장 높은 활성을 보인 ethyl acetate (OJE-E) 층을 대상으로 thin layer chromatography를 실시하여 높은 항산화 활성을 나타낸 밴드를 확인하고 HPLC로 분리하였다. 미나리의 활성물질은 caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid] 로 확인되었으며, caffeic acid 는 항산화 활성을 가진 물질의 하나로 커피콩, 감자, 채소 등에도 들어있는 것으로 알려져 있다. Caffeic acid 의 산화적 스트레스에 대한 간 보호효과를 알아보기 위해 HepG2 세포를 이용하여 in vitro assay 를 수행하였다. 세포생존 (viability) 정도를 측정하기 위해 XTT assay를 수행한 결과 caffeic acid (30~50 ug/mL)를 처리한 세포는 활성산소의 하나인 H₂O₂ 처리에 의한 세포 독성을 유의적으로 억제 하였으며, ROS 농도가 현저히 저하되었다. 산화적 스트레스에 대한 항산화 효과를 알아보기 위하여, MDA, GSH 함량과 SOD, GPx, Catalase, GST 활성을 측정하였다. 무처리 대조군과 H₂O₂ 만 처리한 군 (HPO), caffeic acid 20 ug/mL 과 H₂O₂ 를 처리한 군 (CAL) 그리고 caffeic acid 50 ug/mL 과 H₂O₂ 를 처리한 군(CAH)으로 나누어 실험하였다. 지질 과산화물인 MDA는 H₂O₂ 만을 처리한 HPO 군에서는 다른 실험군 들에 비하여 유의하게 증가하였으며 대조군과 caffeic acid 를 처리한 후 H₂O₂를 처리한 군(CAL, CAH) 간에는 유의차가 없었다. GSH 함량과 SOD, GPx, Catalase, GST 활성은 H₂O₂ 만을 처리한 HPO 군에서는 유의하게 낮은 활성을 보였고, Caffeic acid를 전처리 한 군은 대조군과 유의한 차이가 없었다. 이와 같은 결과는 caffeic acid의 처리가 세포의 산화적 스트레스를 억제하고 있음을 시사한다. 위와 같은 결과를 토대로 동물 실험을 진행하기위하여, 24마리의 8주령 수컷 rat을 대상으로 in vivo assay 를 수행하였다. Caffeic acid (10 mg/kg, 40 mg/kg)를 2주 동안 경구 투여하였으며, 마지막 투여 세 시간 후 CCl₄ 를 경구 투여하여 간독성을 유발시켰다. Rat 에서 수집한 혈액 분석 결과, CCl₄ 만 투여한 Rat 에서는 간독성 지표인 GOT 와 GPT 수치가 증가하였고 CCl₄ 투여 전 caffeic acid 를 경구 투여한 rat 에서는 그 수치가 유의적으로 감소하였으며, 세포 괴사도 감소하였다. Rat 의 간과 신장을 균질화 한 후 MDA, GSH 함량과 SOD, GPx, catalase, GST 활성을 측정하여 본 결과, MDA 함량은 CCl₄ 만 처리한 군에서 유의적으로 증가하였으며, GSH 함량과 SOD, GPx, catalase, GST 활성은 유의적으로 낮게 나타났다. 그러나 CCl₄ 투여 전caffeic acid 를 경구 투여한 rat 에서는 이 같은 효과가 보이지 않았으며, 대조군과의 유의차가 없었다. 본 연구의 결과로부터, 불미나리로부터 분리된 caffeic acid는 산화적손상으로부터 세포를 보호하며, CCl₄ 투여로 인해 발생하는 간독성에 대한 억제효과를 갖고있음을 확인하였다. 따라서, 불미나리 에탄올 추출물 또는 이로부터 분리된caffeic acid는 간세포를 보호하는 천연 항산화제로 활용될 수 있을 것으로 기대된다.
The present study was conducted to investigate the hepatoprotective effects of Oenanthe javanica DC. (OJ) against oxidative stress in two experimental models, hepG2 cells for in vitro and rats for in vivo. The OJ was extracted by hot water (OJH), cold water (OJC), 80% EtOH (OJE) and MeOH (OJM). Of f...
The present study was conducted to investigate the hepatoprotective effects of Oenanthe javanica DC. (OJ) against oxidative stress in two experimental models, hepG2 cells for in vitro and rats for in vivo. The OJ was extracted by hot water (OJH), cold water (OJC), 80% EtOH (OJE) and MeOH (OJM). Of four extracts, OJE contained the relatively large phenolic compound. It also exihibited the high DPPH radical- and superoxide anion radical- scavenging activities with the inhibition of lipid peroxidation. To determine the in vitro hepatoprotective effects of OJE and OJH, they were treated to HepG2 cells in the presence of H₂O₂. Compared to the cells with the treatment of OJH, OJE treated cells showde the high viability, suggesting OJE as a better hepatoprotectant. Since OJE had the high hepatoprotective pontential, it was further fractionated by partitioning with n-hexane (OJE-H), chloroform (OJE-C), ethyl acetate (OJE-E) and water (OJE-W). In this stage, OJE-E showed the highest amount of phenolic compound and the strongest antioxidative activities among the other fractions. The fact that OJE and OJE-E revealed the high antioxidant activities, indicates that they have a high concentration of phenolic compounds, which are responsible for their high antioxidant activities. Also, OJE-E exhibited the protective effect against oxidative stress induced by 2mM H₂O₂ in HepG2 cells. Therefore, OJE-E was employed to the next study for the purification of hepatoprotectant by TLC and HPLC. The hepatoprotectant from OJ was identified as caffeic acid (CA). Caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid], which is a hydroxycinamic acid derivative, is one of the antioxidative compounds in various agriculture products such as coffee beans, potatoes, grains and vegetables. To determine the antioxidant activities of CA, the DPPH radical- and superoxide anion radical-scavenging activities and the TBARS generation inhibitory activities were examind. In DPPH radical scavenging activity assay, IC50 was 5.35 ug/mL, and the inhibitory effects on TBARS generation was 76.8% at a dose of 80 ug/mL. To investigate the hepatoprotective effects of CA, assays for the H₂O₂-induced cytotoxicity and the formation of intracellular reactive oxygen species were performed in HepG2 cells. The pretreatment for 24 h with 30~50 ug/mL of CA completely prevented the oxidative stress induced by H₂O₂. Also, less reactive oxygen species were intracellularly formed with the treatment of CA. When CA was treated in HepG2 cells, the changes in enzymatic antioxidative activities such as superoxide dismutase (SOD), catalase, glutathione-S-transferase (GST), and glutathione peroxidase (GSH-Px) and non-enzymatic antioxidative activities such as glutathion (GSH), and malondialdehyde (MDA) were examined. The cells were divided into 4 groups; ⅰ) control, ⅱ) 2 mM H₂O₂, and ⅲ) 2mM H₂O₂ plus 10 ug/mL of CA, and ⅳ) 2 mM H₂O₂ plus 40 ug/mL of CA. The activities of SOD, catalase, GST, GSH-Px and GSH levels in the H₂O₂ group were significantly lower than those of the control group, while H₂O₂ plus CA group stimulated the hepatic enzyme activities and GSH level. The level of MDA which had been increased by H₂O₂ were markedly decreased in the CA pre-treated group. From these results, we recognized that CA might be considered as a useful agent in the prevention of various liver injuries induced by oxidative stress. Therefore, the capability of CA to protect against CCl₄-induced hepatotoxicity and oxidative stress was investigated in rats. In this study, the model of CCl₄-induced acute hepatotoxicity in rats, was employed because this chemical is a potent hepatotoxin and a single exposure can rapidly lead to severe hepatic necrosis and steatosis. As expected, a single oral dose of CCl₄ at 1.25 mL/kg showed the significant hepatotoxicity, as evidenced by a dramatic elevation in the serum GOT and GPT activities and an increased incidence and severity of histopathological hepatic lesions in rats. In addition, CCl₄ treatment produced high levels of oxidative damage, as evidenced by a significant elevation in hepatic MDA level and a significant decrease in GSH concentration and catalase, SOD, GST, and GPx activities, which suggest a role of oxidative stress in CCl₄ hepatotoxicity. However, pre-treatment with CA showed a significant protective effect against CCl₄-induced acute hepatotoxicity and oxidative stress in rats. A single oral dose of CCl₄ at 1.25 mL/kg resulted in a significant increase in the hepatic MDA concentration, indicating increased lipid peroxidation caused by administration of CCl₄. The significant decrease in the hepatic MDA concentration confirms that pretreatment with CA could effectively protect against the hepatic lipid peroxidation induced by CCl₄. Based upon these results, CA from OJ had protective action against H₂O₂-induced hepatotoxicity in vitro and CCl₄-induced hepatotoxicity in vitro, indicating CA as a useful hepatoprotectant against various liver diseases induced by oxidative stress.
The present study was conducted to investigate the hepatoprotective effects of Oenanthe javanica DC. (OJ) against oxidative stress in two experimental models, hepG2 cells for in vitro and rats for in vivo. The OJ was extracted by hot water (OJH), cold water (OJC), 80% EtOH (OJE) and MeOH (OJM). Of four extracts, OJE contained the relatively large phenolic compound. It also exihibited the high DPPH radical- and superoxide anion radical- scavenging activities with the inhibition of lipid peroxidation. To determine the in vitro hepatoprotective effects of OJE and OJH, they were treated to HepG2 cells in the presence of H₂O₂. Compared to the cells with the treatment of OJH, OJE treated cells showde the high viability, suggesting OJE as a better hepatoprotectant. Since OJE had the high hepatoprotective pontential, it was further fractionated by partitioning with n-hexane (OJE-H), chloroform (OJE-C), ethyl acetate (OJE-E) and water (OJE-W). In this stage, OJE-E showed the highest amount of phenolic compound and the strongest antioxidative activities among the other fractions. The fact that OJE and OJE-E revealed the high antioxidant activities, indicates that they have a high concentration of phenolic compounds, which are responsible for their high antioxidant activities. Also, OJE-E exhibited the protective effect against oxidative stress induced by 2mM H₂O₂ in HepG2 cells. Therefore, OJE-E was employed to the next study for the purification of hepatoprotectant by TLC and HPLC. The hepatoprotectant from OJ was identified as caffeic acid (CA). Caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid], which is a hydroxycinamic acid derivative, is one of the antioxidative compounds in various agriculture products such as coffee beans, potatoes, grains and vegetables. To determine the antioxidant activities of CA, the DPPH radical- and superoxide anion radical-scavenging activities and the TBARS generation inhibitory activities were examind. In DPPH radical scavenging activity assay, IC50 was 5.35 ug/mL, and the inhibitory effects on TBARS generation was 76.8% at a dose of 80 ug/mL. To investigate the hepatoprotective effects of CA, assays for the H₂O₂-induced cytotoxicity and the formation of intracellular reactive oxygen species were performed in HepG2 cells. The pretreatment for 24 h with 30~50 ug/mL of CA completely prevented the oxidative stress induced by H₂O₂. Also, less reactive oxygen species were intracellularly formed with the treatment of CA. When CA was treated in HepG2 cells, the changes in enzymatic antioxidative activities such as superoxide dismutase (SOD), catalase, glutathione-S-transferase (GST), and glutathione peroxidase (GSH-Px) and non-enzymatic antioxidative activities such as glutathion (GSH), and malondialdehyde (MDA) were examined. The cells were divided into 4 groups; ⅰ) control, ⅱ) 2 mM H₂O₂, and ⅲ) 2mM H₂O₂ plus 10 ug/mL of CA, and ⅳ) 2 mM H₂O₂ plus 40 ug/mL of CA. The activities of SOD, catalase, GST, GSH-Px and GSH levels in the H₂O₂ group were significantly lower than those of the control group, while H₂O₂ plus CA group stimulated the hepatic enzyme activities and GSH level. The level of MDA which had been increased by H₂O₂ were markedly decreased in the CA pre-treated group. From these results, we recognized that CA might be considered as a useful agent in the prevention of various liver injuries induced by oxidative stress. Therefore, the capability of CA to protect against CCl₄-induced hepatotoxicity and oxidative stress was investigated in rats. In this study, the model of CCl₄-induced acute hepatotoxicity in rats, was employed because this chemical is a potent hepatotoxin and a single exposure can rapidly lead to severe hepatic necrosis and steatosis. As expected, a single oral dose of CCl₄ at 1.25 mL/kg showed the significant hepatotoxicity, as evidenced by a dramatic elevation in the serum GOT and GPT activities and an increased incidence and severity of histopathological hepatic lesions in rats. In addition, CCl₄ treatment produced high levels of oxidative damage, as evidenced by a significant elevation in hepatic MDA level and a significant decrease in GSH concentration and catalase, SOD, GST, and GPx activities, which suggest a role of oxidative stress in CCl₄ hepatotoxicity. However, pre-treatment with CA showed a significant protective effect against CCl₄-induced acute hepatotoxicity and oxidative stress in rats. A single oral dose of CCl₄ at 1.25 mL/kg resulted in a significant increase in the hepatic MDA concentration, indicating increased lipid peroxidation caused by administration of CCl₄. The significant decrease in the hepatic MDA concentration confirms that pretreatment with CA could effectively protect against the hepatic lipid peroxidation induced by CCl₄. Based upon these results, CA from OJ had protective action against H₂O₂-induced hepatotoxicity in vitro and CCl₄-induced hepatotoxicity in vitro, indicating CA as a useful hepatoprotectant against various liver diseases induced by oxidative stress.
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