• 한국식품과학회지
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• 제37권6호
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• pp.983-988
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• 2005

제주자생 재래감귤종이 하나인 진귤(Citrus sunki Hort. ex Tanaka)의 과피는 전통적으로 매우 중요한 한약재 성분으로 사용되어 왔으나 그 약리학적 효과에 대해서는 과학적인 분석이 되어 있지 못하다. 본 연구에서는 진귤과피추출물과 과피발효 추출물의 1차 항산화활성을 검색하여 발효 후 추출물이 더욱 효과적인 활성을 가지고 있는 사실을 발견하였고 이를 바탕으로 대식세포인 Raw264.7세포에서 산화질소의 생성, 염증유발 단백질(NOS2, Cox-2)의 수준을 억제할 뿐 아니라 동 세포의 생존능을 개선시키는 결과를 얻었다. 그러나 상피세포유래 세포주인 CHO-IR 세포 및 사람의 간암세포주인 HepG2 세포의 생존능은 반대로 발효후 추출물에 의해 억제되는 것으로 나타났다. 이러한 결과들은 진피의 발효후 추출물이 대식세포의 항염증활성을 증가시키는 반면, 종양세포의 증식을 억제하고 세포사멸을 유도하는 다양한 약리효과를 가지고 있음을 의미한다.

• 한방안이비인후피부과학회지
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• 제34권4호
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• pp.24-36
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• 2021

Objectives : This study was conducted to confirm the anti-oxidative effect of Chungpyesagan-tang(CPSGT) extract. Methods : In this study, MTT assay was performed to confirm cell viability, and DPPH and ABTS were performed to confirm radical scavenging ability. The ROS scavenging ability and the protective effect against DNA damage were confirmed by 2,7-dichlorofluorescin diacetate(DCF-DA) and 4',6-diamidino-2-phenylindole(DAPI) staining and comet assay. mRNA expression of Heme oxygenase-1(HO-1) was measured by real-time PCR, and expression of HO-1 and Kelch-like ECH-associated protein 1(Keap1) proteins was measured by western blot. Results : CPSGT was not cytotoxic at 50-400㎍/㎖. The radical scavenging activity was increased, and the ROS scavenging activity and the protective effect against DNA damage were increased compared to the LPS-treated group. The mRNA expression and protein expression of HO-1 were increased in a concentration-dependent manner. The protein expression level of Keap1 was decreased in a concentration-dependent manner. Conclusion : This suggests that CPSGT has an antioxidant effect and can be used as a potential material for skin diseases.

• Journal of Applied Biological Chemistry
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• 제63권3호
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• pp.283-290
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• 2020

Oxidative stress is one of the contributors of neurodegenerative disorders including Alzheimer's disease. According to previous studies, Aster yomena (Kitam.) Honda (AY) possesses variable pharmacological activities including anti-coagulant and anti-obesity effect. In this study, we aimed to determine the neuroprotective effect of ethyl acetate fraction from Aster yomena (Kitam.) Honda (EFAY) against oxidative stress. Therefore, we carried out 3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyl tetrazolium bromide, lactate dehydrogenase (LDH), and 2',7'-dichlorofluorescin diacetate assays in SH-SY5Y neuronal cells treated with hydrogen peroxide (H₂O₂). H₂O₂-treated control cells exhibited reduced viability of cells, and increased LDH release and reactive oxygen species (ROS) production compared to normal cells. However, treatment with EFAY restored the cell viability and inhibited LDH release and ROS production. To investigate the underlying mechanisms by which EFAY attenuated neuronal oxidative damage, we measured protein expressions using Western blot analysis. Consequently, it was observed that EFAY down-regulated cyclooxygenase-2 and interleukin-1β protein expressions in H₂O₂-treated SH-SY5Y cells that mediated inflammatory reaction. In addition, apoptosis-related proteins including B-cell lymphoma-2-associated X protein/B-cell lymphoma-2 ratio, cleaved caspase-9, and cleaved-poly (ADP-ribose) polymerase protein expressions were suppressed when H₂O₂-treated cells were exposed to EFAY. Our results indicate that EFAY ameliorated H₂O₂-induced neuronal damage by regulating inflammation and apoptosis. Altogether, AY could be potential therapeutic agent for neurodegenerative diseases.

• 농업과학연구
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• 제48권1호
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• pp.119-131
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• 2021

Over-produced reactive oxygen species (ROS) exert oxidative damage on lipids, proteins, and DNA in the human body, which leads to the onset of neurodegenerative diseases such as Alzheimer's disease (AD). In this study, we explored the cellular antioxidant effect of Cirsium japonicum var. maackii (CJM) against hydrogen peroxide (H₂O₂)-induced oxidative stress in neuronal cells. The antioxidant activity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 2',7'-dichlorofluorescin diacetate and nitric oxide (NO) assays, and the molecular mechanisms were examined by Western blot analysis. H₂O₂ treatment of SH-SY5Y cells decreased cell viability and increased ROS and NO production compared to H₂O₂-untreated cells. However, CJM increased cell viability and decreased ROS and NO accumulation in the H₂O₂-treated SH-SY5Y cells compared to H₂O₂-treated control cells. Especially, the EtOAc fraction from CJM showed the strongest antioxidant effect compared with the other extracts and fractions. Therefore, we further examined the CJM mechanism against oxidative stress using the EtOAc fraction from CJM. The EtOAc fraction up-regulated the expressions of heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and thioredoxin reductase 1. These results indicate that CJM promotes the activation of antioxidative enzymes, which eliminate ROS and NO, and further leads to an increase in the cell viability. Taken together, our results show that CJM exhibited an antioxidant activity in H₂O₂-treated SH-SY5Y cells, and it could be a novel antioxidant agent for the prevention or treatment of neurodegenerative disease such as AD.

• 대한화장품학회지
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• 제28권1호
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• pp.42-57
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• 2002

본 연구는 널리 알려져 있는 항산화제들의 세포 수준에서의 anti-oxidative stress 효과 및 그 기작을 알아보기 위한 연구이다. 연구에 사용한 항산화제로는 지용성인 retinol, $\alpha$-tocopherol, propyl gallate(PG) 및 butylated hydroxy toluene(BHT)과 수용성인 ascorbic acid, $\alpha$-glucosyl rutin 및 green tea extract를 사용하였으며 이들 항산화제들의 시간별 세포 생존율을 NR assay 로 측정한 후 적정 농도에서 DCFH-DA(2', 7'-dichlorofluorescin-diacetate) 를 이용하여 항산화제들의 anti-oxidative stress 억제 효과를 시간별로 측정하였다. 또한 이들 항산화제의 항산화 기작을 알아보기 위하여 NBT(Nitro-blue-tetrazolium) 및 DPPH(Diphenyl-picry-hydrazl)도 병행하여 실시하였다. Anti-oxidative stress 실험에서 지용성 항산화제들은 전반적으로 수용성 항산화제에 비하여 세포에 대한 독성이 상대적으로 강하여 retinol 의 경우에는 0.01%에서 oxidative stress 억제 효과를 관할할 수 있었으며 1 시간경과 후 측정시 53.1%의 억제 효과를 보여 주었다. PG 의 경우에는 0.1%에서 2 시간 경과 후 측정시 50%의 oxidative stress 억제 효과를 보여주었다. 수용성 항산화제인 green tea extract 및 $\alpha$-glucosyl rutin의 경우에는 1%에서 1시간 경과 후 측정시 각 각 51.6% 및 69.7%의 oxidative stress 억제 효과를 관찰할 수 있었다. 또한 시료처리 후 자외선 조사시 oxidative stress 억제 효과의 경우 수용성 항산화제인 ascorbic acid, $\alpha$-glucosyl rutin 및 green tea extract 와 지용성 항산화제 중에서는 $\delta$-tocopherol 에서만 oxidative stress 억제 효과가 관찰되었으나 자외선을 조사 하지 않았을 때 보다 약 20%-40%까지 억제 효과가 감소되었다. 그리고 PG 및 retinol 의 경우에는 자외선 조사시 독성이 증가하여 oxidative stress 억제 효과를 측정할 수 없었다. NBT실험에서 $\alpha$-glucosyl rutin, $\alpha$-tocopherol 및 PG 1%에서 70%이상의 superoxide anion 생성 억제 효과를 보였으며 DPPH 실험에서는 ascorbic acid 와 PG 1%에서 98%의 hydroxyl radical 생성 억제 효과를 보여 주었다. 본 실험을 통하여 BHT 를 제외하고 전반적으로 세포 수준에서의 oxidative stress 에 대한 억제 효과를 확인해 볼 수 있었으며 특히 수용성 항산화제들에서 두드러진 효과를 보여 주었다.

• Preventive Nutrition and Food Science
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• 제9권2호
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• pp.150-155
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• 2004

The free radical scavenging activities and the protective effects of Rhus javanica extracts against oxidative damage induced by reactive oxygen species (ROS) were investigated. n-Hexane, ethyl acetate and water fractions were prepared from a methanol extract. DPPH radical, superoxide anion and hydroxyl radical scavenging activities were estimated. Intracellular ROS formation was quantified using fluorescent probes, 2', 7'-dichlorofluorescin diacetate (DCFH-DA) for hydroxyl radical and dihydroethidium (DHE) for superoxide anion. The oxidative DNA damage was investigated by the comet assay in HepG$_2$ cells exposed either to $H_2O$$_2$ or to menadione. The highest $IC_{50}$/ values for DPPH radical scavenging activity was found in the ethyl acetate fraction with a value of 5.38 $\mu\textrm{g}$/mL. Cells pretreated with $\geq$ 1 $\mu\textrm{g}$/mL of the ethyl acetate extract had significantly increased cell viability compared to control cells, which were not pretreated with the extract. Intracellular ROS formation and DNA damage in HepG$_2$ cells, which were pretreated with the various concentrations of Rhus javanica ethyl acetate extract and then incubated either with $H_2O$$_2$ or with menadione, reduced in a dose-dependent manner. These findings suggest that Rhus javanica might have biologically active components which have strong protective effects against ROS induced oxidative damages to the biomolecules, such as cell membranes and DNA.

• The Korean Journal of Physiology and Pharmacology
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• 제15권6호
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• pp.319-326
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• 2011

Quercetin-3-O-${\beta}$-D-glucuronopyranoside (QGC) is a flavonoid glucoside extracted from Rumex Aquaticus Herba. We aimed to explore its protective effect against ethanol-induced cell damage and the mechanism involved in the effect in feline esophageal epithelial cells (EEC). Cell viability was tested and 2',7'-dichlorofluorescin diacetate assay was used to detect intracellular $H_2O_2$ production. Western blotting analysis was performed to investigate MAPK activation and interleukin 6 (IL-6) expression. Exposure of cells to 10% ethanol time-dependently decreased cell viability. Notably, exposure to ethanol for 30 min decreased cell viability to 43.4%. When cells were incubated with $50{\mu}M$ QGC for 12 h prior to and during ethanol treatment, cell viability was increased to 65%. QGC also inhibited the $H_2O_2$ production and activation of ERK 1/2 induced by ethanol. Pretreatment of cells with the NADPH oxidase inhibitor, diphenylene iodonium, also inhibited the ethanol-induced ERK 1/2 activation. Treatment of cells with ethanol for 30 or 60 min in the absence or presence of QGC exhibited no changes in the IL-6 expression or release compared to control. Taken together, the data indicate that the cytoprotective effect of QGC against ethanol-induced cell damage may involve inhibition of ROS generation and downstream activation of the ERK 1/2 in feline EEC.

• International Journal of Oral Biology
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• 제38권1호
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• pp.5-12
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• 2013

Recent studies indicate that reactive oxygen species (ROS) can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In this study, we investigated the effects of NaOCl, a ROS donor, on neuronal excitability and the intracellular calcium concentration ($[Ca^{2+}]_i$) in spinal substantia gelatinosa (SG) neurons. In current clamp conditions, the application of NaOCl caused a membrane depolarization, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN), a ROS scavenger. The NaOCl-induced depolarization was not blocked however by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Confocal scanning laser microscopy was used to confirm whether NaOCl increases the intracellular ROS level. ROS-induced fluorescence intensity was found to be increased during perfusion of NaOCl after the loading of 2',7'-dichlorofluorescin diacetate ($H_2DCF$-DA). NaOCl-induced depolarization was not blocked by pretreatment with external $Ca^{2+}$ free solution or by the addition of nifedifine. However, when slices were pretreated with the $Ca^{2+}$ ATPase inhibitor thapsigargin, NaOCl failed to induce membrane depolarization. In a calcium imaging technique using the $Ca^{2+}$-sensitive fluorescence dye fura-2, the $[Ca^{2+}]_i$ was found to be increased by NaOCl. These results indicate that NaOCl activates the excitability of SG neurons via the modulation of the intracellular calcium concentration, and suggest that ROS induces nociception through a central sensitization.

• Journal of Applied Biological Chemistry
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• 제62권4호
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• pp.347-353
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• 2019

Stings of jellyfish, which frequently occur in a warm season, cause severe pain, inflammation and sometimes irreversible results such as the death. Harmful venoms from jellyfish, therefore, have been studied for finding the therapeutic agents to relieve pain or to neutralize toxic components. However, it is still unclear if and how jellyfish venom reveal neuronal toxicity even though pain induction seems to result from the activation of nociceptors such as nerve endings. In this study, using HT-22 cell line, we investigated neurotoxic effects of the venom of Chrysaora pacifica (CpV) which appears in South-East ocean of Korea. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, CpV significantly reduced the viability of HT-22 cells in a dose-dependent manner. Additionally, in 2',7'-Dichlorofluorescin diacetate fluorescence test under the culture condition lacking dominant inflammatory factors, CpV remarkably increased the production of intracellular reactive oxygen species (ROS). Reduced responsive fluorescence to Rhodamine123 and increased expression of intracellular cytochrome c were also observed in HT-22 cells treated with CpV. These indicate that CpV-reduced viability of HT-22 cells may be due to the activation of apoptotic signalings mediated with oxidative stress and mitochondrial dysfunction. Furthermore, removing Ca²⁺ ion or adding N-acetyl-Lcystein remarkably blocked the CpV effect to reduce the viability of HT-22 cells. The findings in this study clearly demonstrate that CpV may activate Ca²⁺-mediated ROS signalings and mitochondrial dysfunction resulting in neuronal damage or death, and suggest that blocking Ca²⁺ pathway is a therapeutic approach to possibly block toxic effects of jellyfish venoms.

• Journal of Nutrition and Health
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• 제48권5호
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• pp.451-456
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• 2015

Purpose: Neuronal apoptotic events induced by aging and hypoxic/ischemic conditions is an important risk factor in neurodegenerative diseases such as ischemia stroke and Alzheimer's disease. The peel of Citrus sunki Hort. ex Tanaka has long been used as a traditional medicine, based on multiple biological activities including anti-oxidant, anti-inflammation, and anti-obesity. In the current study, we examined the actions of fermented C. sunki peel extract against cobalt chloride ($CoCl_2$)-mediated hypoxic death in human neuroblastoma SH-SY5Y cells. Methods: Cell viability was measured by trypan blue exclusion. Expression of apoptosis related proteins and release of cytochrome c were detected by western blot. Production of intracellular reactive oxygen species (ROS) and apoptotic morphology were examined using 2',7'-dichlorofluorescin diacetate (DCF-DA) and 4',6-diamidino-2-phenylindole (DAPI) staining. Results: Exposure to $CoCl_2$, a well-known mimetic agent of hypoxic/ischemic condition, resulted in neuronal cell death via caspase-3 dependent pathway. Extract of fermented C. sunki peel significantly rescued the $CoCl_2$-induced neuronal toxicity with the cell viability and appearance of apoptotic morphology. Cytoprotection with fermented C. sunki peel extract was associated with a decrease in activities of caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). In addition, increase in the intracellular ROS and release of cytochrome c from mitochondria to the cytosol were inhibited by treatment with extract of fermented C. sunki peel. Conclusion: Based on these data, fermented C. sunki peel extract might have a protective effect against $CoCl_2$-induced neuronal injury partly through generation of ROS and effectors involved in mitochondrial mediated apoptosis.