• Molecules and Cells
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• 제24권1호
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• pp.113-118
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• 2007

The brains of Alzheimer's disease (AD) patients are characterized by large deposits of amyloid beta peptide ($A{\beta}$). $A{\beta}$ is known to increase free radical production in nerve cells, leading to cell death that is characterized by lipid peroxidation, free radical formation, protein oxidation, and DNA/RNA oxidation. In this study, we selected an extract of Gardenia jasminoides by screening, and investigated its ameliorating effects on $A{\beta}$-induced oxidative stress using PC12 cells. The effects of the extract were evaluated using the 2',7'-dichlorofluorescein diacetate (DCF-DA) assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. To find the active component, the ethanol extract was partitioned with hexane, chloroform, and ethyl acetate, respectively, and the active component was purified by silica-gel column chromatography and HPLC. The results suggested that Gardenia jasminoides extract can reduce the cytotoxicity of $A{\beta}$ in PC 12 cells, possibly by reducing oxidative stress.

• 대한한의학회지
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• 제32권1호
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• pp.109-120
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• 2011

Objectives: The aim of this investigation was to evaluate the efficacy of KHchunggan-tang aqueous extract on the experimental nonalcoholic fatty liver disease(NAFLD) induced by palmitate. Materials and Methods: To generate a cellular model of NAFLD, we used HepG2 cells, a human hepatoma cell line, treated with 0.5 mM palmitate. By this cellular model, effects of KHchunggan-tang aqueous extract were evaluated. Intracellular lipid accumulation, free radical formation, and apoptosis were detected by Nile red staining, 2',7'-dichloroflourescin diacetate(H2DCF-DA), and 4',6-diamidino-2-phenylindole(DAPI)/propidium iodide(PI) staining, respectively. Some proteins related with NAFLD were determined by western blot. Results: Typical pathological features of NAFLD occurred in the cellular model. Palmitate increased the levels of intracellular lipid vacuoles, decreased cell viability, and increased apoptosis. Palmitate increased free radical formation and lipid peroxidation, too. However, KHchunggan-tang aqueous extract reduced palmitate-induced pathologic features, i.e. steatosis, free radical formation, and apoptosis. In addition, KHchunggan-tang aqueous extract suppressed palmitate-activated c-Jun N-terminal kinase(JNK) signaling, and SP600125, a JNK inhibitor, significantly reversed the palmitate-induced pathologic changes as KHchunggan-tang aqueous extract. It means that the signaling pathway other than JNK can be involved in the KHchunggan-tang mediated cellular protection of palmitate-treated Hep G2 cells. Conclusions: These results suggest that KHchunggan-tang aqueous extract has hepatoprotective effects on NAFLD with combined properties in cellular steatosis, ROS production, and cytoprotection, and thus may have valuable clinical applications for treatment of this chronic liver disease.

• 대한본초학회지
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• 제23권4호
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• pp.121-134
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• 2008

Objectives: Melia toosendan(MT) has been used as a traditional Korean herbal medicine, and today it is used as a medication for colic, side aches, heartache and other disorders of liver. The aim of this study was to determine whether fractionated extracts of MT inhibit free radical generation such as DPPH radical, superoxide radical and nitric oxide, production of nitrite, an index of NO, PGE2, iNOS, COX-2 and proinflammatory cytokines in lipopolysaccharide(LPS)-treated RAW 264.7 macrophages. Methods: MT extract prepared with methanol, and then fractionated with hexane, dichloromethane, ethylacetate, n-butanol and water. Inhibitory effect of MT onto free radical generation was determined by measuring DPPH, superoxide anions and nitric oxide scavenging activities in vitro. Cytotoxic activity of extracts on RAW 264.7 cells was measured using 5-(3-caroboxymeth-oxyphenyl)-2H-tetra-zolium inner salt(MTS) assay. Intracelluar oxidation was analysed by DCF-DA assay. The nitric oxide(NO) production was measured by Griess reagent system. The levels of iNOS and COX-2 expression were confirmed by western blot. And pro inflammatory cytokines were measured by ELISA kit. Results: Our results indicated that fractionated extracts, especially dichloromethane and ethyl acetate extracts, significantly inhibited free radical generation, the LPS-induced $H_2O_2$, NO, $PGE_2$ production and iNOS, COX-2 expression accompanied by an attenuation of TNF-${\alpha}$, IL-1${\beta}$ and IL-6 formation in macrophages. Conclusions: These results indicate that dichloromethane and ethyl acetate extracts of MT have potential as an agent of chronic inflammatory diseases.

• 생약학회지
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• 제43권2호
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• pp.107-121
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• 2012

Twenty-five compounds were isolated from the methanolic extract of Geum japonicum (Rosaceae), and their structures were identified as eleven triterpenoids [ursolic acid 3-acetate (2), cecropiacic acid 3-methyl ester (3), pomolic acid 3-acetate (5), ursonic acid (6), ursolic acid (7), pomolic acid (8), corosolic acid (9), euscaphic acid (11), arjunic acid (16), tormentic acid (18), 23-hydroxytormentic acid (21)], two saponins [rosamultin (22) and kaji-ichigoside $F_1$ (23)], two megastigmanes [blumenol A (14) and (+)-dehydrovomifoliol (15)], three flavonoids [apigenin (13), isoquercitrin (17) and tiliroside (24)], two ellagic acid derivatives [3,3'-di-O-methylellagic acid (12) and ducheside B (25)] and five others [eugenol (1), emodin (4), vanillic acid (10), gallic aldehyde (19), salidroside (20)]. The chemical structures of these compounds were identified on the basis of spectroscopic methods and comparison with literature values. This is the first report of the eleven compounds, 2~6, 10, 15, 16, 20, 23, and 25 from the genus Geum, as well as the first report of apigenin (13) and 3,3'-di-O-methylellagic acid (12) from G. japonicum. The antioxidant properties of 22 isolates (1~11, 14, 16~25) were evaluated by the intracellular reactive oxygen species (ROS) radical scavenging using 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) assay. Among them, isoquercitrin (17) showed significant scavenging activity, and gallic aldehyde (19) and ducheside B (25) showed weak scavenging activity.

• 동의생리병리학회지
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• 제19권6호
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• pp.1615-1621
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• 2005

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are widely implicated in the aging process and age-related diseases. The present study was carried out to investigate scavenging activities of Junglans sinensis extract and its subfraction using fluorescent probes, DCF-DA, DAF-2 and DHR 123. Jungians sinensis was washed and crushed. The crushed Junglans sinensis was extracted 3times, each time with 3 volumes of methyl alcohol at $60^{\circ}C$ for 24 h. The extract was filtered and evaporated under a reduced pressure using a rotary evaporator to yield 16 g. Scavenging activities of $ONOO^-$ was measured by Kooy' method and ROS was measured by DCFDA assay. Junglans sinensis had the marked scavenging activites of $ONOO^-$, NO and $O_2^-$. Junglans sinensis scavenged $ONOO^-$ through electron donation and dose-dependently inhibited the nitration of bovine serum albumin by $ONOO^-$. Junglans sinensis also had ROS scavenging activity. Especially, ethylacetate fraction of Junglans sinensis showed the most effective scavenging activities for ROS and RNS. These results suggest that Junglans sinensis might be developed as an effective ROS and RNS scavenger Therefore, Junglans sinensis might be used as a preventive agent for the aging and relevant to aging of illness.

• Food Science and Biotechnology
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• 제16권6호
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• pp.1035-1040
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• 2007

Epidemiologic studies have shown important relationships between oxidative stress and Alzheimer's disease (AD) brain. In this study, free radical scavenging activity and neuronal cell protection effect of aqueous methanol extracts of Acanthopanax senticosus (A. senticosus) were examined. $H_2O_2$-induced oxidative stress was measured using 2',7'-dichlorofluorescein diacetate (DCF-DA) assay. Pretreatment with the phenolics of A. senticosus prevented oxidative injury against $H_2O_2$ toxicity. Since oxidative stress is known to increase neuronal cell membrane breakdown, leading to cell death, lactic dehydrogenase release, and trypan blue exclusion assays were utilized. We found that phenolics of A. senticosus have neuronal cell protection effects. It suggests that the phenolics of A. senticosus inhibited $H_2O_2$-induced oxidative stress and A. senticosus may be beneficial against the oxidative stress-induced risk in AD.

• International Journal of Oral Biology
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• 제41권3호
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• pp.141-147
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• 2016

Reactive oxygen species (ROS) and nitrogen species (RNS) are both important signaling molecules involved in pain transmission in the dorsal horn of the spinal cord. Xanthine oxidase (XO) is a well-known enzyme for the generation of superoxide anions ($O_2^{\bullet-}$), while S-nitroso-N-acetyl-DL-penicillamine (SNAP) is a representative nitric oxide (NO) donor. In this study, we used patch clamp recording in spinal slices of rats to investigate the effects of $O_2^{\bullet-}$ and NO on the excitability of substantia gelatinosa (SG) neurons. We also used confocal scanning laser microscopy to measure XO- and SNAP-induced ROS and RNS production in live slices. We observed that the ROS level increased during the perfusion of xanthine and xanthine oxidase (X/XO) compound and SNAP after the loading of 2',7'-dichlorofluorescin diacetate ($H_2DCF-DA$), which is an indicator of intracellular ROS and RNS. Application of ROS donors such as X/XO, ${\beta}-nicotinamide$ adenine dinucleotide phosphate (NADPH), and 3-morpholinosydnomimine (SIN-1) induced a membrane depolarization and inward currents. SNAP, an RNS donor, also induced membrane depolarization and inward currents. X/XO-induced inward currents were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger) and manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP; superoxide dismutase mimetics). Nitro-L-arginine methyl ester (NAME; NO scavenger) also slightly decreased X/XO-induced inward currents, suggesting that X/XO-induced responses can be involved in the generation of peroxynitrite ($ONOO^-$). Our data suggest that elevated ROS, especially $O_2^{\bullet-}$, NO and $ONOO^-$, in the spinal cord can increase the excitability of the SG neurons related to pain transmission.

• 대한예방한의학회지
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• 제12권2호
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• pp.51-59
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• 2008

Objectives : Buplueri Radix (BR), used medical plant in Korea traditional medicine, contains various compounds, including a series of triterpene saponins known as saikosaponins. We performed this study for the protective effect of BR against oxidative damage induced by 1,2,4-benzentriol(BT) in human lymphocytes. Methods : In order to investigate the protective effect of BR against carcinogens, genotoxicity induced by benzene metabolite, BT were performed using cytokinesis-block micronucleus(CBMN) assay and comet assay. Results : The frequency of micronucleus at 25, 50 and $100{\mu}M$ concentration of BT were $8{\pm}2.36$, $23{\pm}2.31$, $35{\pm}4.17$ respectively. In addition of BR with concentration of 25 and $50{\mu}g/mL$, MN frequencies were significantly decreased. According to comet assay, BT induced DNA damage in a dose-dependent manner at concentration of 10 and 50 while BT with BR treatment decreased DNA breakage. No genotoxicity was observed by BR($25{\sim}50{\mu}g/mL$) treatment alone on DNA breakage. Since BT can induce DNA damage through the generation of reactive oxygen species(ROS), we examined the level of ROS in human lymphocytes treated with BT and/or BR using DCF-DA, ROS-sensitive probe. The generation of ROS in BT-treated cells was also observed, and BR addition inhibited the level of BT-induced DNA damage. Conclusions : From above results it is suggested that BR could protect the cell and DNA from pro-oxidant effect by ROS by BT

• 한국약용작물학회지
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• 제23권2호
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• pp.138-143
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• 2015

This study was carried out to evaluate the preventive effect of three forms of Korean ginseng roots (fresh, white and red) against bisphenol A (BPA) toxicity in mouse male germ cells (GC-2spd, TM3, TM4). ROS (reactive oxygen species) generation were measured by DCF-DA (2',7'-dichlorohydrofluorescein diacetate) assay. Also, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was performed to quantify the mRNA expression levels of apoptosis-related genes, Bax (pro-apoptotic gene) and Bcl2 (anti-apoptotic gene). ROS generation was increased by $50{\mu}M$ BPA, but definitely decreased by treatment with Korean ginseng extracts (fresh, white and red) in mouse male germ cells. In especial, Korean fresh ginseng extract reduced significantly ROS production to normal control. In addition, Korean fresh and white ginseng extracts suppressed the apoptosis of mouse male germ cells by fine-tuning mRNA levels of apoptotic genes changed by BPA. In general, Korean fresh ginseng extract was more effective than white ginseng extract for reducing BPA-induced oxidative stress and apoptosis in mouse male germ cells. Therefore, Korean fresh and white ginseng may help to alleviate biphenol A toxicity in mouse male germ cells.

• Molecules and Cells
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• 제43권9호
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• pp.813-820
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• 2020

NB4 cell, the human acute promyelocytic leukemia (APL) cell line, was treated with various concentrations of arsenic trioxide (ATO) to induce apoptosis, measured by staining with 7-amino-actinomycin D (7-AAD) by flow cytometry. 2', 7'-dichlorodihydro-fluorescein-diacetate (DCF-DA) and MitoSOX™ Red mitochondrial superoxide indicator were used to detect intracellular and mitochondrial reactive oxygen species (ROS). The steady-state level of SO₂ (Cysteine sulfinic acid, Cys-SO₂H) form for peroxiredoxin 3 (PRX3) was measured by a western blot. To evaluate the effect of sulfiredoxin 1 depletion, NB4 cells were transfected with small interfering RNA and analyzed for their influence on ROS, redox enzymes, and apoptosis. The mitochondrial ROS of NB4 cells significantly increased after ATO treatment. NB4 cell apoptosis after ATO treatment increased in a time-dependent manner. Increased SO₂ form and dimeric PRX3 were observed as a hyperoxidation reaction in NB4 cells post-ATO treatment, in concordance with mitochondrial ROS accumulation. Sulfiredoxin 1 expression is downregulated by small interfering RNA transfection, which potentiated mitochondrial ROS generation and cell growth arrest in ATO-treated NB4 cells. Our results indicate that ATO-induced ROS generation in APL cell mitochondria is attributable to PRX3 hyperoxidation as well as dimerized PRX3 accumulation, subsequently triggering apoptosis. The downregulation of sulfiredoxin 1 could amplify apoptosis in ATO-treated APL cells.