• The Journal of Internal Korean Medicine
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• v.31 no.1
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• pp.54-65
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• 2010

Objective : The water extract of Chungsimyeonja-eum (CSYJE) has traditionally been used in treatments of heart diseases and brain diseases in Oriental medicine. However, little is known about the mechanism by which CSYJE protects neuronal cells from injury damages. Therefore, in this study we attempted to elucidate the mechanism of the cytoprotective effect of the CSYJE extract on glutamate-induced C6 glial cell death. Methods : Cultured cells were pretreated with CSYJE and exposed to glutamate, cell damage was assessed by using MTT assay and propidium iodide (PI), probe 2',7'-dichlorofluorescein diacetate (DCF-DA) staining. Western blotting was performed using anti-procaspase-3 and anti-PARP, respectively. Result : We determined the elevated cell viability by CSYJE extract on glutamate-induced C6 glial cell death. Glutamate induced DNA fragmentation on C6 glial cells but pre-treatment with CSYJE inhibited DNA fragmentation. One of the main mediators of glutamate-induced cytotoxicity was known to generation of reactive oxygen species (ROS). Pre-treatment with CSYJE inhibited this ROS generation from glutamate-stimulated C6 glial cells. Also, we identified that the ROS-induced DCF-DA green fluorescence was reduced by CSYJE pre-treatment. The critical markers of apoptotic cell death are the cleavages of procaspase-3 protease and PARP proteins, so we checked the expression level and cleavages of procaspase-3 protease and PARP proteins. Glutamate-treated C6 glial cells showed the cleavages of procaspase-3 protease and PARP proteins and followed the reduction of expression of these proteins. Conclusion : These findings indicate that CSYJE may prevent cell death from glutamate-induced C6 glial cell death by inhibiting the ROS generation and procaspase-3 and PARP expression.

• Korean Journal of Medicinal Crop Science
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• v.27 no.6
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• pp.365-375
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• 2019

Background: Oxidative stress plays an important role in neuro-degenerative disorders such as Alzheimer's disease. Oxidative stress is mediated by reactive oxygen species (ROS), which are implicated in the pathogenesis of numerous diseases, and account for the toxicity of a wide range of compounds. Methods and Results: In order to study the neuro-protective effect of the complex extracts of Aster scaber Thunberg (AS) and Chaenoleles sinensis Koehne (CSK) against hydrogen peroxide in PC12 cells, cell viability was evaluated by the MTT assay using tetrazole, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the intracellular ROS levels were determined the by 2',7'-dichlorofluorescein diacetate (DCF-DA) assay. In order to examine the anti-amnesic effects of the complex extracts of AS and CSK, behavioral tests were performed on male ICR mice. The ameliorating effect of the complex extracts against Aβ_1-42-induced learning and memory impairment was analyzed by y-maze and passive avoidance tests. The AS and CSK extracts showed neuro-protective activity both in vitro and in vivo, and the neuro-protective effect of their 60 : 40 (AS : CSK) mixture was better than that of the other mixtures. Moreover, the complex extracts synergistically inhibited acetylcholinesterase activity and rapid peroxidation. Conclusions: A mixture of the AS and CSK extracts could be used to develop functional foods and serve as raw materials for the development of therapeutics against Alzheimer's disease.

• Journal of Microbiology and Biotechnology
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• v.28 no.1
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• pp.40-49
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• 2018

We evaluated the antioxidant activity and neuronal cell-protective effect of fucoidan extract from Ecklonia cava (FEC) on hydrogen peroxide ($H_2O_2$)-induced cytotoxicity in PC-12 and MC-IXC cells to assess its protective effect against oxidative stress. Antioxidant activities were examined using the ABTS radical scavenging activity and malondialdehyde-inhibitory effect, and the results showed that FEC had significant antioxidant activity. Intracellular ROS contents and neuronal cell viability were investigated using the DCF-DA assay and MTT reduction assay. FEC also showed remarkable neuronal cell-protective effect compared with vitamin C as a positive control for both $H_2O_2$-treated PC-12 and MC-IXC cells. Based on the neuronal cell-protective effects, mitochondrial function was analyzed in PC-12 cells, and FEC significantly restored mitochondrial damage by increasing the mitochondrial membrane potential (${\Delta}{\Psi}m$) and ATP levels and regulating mitochondrial-mediated proteins (p-AMPK and BAX). Finally, the inhibitory effects against acetylcholinesterase (AChE), which is a critical hydrolyzing enzyme of the neurotransmitter acetylcholine in the cholinergic system, were investigated ($IC_{50}$ value = 1.3 mg/ml) and showed a mixed (competitive and noncompetitive) pattern of inhibition. Our findings suggest that FEC may be used as a potential material for alleviating oxidative stress-induced neuronal damage by regulating mitochondrial function and AChE inhibition.

• Molecules and Cells
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• v.24 no.1
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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.

• The Korea Journal of Herbology
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• v.23 no.4
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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.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.34 no.4
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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.

• Korean Journal of Food Science and Technology
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• v.48 no.2
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• pp.172-177
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• 2016

The phenolic compounds, antioxidant activity and neuronal cell protective effect of Cirsium japonicum extract were evaluated in this study. High performance liquid chromatography mass analysis showed that C. japonicum was composed of chlorogenic acid, linarin, and pectolinarin. C. japonicum extract showed its antioxidant activity with half-maximal inhibitory concentrations of 567 and $130{\mu}g/mL$ by DPPH and ABTS radical scavenging activity, respectively. The total antioxidant capacities of C. japonicum via DPPH, ABTS, and FRAP assays were 11.32, 100.15, and $12.76{\mu}g/mL$ trolox equivalents, respectively. In addition, the neuroprotective effect of C. japonicum extract was investigated by measuring cell viability via MTT, LDH and DCF-DA assay using $H_2O_2-damaged$ PC12 cells. C. japonicum extract showed neuronal cell protective effects in a dose-dependent manner. These results indicated that C. japonicum extract has potent antioxidant and neuronal protective effects. Therefore, C. japonicum can be regarded as an effective and safe functional food resource as natural antioxidants, and may decrease the risk of neurodegenerative disorders.

• Journal of Pharmacopuncture
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• v.21 no.1
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• pp.18-25
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• 2018

Objective: D-galactose (D-gal) is well-known agent to induce aging process. In the present study, we selected crocin, the main constituent of Crocus sativus L. (saffron), against D-gal- induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Methods: Pretreated cells with crocin ($25-500{\mu}M$, 24 h) were exposed to D-gal (25-400 mM, 48 h). The MTT assay was used for determination cell viability. Dichlorofluorescin diacetate assay (DCF-DA) and senescence associated ${\beta}$-galactosidase staining assay (SA-${\beta}$-gal) were used to evaluate the generation of reactive oxygen species and beta-galactosidase as an aging marker, respectively. Also advanced glycation end products (AGEs) expression which is known as the main mechanism of age-related diseases was measured by western blot analysis. Results: The findings of our study showed that treatment of cells with D-gal (25-400 mM) for 48h decreased cell viability concentration dependency. Reactive oxygen species (ROS) levels which are known as main factors in age-related diseases increased from $100{\pm}8%$ in control group to $132{\pm}22%$ in D-gal (200 mM) treated cells for 48h. The cytotoxic effects of D-gal decreased with 24h crocin pretreatment of cells. The cell viability at concentrations of $100{\mu}M$, $200{\mu}M$ and $500{\mu}M$ increased and ROS production decreased at concentrations of 200 and $500{\mu}M$ to $111.5{\pm}6%$ and $108{\pm}5%$, respectively. Also lysosomal biomarker of aging and carboxymethyl lysine (CML) expression as an AGE protein, significantly increased in D-gal 200 mM group after 48h incubation compare to control group. Pre-treatment of SHSY-5Y cells with crocin ($500{\mu}M$) before adding D-gal significantly reduced aging marker and CML formation. Conclusion: Treatment of SH-SY5Y cells with crocin before adding of D-gal restored aging effects of D-gal concentration dependency. These findings indicate that crocin has potent anti- aging effects through inhibition of AGEs and ROS production.

• Journal of the East Asian Society of Dietary Life
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• v.21 no.1
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• pp.24-30
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• 2011

The anti-cancer effects of Angelica keiskei ethanol extract were evaluated in human breast cancer MDA-MB-231 cells. The concentrations of extract were 1, 2, 3, 4 and 5 mg/mL. Dose-dependent reductions in the number of cells with altered cell shape and pyknotic nuclei were observed at 48 h after treatments. MTT assay also exhibited a similar dose-dependent reduction in mitochondrial reductase activity (p<0.05), in particular, with a rapid reduction in the activity of the 5 mg/mL group. Analysis of cell death with propidium iodide (PI) staining revealed only a slight increase in cell death in the 5 mg/mL group. Analysis of bromodeoxyuridine (BrdU) incorporations also showed a dose-dependent reduction in cell proliferation (p<0.05). Finally, increases in total radical oxygen species (ROS) accumulation in cells, as revealed by DCF-DA staining, were observed in the treated groups in a similar dose-dependent fashion (p<0.05). These results indicate that Angelica keiskei ethanol extract exhibiting anti-cancer effects in MDA-MB-231 cells causes multiple changes in cell shape, enzyme activity, and ROS accumulation, thereby inducing cell death.

• ALGAE
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• v.27 no.3
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• pp.205-213
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• 2012

The thermostability of antioxidant activity of dieckol, a phlorotannin isolated from brown seaweed Ecklonia cava was investigated. The thermostable antioxidant properties of dieckol were evaluated at 30, 60, and $90^{\circ}C$ for 7 days using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activities, and comparing its performance to that of ascorbic acid. The intracellular reactive oxygen species (ROS) scavenging activity and apoptotic body formation were investigated using DCF-DA assay and nuclear staining with Hoechst 33342, propidium iodide and flow cytometry. Dieckol treated at different temperatures during 7 days showed stable scavenging activities on towards DPPH and hydroxyl radicals. In addition, dieckol showed a stable protective effect against $H_2O_2$-induced apoptotic body formation in Vero cells. On the other hand, the radical scavenging activities and intracellular ROS scavenging activities of ascorbic acid, used as a positive control, were significantly decreased at $60^{\circ}C$ and $90^{\circ}C$ from on the 4th day and 3rd days, respectively. In conclusion, the results indicated that food grade antioxidant extracts containing dieckol derived from E. cava remain a stable during the temperatures encountered during the processing of food and cosmetics.