• Archives of Pharmacal Research
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• 제26권4호
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• pp.279-285
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• 2003

In our ongoing study to identity antioxidants from natural sources, the antioxidant activity of Nelumbo nucifera stamens was evaluated for their potential to scavenge stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, inhibit total reactive oxygen species (ROS) generation, in kidney homogenates using 2 ,7 -dichlorodihydrofluorescein diacetate (DCHF-DA), and scavenge authentic peroxynitrites ($ONOO^-$). A methanol (MeOH) extract of the stamens of N. nucifera showed strong antioxidant activity in the $ONOO^-$system, and marginal activity in the DPPH and total ROS systems, so were therefore fractionated with several organic solvents, such as dichloromethane ($CH_2 Cl_2$), ethyl acetate (EtOAc) and n-butanol (n-BuOH). The EtOAc soluble fraction, which exhibited strong antioxidant activity in all the model systems tested, was further purified by repeated silica gel and Sephadex LH-20 column chromatographies. Seven known flavonoids [kaempferol (1), kaempferol 3-Ο-$\beta$-D-glucuronopyranosyl methylester (2), kaempferol 3-Ο-$\beta$-D-glucopyranoside (3), kaempferol 3-Ο-$\beta$-D-galactopyranoside (4), myricetin 3 ,5 -dimethylether 3-Ο-$\beta$-D-glucopyranoside (5), kaempferol 3-Ο-$\alpha$-L-rhamnopyranosyl-(1$\rightarrow$6)-$\beta$-D-glucopyranoside (6) and kaempferol 3-Ο-$\beta$-D-glucuronopyranoside (7)], along with $\beta$-sitosterol glucopyranoside (8), were isolated. Compound 1 possessed good activities in all the model systems tested. Compounds 2 and 7 showed scavenging activities in the DPPH and $ONOO^-$ tests, while compounds 3 and 4 were only active in the $ONOO^-$ test. Conversely, compound 8 showed no activities in any of the model systems tested.

• 농업과학연구
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• 제39권4호
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• pp.467-471
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• 2012

In this study, we investigated the antioxidative and antiaging activity of Perilla frutescens using LLC-$PK_1$ porcine renal epithelial cell and WI-38 human diploid fibroblast cell. The extract from Perilla frutescens showed strong protective effect against nitric oxide (NO) and superoxide ($O_2{^-}$)-induced oxidative stress generated by sodium nitroprusside (SNP) and pyrogallol, respectively. The result showed that P. frutescens increased the cell viability and showed scavenging activity of NO and $O_2{^-}$. In addition, the extract of P. frutescens exerted the protective effect against peroxynitrite ($ONOO^-$) induced by 3-morpholinosydnonimine. It suggests that P. frutescens would have the protective role against $ONOO^-$ itself and its precursors, NO and $O_2{^-}$. Furthermore, the aging model of hydrogen peroxide ($H_2O_2$)-treated WI-38 human diploid fibroblast was employed to investigate the anti-aging effect of P. frutescens. $H_2O_2$-treated WI-38 cells showed the loss of cell viability, however before-treatment with P. frutescens to WI-38 cells under premature senescence could delay the cellular aging process. The present study suggests the antioxidative and antiaging potential against free radical-induced oxidative damage of P. frutescens.

• Archives of Pharmacal Research
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• 제28권10호
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• pp.1127-1130
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• 2005

A new phenolic glucoside (1), pursargentoside, was isolated from the leaves of Prunus serrulata var. spontanea, along with three other known compounds, orobol 7-O-glucoside (2), $1{\beta}$, $2{\alpha}$, $3{\alpha}$, 24-tetrahydroxy-urs-12-en-28-oic acid (3), and chlorogenic acid (4). The structure of pursargentoside (1) was identified by spectroscopic data analysis including 1D and 2D NMR spectroscopy, as 2-O-${\beta}$-(6'-benzoyl)-glucopyranosyl o-(Z)-coumaric acid. Compounds 1, 2, and 4 exhibited ONOO- scavenging activity, whereas compound 3 was determined to be virtually inactive.

• 동의생리병리학회지
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• 제23권2호
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• pp.464-472
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• 2009

The aim of this study was to investigate the effects of Cheonghyul-san on the generation of peroxynitrite ($ONOO^-$), nitric oxide (NO) and superoxide anion radical ( ${\cdot}\;O_2^-$), and on the expression of NF-${\kappa}$B-dependent proinflammatory proteins in ob/ob mice. Mice were grouped and treated for 5 weeks as follows. Both the normal lean (C57BL/6J black mice) and control obese (ob/ob mice) groups have received the standard chow. The experimental groups were fed with a diet of chow supplemented with 7.5, 15 and 30 mg Cheonghyul-san per 1 kg of body weight for 14 days. For this study, the fluorescent probes, namely 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), 4,5-diaminofluorescein-2 (DAF-2) and dihydrorhodamine 123 (DHR 123) were used. Western blot was performed using anti-IKK-${\alpha}$, anti-phospho I${\kappa}$B-${\alpha}$, anti-NF-${\kappa}$B (p50, p65), anti-COX-2, anti-iNOS, anti-VCAM-1 antibodies, respectively. Cheonghyul-san prevented $H_2O_2$-induced cell death. Cheonghyul-san inhibited the generation of $ONOO^-$, NO and ${\cdot}\;O_2^-$ in the $H_2O_2$-treated LLC-$PK_1$ cells. The generation of $ONOO^-$, NO and ${\cdot}\;O_2^-$ were inhibited in the Cheonghyul-san-administered ob/ob mice groups. The GSH/GSSG ratio was decreased in the ob/ob mice, whereas the ratio was improved in the Cheonghyul-san-administered groups. Cheonghyul-san inhibited the protein expression levels of phospho-I${\kappa}$B-${\alpha}$, IKK-${\alpha}$, NF-${\kappa}$B (p50, p65), COX-2, iNOS and VCAM-1 genes. These results suggest that Cheonghyul-san is an effective scavenger of $ONOO^-$, ${\cdot}\;O_2^-$ and NO, and has an inhibitory effect on the expression of NF-${\kappa}$B-dependent inflammatory genes in ob/ob mice. Therefore, Cheonghyul-san might be used as a potential therapeutic drug against the diabetes- and obesity-related proinflammatory diseases.

• 한국식품영양과학회지
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• 제39권10호
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• pp.1433-1438
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• 2010

본 연구는 해양생물 중 페기되어지인 군소내장을 추출하고 각 용매별로 분획하여 항산화 효과 및 항균활성을 측정하였다. DPPH free radical 소거활성을 측정하여 항산화 활성효과를 알아 본 결과 AKMB층이 $286.66\;{\mu}g/mL$에서 50%의 DPPH free radical의 소거활성을 보여 가장 높은 항산화 효과를 나타내었다. ROS 제거활성 측정 결과에서도 AKMB층이 가장 높은 항산화 효과를 나타내었으며 $54.73\;{\mu}g/mL$에서 50%의 ROS 제거 활성을 나타내었고, $ONOO^-$ 제거활성 역시 AKMB층이 가장 높게 나타났으며, $65.64\;{\mu}g/mL$에서 50%의 $ONOO^-$ 제거활성을 나타냈다. 또한 paper disc method를 이용한 항균효과 실험결과, Proteus vulgaris의 경우는 AKMH층과 AKMM층의 두층에서 높은 항균활성을 나타내었고, 그 외 사용한 모든 균주에서는 AKMM층에서 가장 높은 항균활성을 나타냄을 알 수 있었다. 이상의 결과로 항산화효과는 군소내장의 부탄올 분획층인 AKMB층에서 가장 높았으며, 항균효과는 주로 메탄올 분획층인 AKMM층에서 높은 항균활성을 나타내었다. 따라서 군소내장의 AKMB층과 AKMM층에서 항산화 활성과 항균력을 나타내는 효과적인 생리활성물질이 함유되어 있을 것으로 사료되어지며, 앞으로 더욱 심도 있는 연구를 통하여 이러한 물질들의 분리를 위한 연구가 더 필요한 것으로 판단되며 이러한 활성성분을 이용한 식품 저장성과 안전성 향상을 위한 항산화제, 식품첨가제 및 천연 식품 보존제 등의 개발 가능성이 기대되어진다.

• 대한한방내과학회지
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• 제26권2호
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• pp.379-389
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• 2005

Objectives: It is well known that aging and aging-related diseases are linked to the increased level of oxidative stress caused by reactive oxygen species(ROS) and reactive nitrogen species(RNS). Nonprotein-SH decreases during aging, while substances such as ROS, nitric oxide(NO), peroxynitrite($ONOO^-$), myeloperoxidase(MPO), and dityrosine show a significant increase. This study investigated the effect of Ichungwhan on the aging process by examining its effect on the generation of the above-mentioned substances. Methods: Four comparison groups of SD rats were used. They were 6 month-old rats, 24 month-old rats, and 24 month-old rats fed with food containing 0.1% and 0.3% of Ichungwhan extract. The amount of NO, $ONOO^-$, and ROS in the rats' kidneys were examined using a fluorescence microplate reader. The reagents used for this purpose include: dihydrorhodamine 123 (DHR 123), 2',7' -dichlorodihydrofluorescein, diacetate(DCFDA), and 4,5-diaminofluorescein(DAF-2). A spectrophotometer was used to investigate the reactivity of nonprotein-SH and myeioperoxidase(MPO), using reagents such as trichloroacetic acid(TCA) and tetramethylbenzidine(TMB). The amounts of MPO protein and dityrosine were measued by western blot. Results: The observed effects of Ichungwhan on rats were as follows: increase of nonprotein-SH; effective decrease of RNS level by suppression of the generation system of $ONOO^-$ and NO; decrease of ROS level; decrease of the MPO reactivity and the subsequent reduction of amount of MPO protein; retardation of dityrosine formation. It can be hypothesized, therefore, that Ichungwhan affects both the earlier and later phases of the molecular inflammatory process, and retards the aging process. Conclusions: Empirical evidence in this study supports a role for Ichungwhan in generation mechanisms of aging process-linked substances ROS, NO, $ONOO^-$, nonprotein-SH, MPO and dityrosine. Affects contrary to the aging process observed in rats beg further empiricism to investigate potential application of Ichungwhan as a medication for age-related diseases in humans.

• KSBB Journal
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• 제24권5호
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• pp.469-476
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• 2009

The antioxidant activities of two crude extracts ($CH_2Cl_2$ and MeOH) and their solvent fractions (n-hexane, 85% aq. MeOH, n-BuOH, and $H_2O$ fractions) from Corydalis heterocarpa were determined by evaluating authentic $ONOO^-$ and $ONOO^-$ generated from SIN-1 (3-morpholinsydnonimine) in vitro as well as by measuring the degree of occurrence of intracellular reactive oxygen species (ROS) and nitric oxide (NO). Scavenging activities of solvent fractions on authentic $ONOO^-$ increased in the order of n-BuOH > 85% aq. MeOH > $H_2O$ > n-hexane fractions, while those on $ONOO^-$ generated from SIN-1 increased in the order of n-BuOH > 85% aq. MeOH > $H_2O$ > n-hexane fractions. In addition, all solvent fractions effectively inhibited the intracellular ROS and NO levels. The n-BuOH fraction especially exhibited the strongest ROS scavenging effect. Further purification of n-BuOH fraction led to the isolation of cnidimoside A, which presented the potent ROS scavenging effect at $10\;{\mu}M$. From these results, extracts of C. heterocarpa and its component, cnidimoside A, were predicted to be potentially useful as ingredients for protecting against oxidation.

• Archives of Pharmacal Research
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• 제26권10호
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• pp.809-815
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• 2003

Since reactive oxygen species (ROS) and hydroxyl radicals ($^-OH$) play an important role in the pathogenesis of many human degenerative diseases, much attention has focused on the development of safe and effective antioxidants. Preliminary experiments have revealed that the methanol (MeOH) extract of the stem of Prunus davidiana exerts inhibitory/scavenging activities on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicals, total ROS and peroxynitrites ($ONOO^-$). In the present study, the antioxidant activities of this MeOH extract and the organic solvent-soluble fractions, dichloromethane (CH$_2$Cl$_2$), ethyl acetate (EtOAc), and n-butanol (n-BuOH), and the water layer of P. davidiana stem were evaluated for the potential to inhibit $^-OH$ and total ROS generation in kidney homogenates using 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA), and for the potential to scavenge authentic $ONOO^-$. We also evaluated the inhibitory activity of seven flavonoids isolated from P. davidiana stem, kaempferol, kaempferol 7-Ο-$\beta$-D-glucoside, (+)-catechin, dihydrokaempferol, hesperetin 5-Ο-$\beta$-D-glucoside, naringenin and its 7-Ο-$\beta$-D-glucoside, on the total ROS, $^-OH$ and $ONOO^-$ systems. For the further elucidation of the structure-inhibitory activity relationship of flavonoids on total ROS and 'OH generation, we measured the antioxidant activity of sixteen flavonoids available, including three active flavonoids isolated from P. davidiana, on the total ROS and 'OH systems. We found that the inhibitory activity on total ROS generation increases in strength with more numerous hydroxyl groups on their structures. Also, the presence of an ortho-hydroxyl group, whether on the Aring or S-ring, and a 3-hydroxyl group on the C-ring increased the inhibitory activity on both total ROS and $^-OH$ generation.

• Preventive Nutrition and Food Science
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• 제18권1호
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• pp.18-22
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• 2013

To investigate radical scavenging effects and protective activities of bitter melon (Momordica charantia) against oxidative stress, in vitro and a cellular system using LLC-$PK_1$ renal epithelial cells were used in this study. The butanol (BuOH) fraction of bitter melon scavenged 63.4% and 87.1% of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals at concentrations of 250 and $500{\mu}g/mL$, respectively. In addition, the BuOH fraction of bitter melon effectively scavenged hydroxyl radicals (${\cdot}OH$). At all concentrations tested, the scavenging activity of the BuOH fraction was more potent than that of the positive control, ascorbic acid. Furthermore, under the LLC-$PK_1$ cellular model, the cells showed a decline in viability and an increase in lipid peroxidation through oxidative stress induced by pyrogallol, a generator of superoxide anion ($O_2{^-}$). However, the BuOH fraction of bitter melon significantly and dose-dependently inhibited cytotoxicity. In addition, 3-morpholinosydnonimine (SIN-1), a generator of peroxynitrite ($ONOO^-$) formed by simultaneous releases of nitric oxide and $O_2{^-}$, caused cytotoxicity in the LLC-$PK_1$ cells while the BuOH fraction of bitter melon ameliorated oxidative damage induced by $ONOO^-$. These results indicate that BuOH fraction of bitter melon has protective activities against oxidative damage induced by free radicals.

• 생약학회지
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• 제40권4호
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• pp.365-369
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• 2009

Six caffeoylquinic acids of Cacalia firma (Komarov) Nakai (Compositae) leaves were identified using standard compounds by HPLC. Each content of those compounds in dried weight was determined as follows: 3,4-di-O-caffeoylquinic acid (1.44${\pm}$0.04 mg/g of dried weight), 3,5-di-O-caffeoyl-muce-quinic acid (2.47${\pm}$0.12 mg/g), 3,5-di-O-caffeoylquinic acid (3.74${\pm}$0.24 mg/g), 5-caffeoylquinic acid (chlorogenic acid, 5.20${\pm}$0.09 mg/g), 3-caffeoylquinic acid (1.35${\pm}$0.01 mg/g) and 3-Op-coumaroylquinic acid (3.84${\pm}$0.25 mg/g). The total content of six caffeoylquinic acids in the plant material was calculated as 18.05${\pm}$0.69 mg/g while the percentage of the six compounds in the MeOH extract was calculated as 30.85${\pm}$1.18%. The $IC_{50}$ value of the MeOH extract scavenging peroxynitrite ($ONOO^-$) was shown as 3.22${\pm}$0.57 ${\mu}g$/ml.