• Archives of Pharmacal Research
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• v.25 no.6
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• pp.865-872
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• 2002

In the course of the investigations of natural antioxidants, we examined the antioxidant activities of the methanol (MeOH) extracts of some selected Prunus species, including P. buergeriana, P. davidiana, P padus, P. pendula for. ascendens, P. sargentii, P. serrulata var. spontanea and P. yedoensis by three methods as represented by the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical, total ROS (reactive oxygen species) and the peroxynitrite ($ONOO^-$) scavenging activity tests. We also evaluated the activities of the organic solvent-soluble fractions, including the dichloromethane ($CH_2Cl_2$), ethyl acetate (EtOAc), n-butanol (n-BuOH) fractions and the water ($H_2O$) layer of P. serrulata var. spontanea leaves. By means of bioassay-directed fractionation, we isolated eleven known flavonoids (1-11) from the EtOAc soluble fraction of the MeOH extract of the Prunus serrulata var. spontanea leaves, exhibiting strong antioxidant activity and characterized as prunetin (1), genistein (2), quercetin (3), prunetin $4'-O-{\beta}-glucopyranoside$ (4), kaempferol $3-O-{\alpha}-arabinofuranoside$ (5), prunetin $5-O-{\beta}-glucopyranoside$ (6), kaempferol $3-O-{\beta}-xylopyranoside$ (7), genistin (8), kaempferol $3-O-{\beta}-glucopyranoside$ (9), quercetin $3-O-{\beta}-glucopyranoside$ (10) and kaempferol $3-O-{\beta}-xylopyranosyl-(1{\rightarrow}2)-{\beta}-glucopyranoside$ (11). Compounds 3 and 10 showed good activities in all tested model systems. Compounds 2 and 8 showed scavenging activities in the DPPH and $ONOO^-$ tests, while compounds 5, 7, 9 and 11 were active in the $ONOO^-$ and ROS tests. On the other hand, compounds 1, 4 and 6 did not show any activities in the tested model systems.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.6
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• pp.1475-1481
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• 2008

This study was to investigate the inhibitory effects of Mori Fructus on the generation of peroxynitrite ($ONOO^-$), nitric oxide (NO) and superoxide anion radical (${\cdot}O_2{^-}$) in the endothelial cells of rat vessels. The aim of this study was to investigate the $ONOO^-$, NO, ${\cdot}O_2{^-}$ scavenging and anti-inflammatory activitives of Mori Fructus. For this study, the fluorescent probes, namely dihydrorhodamine 123 (DHR 123), 4,5-diaminofluorescein (DAF-2) and 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) were used. Western blotting was performed using anti-NF-${\kappa}B$ (p50, p65), anti-COX-2, anti-iNOS antibodies, respectively. Mori Fructus prevented lipopolysaccharide (LPS)-induced cell death in YPEN cells. Mori Fructus inhibited the generation of $ONOO^-$, NO and ${\cdot}O_2{^-}$ in the LPS-treated cells. Mori Fructus inhibited the expression of COX-2 and iNOS genes by means of decreasing the NF-${\kappa}B$ activation. These results suggest Mori Fructus is effective on inhibiting the generation of $ONOO^-$, NO and ${\cdot}O_2{^-}$, and that therefore it might have a potential role as a treatment for the inflammatory process and inflammation-related diseases.

• The Journal of Internal Korean Medicine
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• v.26 no.2
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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.

• Archives of Pharmacal Research
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• v.29 no.12
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• pp.1074-1079
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• 2006

As part of our research on phytochemicals that exert protective effects against diseases related to reactive nitrogen species, we have evaluated the scavenging activity of the yellow leaves of Ginkgo biloba on $ONOO^{-}$. The methanol extract and ethyl acetate fraction obtained from yellow leaves of G. biloba evidenced a marked scavenging activity on authentic $ONOO^{-}$. Repeated column chromatography of the active ethyl acetate soluble fraction on silica gel, Sephadex LH-20, and RP-18, resulted in the purification of 15 known compounds, including sciadopitysin (1), ginkgolide B (2), bilobalide (3), isoginkgetin (4), kaempferol (5), luteolin (6), protocatechuic acid (7), bilobetin (8), amentoflavone (9), ${\beta}-sitosterol$ glucopyranoside (10), kaempferol 3-O-rhamnopyranoside (11), kaempferol 3-O-glucopyranoside (12), kaempferol $3-O-[{6^{'}-O-p-coumaroyl-{\beta}-D-glucopyranosyl(1{\rightarrow}2)-{\alpha}-L-rhamnopyranoside]$ (13), kaempferol 3-O-rutinoside (14), and 6-hydroxykynurenic acid (15). Among the compounds isolated, flavonoids (5, 6 and 11-14), protocatechuic acid (7), and 6-hydroxykynurenic acid (15) all exhibited marked scavenging activities on authentic $ONOO^{-}$. The $IC_{50}$ values of 5-7, 11-14 and 15 were as follows: $2.86{\pm}0.70,\;2.30{\pm}0.04,\;2.85{\pm}0.10,\;5.60{\pm}0.47,\;4.16{\pm}1.65,\;2.47{\pm}0.15,\;3.02{\pm}0.48,\;and\;6.24{\pm}0.27\;{\mu}M$, respectively. DL-Penicillamine ($IC_{50}=4.98{\pm}0.27\;{\mu}M$) was utilized as a positive control. However, the other compounds (1-4, 8-10) exerted no effects against $ONOO^{-}$.

• KSBB Journal
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• v.24 no.5
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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.

• Natural Product Sciences
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• v.21 no.3
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• pp.155-161
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• 2015

Peroxynitrite (ONOO⁻)-scavenging activities of nine Compositae herbs consisting of three Ixeris, two Youngia, two Cirsium and one of each Lactuca and Taraxacum species were evaluated. The contents of their ONOO⁻ scavengers in the extracts were also determined on a HPLC using seven standard compounds, chlorogenic acid (CGA), chicoric acid (CA), luteolin 7-glucoside (luteolin-7-glc), luteolin 7-glucuronide (luteolin-7-glcU), luteolin, linarin and pectolinarin. Five of those compounds exhibited potent ONOO⁻-scavenging activities: IC₅₀, CA (0.76 μM), CGA (1.34 μM), luteolin (0.81 μM), luteolin-7-glc (0.86 μM) and luteolin-7-glcU (3.13 μM). Both CA and luteolin-7-glc were highly contained in I. dentata (19.71 mg/g and 13.58 mg/g, respectively), I. dentata var. albiflora (17.58 mg/g and 23.83 mg/g, respectively) and I. sonchifolia (65.71 mg/g and 6.99 mg/g, respectively). Among the nine herbs, those three Ixeris species had very low IC₅₀ values over the range of 0.48 - 1.74 μg/mL, suggesting that they could be potential therapeutic vegetables, particularly for preventing diabetic complications or obesity, which can be caused by an excess production of ONOO⁻.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.6
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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.

• Archives of Pharmacal Research
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• v.26 no.10
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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.

• The Journal of Internal Korean Medicine
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• v.29 no.2
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• pp.421-431
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• 2008

Objectives : This study was to investigate the inhibitory effects of Ulmus davidiana on the generation of peroxynitrite $(ONOO^{-})$, nitric oxide (NO) and superoxide anion radicals $(O_{2}^{-})$ in the endothelial cells of rat vessels. The effects of Ulmus davidiana on the expression of inflammation-related proteins, $NF-{\kappa}B$ (p50, p65), COX-2, and iNOS, were examined by western blotting. Methods : For this study, fluorescent probes, namely dihydrorhodamine 123 (DHR 123), 4,5-diaminofluorescein (DAF-2) and 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) were used. Western blotting was performed via using anti-$NF-{\kappa}B$ (p50, p65), anti-COX-2, and anti-iNOS, respectively. Results : Ulmus davidiana inhibited the generation of $ONOO^{-}$, NO and $(O_{2}^{-})$ in the lipopolysaccharide (LPS)-treated endothelial cells of rat vessels in vitro. Ulmus davidiana inhibited the expression of COX-2 and iNOS genes by means of decreasing the $NF-{\kappa}B$ activation. Conclusions : These results suggest Ulmus davidiana is effective on inhibiting the generation of $ONOO^{-}$, NO and $O_{2}^{-}$, and that therefore it might have a potential role as a treatment for the inflammatory process and inflammation-related diseases.

• Food Science and Biotechnology
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• v.18 no.1
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• pp.167-171
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• 2009

This study examined the antioxidative activity of delphinidin, a kind of anthocyanidin from eggplant. Cellular protective potential from oxidative damage by nitric oxide (NO), superoxide anion ($O_2^-$), and peroxynitrite ($ONOO^-$) using epithelial cell line LLC-PK1 cell as well as in vitro radical scavenging effects were investigated. Delphinidin showed strong in vitro radical scavenging effects against NO, $O_2^-$, and hydroxyl radical (${\cdot}OH$) in dose-dependent manners. In addition, delphinidin increased cell viability in LLC-PK1 cells in a concentration-dependent manner when viability was reduced by $ONOO^-$-induced oxidative damage. To elucidate the protective mechanisms of delphinidin from $ONOO^-$, sodium nitroprusside (SNP), and pyrogallol were also employed to generate NO and $O_2^-$, respectively. The treatment of delphinidin recovered reductions in cell viability caused by SNP and pyrogallol, indicating that delphinidin can attenuate oxidative stress induced by NO and $O_2^-$. The present study suggests that delphinidin is a promising anti oxidative agent.