Sang-Cheol Lim

Simultaneous quantitative determination and validation of quercetin glycosides with peroxynitrite-scavenging effects from Saussurea grandifolia.

The leaves of Saussurea grandifolia (Compositae) are used as chwinamul, a well-known Korean mountainous vegetable, or to treat hepatitis and hematuria. Since the methanolic extract of S. grandifolia leaves exhibit a potent peroxynitrite-scavenging effect, phytochemical and high-performance liquid chromatographic (HPLC) analyses were employed to identify and simultaneously quantify the active components: chlorogenic acid and 3,5-di-O-caffeoylquinic acid (3,5-DQ) as caffeoylquinic acids, and quercetin, isoquercitrin (quercetin-3-glucoside), saxifragin (quercetin-5-glucoside), and rutin (quercetin-3-rutinoside). Validation of HPLC methods was performed to verify the linearity, LOD, LOQ, intra-day and inter-day variabilities, recovery, and repeatability to ensure that this method is selective, sensitive, precise, accurate and reproducible. In particular, leaves contained saxifragin with potent peroxynitrite-scavenging activity (IC(50), 0.67 μM) as 4.65 mg/g dry weight, which is equivalent to 33.74 mg/g extract.

Simultaneous quantification and validation of caffeoylquinic acids and flavonoids in Hemistepta lyrata and peroxynitrite-scavenging activity

Traditionally, Hemistepta lyrata is consumed as a mountainous vegetable or a medicinal herb to treat inflammation, fever, hemorrhage, and hemorrhoids. In order to provide the scientific evidence of traditional uses of this plant, we identified and quantified thirteen active substances (caffeic acid, chlorogenic acid, and 3,5-di-O-caffeoylquinic acid as caffeoylquinic acids; apigenin, isorhoifolin, acacetin, linarin, diosmetin, diosmin, pectolinarigenin, and pectolinarin as flavones or their glycosides; kaempferol 3-O-rutinoside and rutin as flavonol glycosides) from H. lyrata and evaluated their peroxynitrite-scavenging activity. The chromatographic separation was performed on a Capcell Pak C18 column (5μm, 250mm×4.6mm i.d.) with a gradient elution of 0.05% TFA (trifluoroacetic acid) and 0.05% TFA in MeOH-CH(3)CN (60:40). Validation of HPLC methods on the linearity, LOD, LOQ, intra-day and inter-day variabilities, recovery, and repeatability proved that this method is selective, sensitive, precise, accurate, and reproducible. In peroxynitrite-scavenging assay, caffeic acid derivatives (chlorogenic acid, caffeic acid, and 3,5-di-O-caffeoylquinic acid) exhibited relatively lower IC(50) values than other substances tested. And HPLC simultaneous quantification showed that the 70% MeOH extract and the BuOH fraction contain a higher quantity of caffeic acid derivatives (17.82 and 30.09mg/g, consecutively). Therefore, caffeic acid derivatives could be the main contributors to the peroxynitrite-scavenging activity of H. lyrata than other phenolic substances.

Simultaneous quantification and validation of new peroxynitrite scavengers from Artemisia iwayomogi

Abstract Context: Artemisia iwayomogi Kitamura (Compositae) has been very widely used for the treatment of acute or chronic hepatitis, jaundice, and gastritis. In the course of our continuing efforts to identify and quantify peroxynitrite scavengers from Compositae plants, A. iwayomogi was used in this study. Objective: The present study was aimed to identify and quantify the peroxynitrite scavengers of A. iwayomogi. Materials and methods: Silica gel and ODS were used for column chromatography. The isolated compounds were quantified using an HPLC equipped with a Capcell Pak C18 column (5 μm, 250 mm × 4.6 mm i.d.), and the method was validated for the quality control. Peroxynitrite (ONOO−)-scavenging activities of the compounds and extracts were evaluated on the measurement of highly fluorescent rhodamine 123 converted from non-fluorescent dihydrorhodamine (DHR)-123 under the presence of peroxynitrite. Results: Based on the spectroscopic evidences, a new compound, 2″-O-caffeoylrutin (2″-O-trans-caffeic acid ester of quercetin 3-O-α-l-rhamnopyranosyl(1 → 6)-β-d-glucopyranoside) was isolated and determined together with patuletin 3-O-glucoside, scopolin, scopoletin, rutin, 3,4-dicaffeoylquinic acid, and chlorogenic acid. All of them were potent peroxynitrite scavengers (IC50 ≤ 1.88 μg/mL). Discussion and conclusion: The peroxynitrite scavengers were mainly distributed in the EtOAc fraction rather than the ether and BuOH fractions. The 70% MeOH extract exhibited a high peroxynitrite-scavenging activity. Through the validation, the present HPLC method was verified to be sufficiently sensitive, accurate, precise, and stable. Therefore, this method can be used for the quality control of A. iwayomogi.

The Effects of Onion Extracts on Mercury-Induced Toxicity and Lipid Peroxidation in Rat Hepatocyte Primary Culture

The objective of present study was to investigate the effect of onion extracts on mercuryinduced cytotoxicity, lipid peroxidation and antioxidant enzyme activities in primary monolayer cultures of rat hepatocytes. Primary cultures of rat hepatocytes were incubated for 6 hr in the presence of various concentrations (0, 1, 5, 10, 30 or 50 ppm) of . Cytotoxicity and cell viability were determined by measuring glutamic oxaloacetic transaminase (GOT) activity, lactate dehydrogenase (LDH) activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) value. Lipid peroxidation w as evaluated using thiobarbituric acid reactive substances (TBARS) assay. Effects of onion extract on antioxidant system were determined by measuring catalase, glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd) activities as well as DPPH free radical scavenging activity. at the concentration of 10 ppm increased GOT activity and TBARS concentration but decreased %MTT reduction, whereas at the concentration of 30 ppm increased LDH activity, representing that caused cytotoxicity and lipid peroxidation in dose-dependent manner, at the concentration of 30 ppm significantly decreased catalase, GSH-Px and GSH-Rd activities. When primary cultures of rat hepatocytes were incubated with various concentrations (0, 0.01, 0.05, 0.1 or 0.3 mg/ml) of onion extract for 6 hr in the presence of 30 ppm of , onion extracts at the concentration of 0.05 mg/ml decreased GOT activity, but increased %MTT reduction by 30 ppm of . LDH activity and TBARS concentration were decreased by onion extract at the concentration of 0.01 mg/ml. Taken together, onion extract prevented H hepatocyte injury and lipid peroxidation. Onion extracts at the concentration of 0.1 mg/ml almost or completely inhibited catalase and GSB-Px activities. GSH-Rd activity, however, was not affected by onion extract. Free radical scavengjing activity was increased as concentration of onion extract increased. Onion extract at the concentrion of 5 mg/ml possesed mote than 93% scavenging activity comparing to 100% radical scavenging activity by pyrogallol solution as a reference. These results demonstrate that onion extracts suppressed mercury-induced cytoctoxicity and lipid peroxidation by scavenging free radical and increasing catalase and GSH-Px activities.