Superoxide anion scavenging properties of fluvastatin and its metabolites.

We investigated the in vitro superoxide anion scavenging activities of fluvastatin and its metabolites. Fluvastatin showed dose-dependent superoxide anion scavenging activity in the NADH/phenazine methosulphate (PMS)/nitroblue tetrazolium (NBT) system, and the effect was as potent as the reference antioxidant, trolox, which is a water-soluble alpha-tocopherol derivative. The superoxide anion scavenging activities of the major metabolites of fluvastatin (M2, M3, M4, M7) were also determined in this system. All of these metabolites showed the activity. In particular, M2 and M3, which possess a phenolic hydroxyl group at the 5 or 6-position of the indole moiety, respectively, showed 3 times stronger activities than that of fluvastatin. Further, we also determined the effects of fluvastatin, M2 and M3 on phorbol myristate acetate (PMA)-induced superoxide anion generation in human peripheral blood polymorphonuclear leukocytes (PMN). The compounds tested also showed a depressing effect on the amount of superoxide anion in this system. We suggest that fluvastatin and its metabolites have the potential to protect cells or lipids from oxidative modification mediated by superoxide anion.     

Statins: 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) Reductase Inhibitors Demonstrate Anti-Atherosclerotic Character due to Their Antioxidant Capacity

Atherosclerosis is a chronic inflammatory disease of multiple etiologies. It is associated with the accumulation of oxidized lipids in arterial lesions leading to coronary heart disease. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (commonly known as statins) are widely used in cardiovascular disease prevention to lower the cholesterol. The antioxidant activity of HMG-CoA reductase inhibitors was studied by lipid peroxidation inhibition assay, DPPH, and hydroxyl radical scavenging-activity methods. The lovastatin (93%) and simvastatin (96%) showed significant action of lipid peroxidation inhibition compared to other HMG-CoA reductase inhibitors. The DPPH radical and hydroxyl radical scavenging activity of simvastatin was 38% and 33%, respectively. The oxidative modification of serum lipid due to reactive oxygen species causes atherosclerosis. This study revealed the importance of lovastatin and simvastatin to prevent oxidative stress-related cardiovascular diseases.     

Suffruyabiosides A and B, two new monoterpene diglycosides from moutan cortex

Two new monoterpene diglycosides, suffruyabiosides A and B, and seven known compounds 3-9 were isolated from Moutan Cortex (root cortex of Paeonia suffruticosa Andrews). The structures were elucidated on the basis of 2D NMR spectral data. Suffruyabiosides A and B are rare monoterpene diglycosides, including a cellobiose in the molecules. Salicylpaeoniflorin (4) had a antiproliferation effect similar to paeoniflorin (3) on human lung adenocarcinoma epitherial A549 cells. Galloylpaeoniflorin (8) and salicylpaeoniflorin (4) revealed a more pronounced radical scavenging effect than a-tocopherol (positive control). An increase in the number of phenolic hydroxyl groups produced a more effective radical scavenging effect [8 > mudanpioside E (6) > oxypaeoniflorin (5)]. Comparison of the effects of 4 and 5 showed that o-substitution with a phenolic hydroxyl group was more effective than p-substitution. The results suggest that salicylpaeoniflorin (4) may be useful as a cytotoxic and a radical scavenging agent.     

Reactions of indole derivatives with cardioprotective activity with reactive oxygen species. Comparison with melatonin

We have previously reported on the synthesis of novel indole derivatives containing an amine-triazole moiety (1a-d, 2a-c), and their antioxidant activity on in vitro non-enzymatic rat hepatic microsomal lipid peroxidation. Some of the compounds showed protective activity against oxidative injury of ischemic myocardium. In the present paper we investigated the interactions of these derivatives with reactive oxygen species, in order to find a mechanism of their antioxidant capacity and to identify structural characteristics responsible for these properties. These interactions were compared with melatonin, which is also an indole derivative. The antioxidant profiles of the compounds were established by different in vitro protocols as follows: 1) by the interaction of the compounds with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable free radical, 2) their scavenging effects on superoxide anions using an enzymic system of xanthine-xanthine oxidase, 3) their inhibitory effects on xanthine oxidase and 4) their ability to scavenge hydroxyl radicals by comparison with dimethyl sulfoxide (DMSO) for *OH. All compounds were found to interact with DPPH, most of them to be superoxide anion scavengers and to be strong hydroxyl radical scavengers. Derivatives 1a and 1d substituted on the nitrogen of the indolic nucleus were found to have better antioxidant properties than the reference compounds used and melatonin.     

Enhanced Production of Coenzyme Q10 by Overexpressing HMG-CoA Reductase and Induction with Arachidonic Acid in Schizosaccharomyces pombe

Coenzyme Q10 (CoQ10) is a vitamin-like substance which plays a crucial role in the respiratory chain ranging from bacteria to humans and in the radical scavenging in human body. In this study, the full-length hmgR gene (encoding 3-hydroxy-3-methyl-glutaryl-CoA reductase, HMG-CoA reductase) was cloned and overexpressed in Schizosaccharomyces pombe. Using the pREPG yeast depressed under the thiamine as the control, CoQ10 contents increased up to 2.68 and 3.09 times when recombinant cells were incubated without and with arachidonic acid, respectively. It demonstrated that arachidonic acid could upregulate the activity of HMG-CoA reductase and that hmgR gene played a significant role in CoQ10 biosynthesis. So, it has an importance to be utilized for fermentation.     

Reactive ketimino radical acceptors: intermolecular alkyl radical addition to imines with a phenolic hydroxyl group

Intermolecular carbon radical addition to the carbon-nitrogen double bond of ketimines was studied. In the study on the reactivity of various aldimines, we found that the aldimine 7 having a phenolic hydroxyl group shows an excellent reactivity toward nucleophilic carbon radicals. A remarkable effect of phenolic hydroxyl group is assumed to be the stabilization of intermediate aminyl radical provided by a hydroxyl group. The screening of reactive ketimino acceptors showed that ketimines 15, 17, and 19 having a phenolic hydroxyl group exhibit excellent reactivities. The radical addition to ketimines 15, 17, and 19 took place regioselectively at the imino carbon to give the C-alkylated products without the formation of N-alkylated products. Enantioselective ethyl radical addition to ketimine 15 using chiral box ligand and Zn(OTf)2 gave the diethylated product 30 in 80% ee.     

Molecular rationale delineating the role of lycopene as a potent HMG-CoA reductase inhibitor: in vitro and in silico study

This study initially aimed to depict the molecular rationale evolving the role of lycopene in inhibiting the enzymatic activity of β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) reductase via in vitro and in silico analysis. Our results illustrated that lycopene exhibited strong HMG-CoA reductase inhibitory activity (IC₅₀ value of 36 ng/ml) quite better than pravastatin (IC₅₀ = 42 ng/ml) and strong DPPH free radical scavenging activity (IC₅₀ value = 4.57 ± 0.23 μg/ml) as compared to ascorbic acid (IC₅₀ value = 9.82 ± 0.42 μg/ml). Moreover, the K_i value of lycopene (36 ng/ml) depicted via Dixon plot was well concurred with an IC₅₀ value of 36 ± 1.8 ng/ml. Moreover, molecular informatics study showed that lycopene exhibited binding energy of ?5.62 kcal/mol indicating high affinity for HMG-CoA reductase than HMG-CoA (ΔG: ?5.34 kcal/mol). Thus, in silico data clearly demonstrate and support the in vitro results that lycopene competitively inhibit HMG-CoA reductase activity by binding at the hydrophobic portion of HMG-CoA reductase.     

罗丹明6G-锰(Ⅱ)-过氧化体系荧光法测定常见油性种子的抗氧化活性

碱性介质中罗丹明6G能产生特征荧光,其最大激发波长和发射波长分别为350 nm和550 nm;Mn2+-H2O2体系在碱性介质中产生的羟自由基可以迅速氧化罗丹明6G使其荧光猝灭,而油性种子的浸提物可以清除羟自由基,从而使溶液的荧光猝灭程度降低,据此建立了测定油性种子抗氧化性的新方法.实验观察到抗氧化剂维生素C和硫脲等在低浓度范围内对羟自由基的清除率与用量呈上升关系,而在大浓度下反而下降.讨论了样品的水、醋酸、乙醇溶剂浸提物对羟自由基的清除效率.用水、醋酸作提取剂,分别测试了7种常见油性种子的抗氧化活性.     

Abnormal solvent effects on hydrogen atom abstraction. 2. Resolution of the curcumin antioxidant controversy. The role of sequential proton loss electron transfer

The rates of reaction of 1,1-diphenyl-2-picrylhydrazyl (dpph*) radicals with curcumin (CU, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), dehydrozingerone (DHZ, "half-curcumin"), and isoeugenol (IE) have been measured in methanol and ethanol and in two non-hydroxylic solvents, dioxane and ethyl acetate, which have about the same hydrogen-bond-accepting abilities as the alcohols. The reactions of all three substrates are orders of magnitude faster in the alcohols, but these high rates can be suppressed to values essentially equal to those in the two non-hydroxylic solvents by the addition of acetic acid. The fast reactions in alcohols are attributed to the reaction of dpph* with the CU, DHZ, and IE anions (see J. Org. Chem. 2003, 68, 3433), a process which we herein name sequential proton loss electron transfer (SPLET). The most acidic group in CU is the central keto-enol moiety. Following CU's ionization to a monoanion, ET from the [-(O)CCHC(O)-](-) moiety to dpph* yields the neutral [-(O)CCHC(O)-]* radical moiety which will be strongly electron withdrawing. Consequently, a phenolic proton is quickly lost into the alcohol solvent. The phenoxide anion so formed undergoes charge migration to produce a neutral phenoxyl radical and the keto-enol anion, i.e., the same product as would be formed by a hydrogen atom transfer (HAT) from the phenolic group of the CU monoanion. The SPLET process cannot occur in a nonionizing solvent. The controversy as to whether the central keto-enol moiety or the peripheral phenolic hydroxyl groups of CU are involved in its radical trapping (antioxidant) activity is therefore resolved. In ionizing solvents, electron-deficient radicals will react with CU by a rapid SPLET process but in nonionizing solvents, or in the presence of acid, they will react by a slower HAT process involving one of the phenolic hydroxyl groups.     

Assessing the antioxidative properties and chemical composition of Linaria vulgaris infusion

The ability of Linaria vulgaris (Scrophulariaceae) infusion to act as a scavenger of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, reactive oxygen species (superoxide radical, hydroxyl radical, hypochlorous acid (HOCl)) and nitric oxide was investigated. The obtained data indicate that the infusion has a good scavenging activity against superoxide radical and is a very potent nitric oxide and DPPH scavenger. In hydroxyl radical assay a pro-oxidant capacity was noticed, especially for concentrations higher than 31.25 microg mL(-1). No effect was found against HOCl. A phytochemical study of this extract was also performed. The HPLC/UV analysis allowed the identification and quantification of eight organic acids (oxalic, aconitic, citric, ketoglutaric, ascorbic, malic, shikimic and fumaric acids). The phenolic composition of the lyophilised infusion was also determined by HPLC/DAD and four compounds were quantified, but, despite its high content, only linarin was managed to be identified.     

Effect of acid and base catalyzed hydrolysis on the yield of phenolics and antioxidant activity of extracts from germinated brown rice (GBR)

The influence of both acidic and basic hydrolysis on the yield, total phenolic content and antioxidative capacity of methanolic extract of germinated brown rice (GBR) was studied. Total phenolic content (TPC), total flavonoid content (TFC), 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation scavenging, and ferric reducing antioxidant power (FRAP) tests were used for the measurement of antioxidant ability. There was a significant difference p < 0.05) in the TPC and DPPH radical scavenging assay results when comparing neutral with acidic and basic catalysed hydrolysis. The yield of the crude extract was slightly higher in acidic hydrolysis than in basic hydrolysis p > 0.05). The TPC and TFC were highest in acidic hydrolysis. A significant correlation was observed between ABTS radical cation scavenging and FRAP. The antioxidant activity measured using DPPH radical scavenging assay showed high activity in acidic hydrolysis, while the ABTS radical cationscavenging activity and FRAP showed the highest values in basic hydrolysis. The samples were further evaluated using HPLC to determine the individual phenolic concentrations in different hydrolytic media contributing to the antioxidant effects. This study revealed that acidic and basic hydrolysis can improve the yield, phenolic content, and antioxidant activity of germinated brown rice.     

Key role of chemical hardness to compare 2,2-diphenyl-1-picrylhydrazyl radical scavenging power of flavone and flavonol O-glycoside and C-glycoside derivatives

The antioxidant activities of flavonoids and their glycosides were measured with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH radical, DPPH(·)) scavenging method. The results show that free hydroxyl flavonoids are not necessarily more active than O-glycoside. Quercetin and kaempferol showed higher activity than apigenin. The C- and O-glycosides of flavonoids generally showed higher radical scavenging activity than aglycones; however, kaempferol C3-O-glycoside (astragalin) showed higher activity than kaempferol. In the radical scavenging activity of flavonoids, it was expected that OH substitutions at C3 and C5 and catechol substitution at C2 of B ring and intramolecular hydrogen bonding between OH at C5 and ketone at C3 would increase the activity; however, the reasons have yet to be clarified. We here show that the radical scavenging activities of flavonoids are controlled by their absolute hardness (η) and absolute electronegativity (χ) as a electronic state. Kaempferol and quercetin provide high radical scavenging activity since (i) OH substitutions at C3 and C5 strikingly decrease η of flavones, (ii) OH substitutions at C3 and C7 decrease χ and η of flavones, and (iii) phenol or o-catechol substitution at C2 of B ring decrease χ of flavones. The coordinate r(χ,?η) as the electron state must be small to increase the radical scavenging activity of flavonoids. The results show that chemically soft kaempferol and quercetin have higher DPPH radical scavenging activity than chemically hard genistein and daidzein.     

肉桂酸类抗氧化剂结构—活性关系研究

利用邻菲罗啉化学发光法研究了中草药有效成分氯原酸、连翘酯甙等肉桂酸类抗氧化剂对HO·的清除作用。量子化学计算[3-21G、B3LYP/6-31G(d)]对实验结果给予了合理的解释。 计算结果显示抗氧化剂形成半醌式自由基前后的能量之差(ΔE)能较好地衡量其活性的高低;抗氧化剂半醌式自由基与邻位取代基形成分子内氢键或通过共振形成邻苯醌可使内能降低,半醌式自由基更稳定;脱氢酚羟基的对位含有给电子取代基团时,可使半醌式自由基易形成并提高其稳定性。     

Structure of dihydrochalcones and related derivatives and their scavenging and antioxidant activity against oxygen and nitrogen radical species

Quantum mechanical calculations at B3LYP/6-31G** level of theory were employed to obtain energy (E), ionization potential (IP), bond dissociation enthalpy (O-H BDE) and stabilization energies (DE(iso)) in order to infer the scavenging activity of dihydrochalcones (DHC) and structurally related compounds. Spin density calculations were also performed for the proposed antioxidant activity mechanism of 2,4,6-trihydroxyacetophenone (2,4,6-THA). The unpaired electron formed by the hydrogen abstraction from the phenolic hydroxyl group of 2,4,6-THA is localized on the phenolic oxygen at 2, 6, and 4 positions, the C? and C? carbon atoms at ortho positions, and the C? carbon atom at para position. The lowest phenolic oxygen contribution corresponded to the highest scavenging activity value. It was found that antioxidant activity depends on the presence of a hydroxyl at the C2 and C4 positions and that there is a correlation between IP and O-H BDE and peroxynitrite scavenging activity and lipid peroxidation. These results identified the pharmacophore group for DHC.     

Analysis of five HMG-CoA reductase inhibitors-- atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin: pharmacological, pharmacokinetic and analytical overview and development of a new method for use in pharmaceutical formulations analysis and in vitro metabolism studies

A specific, accurate, precise and reproducible high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous quantitation of five 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitors, viz. atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin, in pharmaceutical formulations and extended the application to in vitro metabolism studies of these statins. Ternary gradient elution at a flow rate of 1 mL/min was employed on an Intertisl ODS 3V column (4.6 x 250 mm, 5 microm) at ambient temperature. The mobile phase consisted of 0.01 m ammonium acetate (pH 5.0), acetonitrile and methanol. Theophylline was used as an internal standard (IS). The HMG-CoA reductase inhibitors and their metabolites were monitored at a wavelength of 237 nm. Drugs were found to be 89.6-105.6% of their label's claim in the pharmaceutical formulations. For in vitro metabolism studies the reaction mixtures were extracted with simple liquid-liquid extraction using ethyl acetate. Baseline separation of statins and their metabolites along with IS free from endogenous interferences was achieved. Nominal retention times of IS, atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin were 7.5, 17.2, 21.6, 28.5, 33.5 and 35.5 min, respectively. The proposed method is simple, selective and could be applicable for routine analysis of HMG-CoA reductase inhibitors in pharmaceutical preparations as well as in vitro metabolism studies.     

π–π Stacking Interaction in an Oxidized CuII–Salen Complex with a Side-Chain Indole Ring: An Approach to the Function of the Tryptophan in the Active Site of Galactose Oxidase

In order to gain new insights into the effect of the π–π stacking interaction of the indole ring with the Cu^II–phenoxyl radical as seen in the active form of galactose oxidase, we have prepared a Cu^II complex of a methoxy-substituted salen-type ligand, containing a pendent indole ring on the dinitrogen chelate backbone, and characterized its one-electron-oxidized forms. The X-ray crystal structures of the oxidized Cu^II complex exhibited the π–π stacking interaction of the indole ring mainly with one of the two phenolate moieties. The phenolate moiety in close contact with the indole moiety showed the characteristic phenoxyl radical structural features, indicating that the indole ring favors the π–π stacking interaction with the phenoxyl radical. The UV/Vis/NIR spectra of the oxidized Cu^II complex with the pendent indole ring was significantly different from those of the complex without the side-chain indole ring, and the absorption and CD spectra exhibited a solvent dependence, which is in line with the phenoxyl radical–indole stacking interaction in solution. The other physicochemical results and theoretical calculations strongly support that the indole ring, as an electron donor, stabilizes the phenoxyl radical by the π–π stacking interaction.     

Synthesis and evaluation of the antioxidant properties for some novel aminomethyl derivatives of 2,6-diisobornylphenol bearing a pinane moiety

Some new derivatives based on 2,6-diisobornylphenol and containing amine moieties of the pinane structure at the para-position relative to the phenolic hydroxy group were synthesized. The antioxidant properties of the obtained compounds were estimated using various in vitro models. The introduction of diamine moiety into a starting molecule led to a significant increase in the radical scavenging activity and the ability to chelate Fe²⁺ ions.

     

LC-MS/MS Screening,Total Phenolic,Flavonoid and Antioxidant Contents of Crude Extracts from Three Asclepiadaceae Species Growing in Jordan

This study aimed to evaluate the antioxidant activity and total phenolic content (TPC) and total flavonoid content (TFC) of crude extracts obtained from three Asclepiadaceae species, namely, Calotropis procera L., Peruglaria tomentosa L., and Pentatropis spiralis (Forsk.) Decne. Both butanol and aq. methanol extracts of the three species showed the highest amount of phenol and flavonoid contents, which exhibited the greatest antioxidant activity in the scavenging of 2,2-diphenyl-2-picrylhydrazyl free radical (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS), ferrous chelating effect (FIC), and hydroxyl radical (HDR) assays. Phytochemical screening of the extracts revealed the presence of alkaloids, tannins, sponins, flavonoids, terpenoids, and glycosides. LC-MS analysis was carried out to identify the major compounds from each crude extract. A total of 12 phenolic compounds in the extracts of the 3 species were identified and quantified, including 9 flavonoids, 2 hydroxybenzoic acids, and 3 hydroxycinnamic acids. The current study also revealed a good correlation between total phenolic contents and the observed antioxidant activity of the crude extracts.     

An improved assay of 3-hydroxy-3-methylglutaryl-CoA reductase activity in Reuber H35 hepatoma cells without microsomes isolation

The optimal conditions for measuring 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity in Reuber H35 hepatoma cells are described in this paper. Cells in the exponential phase of growth were lysed by incubation with Brij 97 detergent for 30 min. We used imidazole buffer supplemented with EDTA and leupeptine, two inhibitors of proteases. Disrupted cells were then centrifuged at 12,000 g. Although microsomes are usually reported as enzyme preparations for measuring HMG-CoA reductase, our data showed that hepatoma cells may be used without previous isolation of microsomes. The 12,000 g supernatant showed similar levels of total and specific activities to those found in the microsomal fraction obtained after 105,000 g centrifugation. The soluble fraction showed less than 10% of reductase activity. Reductase activity from Reuber H35 hepatoma cells increased proportionally to the reaction time from 30 to 90 min and to the amount of protein added in a range of 50-500 micrograms. Our modified method was very sensitive and reproducible, because very low specific activity (about 15-100 pmol min-1 per mg protein) could be quantified in different assay conditions obtaining similar values.     

明日叶黄酮类化合物清除羟基自由基活性研究

为了研究明日叶黄酮类化合物对羟基自由基的清除作用,以明日叶（主要取叶片）为原料,用体积分数为65％乙醇提取明日叶总黄酮,测定其总黄酮含量.通过Fenton反应体系产生羟基自由基,利用明日叶提取液中的功能成分黄酮类化合物对羟基自由基的清除作用进行研究.结果表明：明日叶提取物总黄酮质量分数为10.18％,且黄酮类化合物对羟基自由基有较强清除效力,当提取物总黄酮浓度在0.1～1.0 mg/mL范围内,其与清除率呈正相关.明日叶中黄酮类化合物对羟基自由基有较强清除效力,作为天然抗氧化产品开发具有一定价值.