Silibinin phosphodiester glyco-conjugates: Synthesis,redox behaviour and biological investigations

New silibinin phosphodiester glyco-conjugates were synthesized by efficient phosphoramidite chemistry and were fully characterized by 2D-NMR. A wide-ranging study focused on the determination of their pKa and E° values as well as on their radical scavenging activities by different assays (DPPH, ABTS⁺ and HRSA) was conducted. The new glyco-conjugates are more water-soluble than silibinin, and their radical scavenging activities are higher than those of silibinin. The conjugation therefore improves both the water solubilities and antioxidant activities of the flavonolignan moieties. The serum stability was evaluated under physiological conditions, and the glyco-conjugates degraded with half-lives of 40–70 h, making them useful in pro-drug approaches. We started by treating androgen-dependent prostate cancer (PCa) LNCaP cells and then expanded our studies to androgen-independent PCa PC3 and DU145 cells. In most cases, the new derivatives significantly reduced both total and live cell numbers, albeit at different levels. Anti-HIV activities were evaluated and the glucosamine-phosphate silibinin derivative showed higher activity (IC₅₀ = 73 μM) than silibinin.     

Novel thiazolo-pyrazolyl derivatives as xanthine oxidase inhibitors and free radical scavengers

Xanthine oxidase (XO) is a complex metalloflavoprotein, overproduction of which usually leads to a pathological condition called Gout. XO inhibitors may prove to be promising antigout agents. Present investigation describes synthesis, characterization and evaluation of 26 thiazolo-pyrazolyl derivatives V(a-z) for XO inhibitory and free radical scavenging activities. Derivatives Vq, Vo and Vh showed most promising XO inhibitory and free radical scavenging activities on the basis of their IC(50) values ranging from (6.5-9 μM). Significant dock scores compared with Allopurinol have been figured out using molecular docking. Evaluation of Vq, Vo and Vh for both the activities for first time may provide a new approach for antigout research.     

Screening and Inhibition Mechanism of Xanthine Oxidase Inhibitors in Ethanolic Extracts of Chimonanthus salicifolius Hu Leaves

This study aimed to evaluate the inhibition of the ethanol elutions of Chimonanthus salicifolius Hu leaves (CsHL) against xanthine oxidase (XO). The results of XO inhibition assay and enzymatic superoxide free radical scavenging assay in vitro showed that 70 % ethanol eluate (EE) had the best inhibitory effect and followed by 40 % EE. High performance liquid chromatograph analysis showed that quercetin and kaempferol were the potential active components of XO inhibition. The inhibition mechanism of quercetin and kaempferol on XO was investigated by kinetic analysis and fluorescence quenching titration assay. The molecular simulation further revealed that quercetin and kaempferol bind to XO mainly by hydrogen bonding and van der Waals, blocking the entry of substrates and leading to the inhibition of XO. In conclusion, the CsHL have inhibitory effects on XO activity, which provides a theoretical basis for relieving or preventing hyperuricemia and gout as a natural food or medicinal plant in the future.     

ortho-dihydroxyisoflavone derivatives from aged Doenjang (Korean fermented soypaste) and its radical scavenging activity

One new ortho-dihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone (2), and two known ortho-dihydroxyisoflavone derivatives were isolated from 5-year-old Doenjang (Korean fermented soypaste), and evaluated as potent antioxidant by comparing with other known isoflavones. 7,8,4'-Trihydroxyisoflavone (1), 7,3',4'-trihydroxyisoflavone (2), and 6,7,4'-trihydroxyisoflavone (3) inhibited DPPH (Diphenyl-1-picryl hydrazyl) formation by 50% at a concentration of 21.5+/-0.2, 28.7+/-0.4 and 32.6+/-0.6 (IC(50)), respectively, whereas three isoflavones showed weak DPPH radical scavenging activity. In xanthine oxidase (XO) system, in which both inhibition of xanthine oxidase and superoxide scavenging effect were measured in one assay. Compound 1 (IC(50)= 6.6+/-0.4 microM) and 2 (IC(50)=16.8+/-1.2 microM) show significant inhibitory activity and greater effect than allopurinol. But, compound 3 and other isoflavones showed lower inhibition activity. This study shows that the position of hydroxyl substituent at the aromatic ring of isoflavone plays an important role in radical scavenging effect.     

Antioxidant and free radical scavenging activities of an exopolysaccharide from a probiotic bacterium

Probiotic bacteria synthesize extracellular polysaccharides (EPSs) with commercially significant physiological and therapeutic activities. This important class of biomolecules is also characterized by their ability to remove reactive oxygen species (ROS) that are formed in the intestine by various metabolic reactions; hence, they exhibit antioxidant activities. Our probiotic bacterium, Bacillus coagulans RK-02, produces an EPS during the exponential and stationary growth phases when grown in a glucose mineral salts medium. The time course of EPS synthesis was studied with respect to biomass growth. The antioxidant and free radical scavenging potential of isolated EPS were studied by various methods, including the beta-carotene-linoleic acid model system, a superoxide radical scavenging assay using the PMS-NADH-nitroblue tetrazolium system, the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, a hydroxyl radical scavenging assay using the ascorbic acid-Cu(2+)-cytochrome c system and an in vitro microsome peroxidation inhibition study using a thiobarbituric acid assay. The antioxidant activities were compared to known antioxidants vitamin C and E, which were used as reference standards. The results showed that the EPS, which is a heteropolymer composed of four monosaccharides, produced by B. coagulans RK-02 had significant antioxidant and free radical scavenging activities.     

Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and radical scavengers

In this study, we evaluated the abilities of a series of chalcones to inhibit the activity of the enzyme xanthine oxidase (XO) and to scavenge radicals. 20 mono- and polyhydroxylated chalcone derivatives were synthesized by Claisen–Schmidt condensation reactions and then tested for inhibitory potency against XO, a known generator of reactive oxygen species (ROS). In parallel, the ability of the synthesized chalcones to scavenge a stable radical was determined. Structure–activity relationship analysis in conjunction with molecular docking indicated that the most active XO inhibitors carried a minimum of three hydroxyl groups. Moreover, the most effective radical scavengers had two neighboring hydroxyl groups on at least one of the two phenyl rings. Since it has been proposed previously that XO inhibition and radical scavenging could be useful properties for reduction of ROS-levels in tissue, we determined the chalcones’ effects to rescue neurons subjected to ROS-induced stress created by the addition of β-amyloid peptide. Best protection was provided by chalcones that combined good inhibitory potency with high radical scavenging ability in a single molecule, an observation that points to a potential therapeutic value of this compound class.     

Measurement of relative antioxidant activity of compounds: a methodological note

The relative activities of some hydrogen-donating antioxidants were assessed by comparing their activities with that of Trolox (Trolox equivalent antioxidant capacity, TEAC) for scavenging the ABTS radical cation (ABTS.+) generated in the aqueous phase. We have verified, however, that TEAC values may change with the concentration of compounds and with the measuring times used. Not withstanding, TEAC values do not differ significantly if the compounds have kinetic curves of ABTS.+ formation similar to that of Trolox. This is the case with ascorbic acid, whose TEAC values, determined by using five concentrations at three different measuring times, are very close. For the flavonoids studied (catechin, rutin, naringenin and silibinin) which have kinetic curves of ABTS.+ formation different from that of Trolox, the TEAC values decrease with increasing concentrations of the compounds for each measuring time, and increase with increasing measuring times for each concentration. In the present study, we conclude that, in order to evaluate relative antioxidant activities of compounds by the ABTS assay, it is essential to perform kinetic studies to assess scavenging of ABTS.+ by these compounds. Therefore, when the TEAC values of compounds are determined for more than one measuring time, we may be sure that all the antioxidant potential of compounds is being considered and whether or not it is possible to establish a hierarchy for their antioxidant activities.     

Enzyme inhibition and radical scavenging activities of aerial parts of Paeonia emodi Wall (Paeoniaceae)

The ethanolic extract derived from aerial parts of an indigenous medicinal plant Paeonia emodi was screened for enzyme inhibition activities against Urease (jack bean and Bacillus pasteurii) and alpha-Chymotrypsin. The extract was also investigated for its radical scavenging activity using DPPH assay. The crude extract was found to possess significant enzyme inhibition activities against jack bean (74%) and Bacillus pasteurii (80%) urease and a moderate activity (54%) against alpha-Chymotrypsin. The extract also displayed excellent (83%) radical scavenging activity. On the basis of these results, the crude extract was subsequently fractionated into n-hexane, chloroform, ethyl acetate, n-butanol and water fractions and tested independently for the aforesaid activities. Significant inhibitory activity against urease enzyme was observed for the ethyl acetate, n-butanol and water fractions while the n-hexane and chloroform fractions were devoid of any such activity. In the alpha-Chymotrypsin enzyme inhibition studies the activity was concentrated into the ethyl acetate fraction. All the fractions displayed potent radical scavenging activity. The crude extract and fractions thereof were also subjected to total phenolic content determination. A correlation between radical scavenging capacities of extracts and total phenolic content was observed in the majority of cases.     

In vitro antioxidant activity of silymarin

Silymarin, a known standardized extract obtained from seeds of Silybum marianum is widely used in treatment of several diseases of varying origin. In the present paper, we clarified the antioxidant activity of silymarin by employing various in vitro antioxidant assay such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH(.)) scavenging, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by Fe3+ - Fe2+ transformation method and Cuprac assay, superoxide anion radical scavenging by riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe2+) chelating activities. Silymarin inhibited 82.7% lipid peroxidation of linoleic acid emulsion at 30 microg/mL concentration; butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol and trolox indicated inhibition of 83.3, 82.1, 68.1 and 81.3% on peroxidation of linoleic acid emulsion at the same concentration, respectively. In addition, silymarin had an effective DPPH(.) scavenging, ABTS(.)+ scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe3+) reducing power by Fe3+-Fe2+ transformation, cupric ions (Cu2+) reducing ability by Cuprac method, and ferrous ions (Fe2+) chelating activities. Also, BHA, BHT, alpha-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. Moreover, this study, which clarifies antioxidant mechanism of silymarin, brings new information on the antioxidant properties of silymarin. According to the present study, silymarin had effective in vitro antioxidant and radical scavenging activity. It could be used in the pharmacological and food industry because of its antioxidant properties.     

Superoxide anion scavenging and xanthine oxidase inhibition of (+)-catechin-aldehyde polycondensates. Amplification of the antioxidant property of (+)-catechin by polycondensation with aldehydes

In this study, the antioxidant property of (+)-catechin-aldehyde polycondensates has been examined. Superoxide anions are one of the most typical reactive oxygen species (ROS) and generated by xanthine oxidase (XO). The measurements of the superoxide anion scavenging and XO inhibition activity showed that catechin had pro-oxidant properties in lower concentrations and little XO inhibition. On the other hand, the polycondensates exhibited much higher effects compared to the catechin monomer, and their physiological activities were greatly affected by the structure of polycondensates. Steady-state analysis of the inhibition against XO showed that the inhibition type of the polycondensate was uncompetitive. Furthermore, the results of the circular dichroism and UV-visible measurements of a mixture of the polycondensate and XO were in good agreement with that of the steady-state analysis; the spectral changes due to the chelation of the polycondensate onto the Fe/S and/or the FAD center of XO were observed. These data strongly suggest that the polycondensates possess a great potential as antioxidant for various applications.     

Measurement of relative antioxidant activityof compounds: a methodological note

Abstract

The relativeThe relative activities of some hydrogen-donating antioxidants were assessed by comparing their activities with that of Trolox (Trolox equivalent antioxidant capacity, TEAC) for scavenging the ABTS radical cation (ABTS^?+) generated in the aqueous phase. We have verified, however, that TEAC values may change with the concentration of compounds and with the measuring times used. Not withstanding, TEAC values do not differ significantly if the compounds have kinetic curves of ABTS^?+ formation similar to that of Trolox. This is the case with ascorbic acid, whose TEAC values, determined by using five concentrations at three different measuring times, are very close. For the flavonoids studied (catechin, rutin, naringenin and silibinin) which have kinetic curves of ABTS^?+ formation different from that of Trolox, the TEAC values decrease with increasing concentrations of the compounds for each measuring time, and increase with increasing measuring times for each concentration. In the present study, we conclude that, in order to evaluate relative antioxidant activities of compounds by the ABTS assay, it is essential to perform kinetic studies to assess scavenging of ABTS^?+ by these compounds. Therefore, when the TEAC values of compounds are determined for more than one measuring time, we may be sure that all the antioxidant potential of compounds is being considered and whether or not it is possible to establish a hierarchy for their antioxidant activities.     

Enzyme inhibition and radical scavenging activities of aerial parts of Paeonia emodi Wall. (Paeoniaceae)

The ethanolic extract derived from aerial parts of an indigenous medicinal plant Paeonia emodi was screened for enzyme inhibition activities against Urease (jack bean and Bacillus pasteurii) and α-Chymotrypsin. The extract was also investigated for its radical scavenging activity using DPPH assay. The crude extract was found to possess significant enzyme inhibition activities against jack bean (74%) and Bacillus pasteurii (80%) urease and a moderate activity (54%) against α-Chymotrypsin. The extract also displayed excellent (83%) radical scavenging activity. On the basis of these results, the crude extract was subsequently fractionated into n-hexane, chloroform, ethyl acetate, n-butanol and water fractions and tested independently for the aforesaid activities. Significant inhibitory activity against urease enzyme was observed for the ethyl acetate, n-butanol and water fractions while the n-hexane and chloroform fractions were devoid of any such activity. In the α-Chymotrypsin enzyme inhibition studies the activity was concentrated into the ethyl acetate fraction. All the fractions displayed potent radical scavenging activity. The crude extract and fractions thereof were also subjected to total phenolic content determination. A correlation between radical scavenging capacities of extracts and total phenolic content was observed in the majority of cases.     

Evaluation of free radical scavenging activity of Butea monosperma Lam

In the present study, ethyl acetate, butanol and aqueous fractions derived from total methanol extract of Butea monosperma flowers were evaluated for radical scavenging activities using different in vitro models like reducing power assay, scavenging of 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical, nitric oxide radical, superoxide anion radical, hydroxyl radical and inhibition of erythrocyte hemolysis using 2, 2' azo-bis (amidinopropane) dihydrochloride (AAPH). Methanol extract along with its ethyl acetate and butanol fractions showed potent free radical scavenging activity, whereas aqueous fraction was found to be devoid of any radical scavenging properties. The observed activity could be due to the higher phenolic content in the extracts (16.1, 25.29, and 17.74% w/w in methanol extract, ethyl acetate and butanol fractions respectively). HPTLC fingerprint profile of the ethyl acetate and butanol fractions were developed which would serve as reference standard for quality control of the extracts.     

Dual-acting agents that possess free radical scavenging and antithrombotic activities: design, synthesis, and evaluation of phenolic tetrahydro-beta-carboline RGD peptide conjugates

A new approach to construct a single dual-acting agent is described. Compounds 6a-c are potent free radical scavengers as demonstrated by the EC(50) values in PC12 cell survival assay in term of NO, H(2)O(2), and ()OH scavenging activity. The Ach-induced vaso-relaxation assay further confirms the potent NO scavenging activity of compounds 6a-c. In addition, 6a-c are efficacious in a rat arterial thrombosis, and are active in ADP- or PAF-induced in vitro platelet aggregation assay, suggesting that compounds 6a-c also possess anti-thrombotic activities. Since both free radical and thrombogenesis are important risk factors in myocardial ischemic/reperfusion injuries, these dual-acting agents having both free radical scavenging and antithrombolic activities may potentially be beneficial toward their treatment.     

Honey as an apitherapic product: its inhibitory effect on urease and xanthine oxidase

The aim of this study was to evaluate new natural inhibitor sources for the enzymes urease and xanthine oxidase (XO). Chestnut, oak and polyfloral honey extracts were used to determine inhibition effects of both enzymes. In addition to investigate inhibition, the antioxidant capacities of these honeys were determined using total phenolic content (TPC), ferric reducing antioxidant power (FRAP), and DPPH radical scavenging activity assays. Due to their high phenolic content, chestnut and oak honeys are found to be a powerful source for inhibition of both enzymes. Especially, oak honeys were efficient for urease inhibition with 0.012–0.021?g/mL IC₅₀ values, and also chestnut honeys were powerful for XO inhibition with 0.028–0.039?g/mL IC₅₀ values. Regular daily consumption of these honeys can prevent gastric ulcers deriving from Helicobacter pylori and pathological disorders mediated by reactive oxygen species.     

Bioorganic synthesis, characterization and antioxidant activity of esters of natural phenolics and α-lipoic acid

Chemo-enzymatic synthesis of six esters of natural phenolics and α-lipoic acid was carried to produce novel compounds with potential bioactivity. The synthetic route was mild, simple, and efficient with satisfactory yields. The synthesized compounds were screened for antioxidant activities. The prepared derivatives exhibited very good antioxidant activities as determined by DPPH radical scavenging assay and inhibition of lipid oxidation in fish oil emulsion system. Among the prepared derivatives, three compounds exhibited radical scavenging activity similar to the reference antioxidants, BHT and alpha-tocopherol in the DPPH radical scavenging assay, where as in fish oil emulsion system, two derivatives showed activity, which was similar to the reference antioxidants.     

In vitro antioxidant activity of silymarin

Silymarin, a known standardized extract obtained from seeds of Silybum marianum is widely used in treatment of several diseases of varying origin. In the present paper, we clarified the antioxidant activity of silymarin by employing various in vitro antioxidant assay such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH·) scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by Fe³⁺ ? Fe²⁺ transformation method and Cuprac assay, superoxide anion radical scavenging by riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe²⁺) chelating activities. Silymarin inhibited 82.7% lipid peroxidation of linoleic acid emulsion at 30 μg/mL concentration; butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox indicated inhibition of 83.3, 82.1, 68.1 and 81.3% on peroxidation of linoleic acid emulsion at the same concentration, respectively. In addition, silymarin had an effective DPPH· scavenging, ABTS^√+ scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe³⁺) reducing power by Fe³⁺ ? Fe²⁺ transformation, cupric ions (Cu²⁺) reducing ability by Cuprac method, and ferrous ions (Fe²⁺) chelating activities. Also, BHA, BHT, α-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. Moreover, this study, which clarifies antioxidant mechanism of silymarin, brings new information on the antioxidant properties of silymarin. According to the present study, silymarin had effective in vitro antioxidant and radical scavenging activity. It could be used in the pharmacological and food industry because of its antioxidant properties.     

Comparison of in vitro antioxidant and antiradical activities of L-tyrosine and L-Dopa

Summary. Phenolic compounds are interesting because of their antioxidant properties. In the present study, the antioxidant properties of L-tyrosine as a monophenolic and L-Dopa as a diphenolic amino acid were investigated by using different antioxidant assays: (i) 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH^•) scavenging; (ii) 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization assay; (iii) total antioxidant activity by ferric thiocyanate method; (iv) ferric ions (Fe³⁺) reducing power; (v) superoxide anion radical (O₂ ^•−) scavenging; (vi) hydrogen peroxide (H₂O₂) scavenging, and (vii) ferrous ions (Fe²⁺) chelating activities. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox, a water-soluble analogue of tocopherol, were used as the reference antioxidant compounds. At the same concentration (20 μg/mL), L-tyrosine and L-Dopa showed 30.6 and 67.9% inhibition of lipid peroxidation of linoleic acid emulsion, respectively. On the other hand, BHA, BHT, α-tocopherol and trolox indicated inhibitions of 74.4, 71.2, 54.7 and 20.1% on the peroxidation of linoleic acid emulsion, respectively, at the above-mentioned concentration. In addition, L-tyrosine and L-Dopa had an effect on DPPH radical scavenging, ABTS radical scavenging, superoxide anion radical scavenging, H₂O₂ scavenging, total ferric ions reducing power and metal chelating on ferrous ions activities.     

Synthesis and characterization of CAPE derivatives as xanthine oxidase inhibitors with radical scavenging properties

Inhibitors of the enzyme xanthine oxidase (XO) with radical scavenging properties hold promise as novel agents against reperfusion injuries after ischemic events. By suppressing the formation of damaging reactive oxygen species (ROS) by XO or scavenging ROS from other sources, these compounds may prevent a buildup of ROS in the aftermath of a heart attack or stroke. To combine these two properties in a single molecule, we synthesized and characterized the non-purine XO inhibitor caffeic acid phenethylester (CAPE) and 19 derivatives using a convenient microwave-assisted Knoevenagel condensation protocol. Varying systematically the number and positions of the hydroxyl groups at the two phenyl rings, we derived structure-activity relationships based on experimentally determined XO inhibition data. Molecular docking suggested that critical enzyme/inhibitor interactions involved π-π interactions between the phenolic inhibitor ring and Tyr914, hydrogen bonds between inhibitor hydroxyl groups and Glu802, and hydrophobic interactions between the CAPE phenyl ring and non-polar residues located at the entrance of the binding site. To effectively scavenge the stable radical DPPH, two hydroxyl groups in 1,2- or 1,4-position at the phenyl ring were required. Among all compounds tested, E-phenyl 3-(3,4-dihydroxyphenyl)acrylate, a CAPE analog without the ethyl tether, showed the most promising properties.     

Antioxidant activities of enzymatic extracts from brown seaweeds

Potential antioxidative activities of enzymatic extracts from seven species of brown seaweeds were evaluated using four different reactive oxygen species (ROS) scavenging assays containing DPPH (1,1-diphenyl-2-pricrylhydrazyl) free radical, superoxide anion, hydroxyl radical and hydrogen peroxide scavenging assay. The brown seaweeds were enzymatically hydrolyzed to prepare water-soluble extracts by using five carbohydrate degrading enzymes (Viscozyme, Celluclast, AMG, Termamyl and Ultraflo) and five proteases (Protamex, Kojizyme, Neutrase, Flavourzyme and Alcalase) of commercial and inexpensive enzymes obtained from Novozyme Co. (Novozyme Nordisk, Bagsvaerd, Denmark). The enzymatic extracts exhibited more prominent effects in hydrogen peroxide scavenging activity (approximately 90%) compared to the other scavenging activities and the activity of enzymatic extracts was even higher than that of the commercial antioxidants. In particular, Ultraflo and Alcalase extracts of S. horneri were dose-dependent and thermally stable. Moreover the two enzymatic extracts strongly inhibited DNA damage (approximately 50%). Those extracts showed significantly (p<0.05) remarkable scavenging effects in DPPH free radical scavenging assay and the activity indicated a marked correlation with phenolic contents. From the results, enzymatic extracts of the brown seaweeds might be valuable antioxidative sources.