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Antioxidative polyphenols from berries of Pimenta dioica.

The ethyl acetate-soluble part of allspice, berries of Pimenta dicica, showed strong antioxidant activity and radical-scavenging activity against 1,1diphenyl-2-picrylhydrazl (DPPH) radical. From the ethyl acetate-soluble part, two new compounds, 5-galloyloxy-3-4-dihydroxypentanoic acid and 5-(5-carboxmethyl-2-oxocyclopenty)3Z-penteny 6-O-galloy-beta-D-glucoside were isolated together with 11 known polyphenols by repeated column chromatography. Their structures were elucidated on the basis of MS and various NMR spectroscopic data. All isolated compounds were evaluated for antioxidative effects on oxidation of methyl linoleate under aeration and heating, anf on peroxidation of liposome induced by 2-2'-azobis-(2-amidinopropane)dihydrocloride (AAPH) as water-soluble initiator along with their radical-scavenging activity against DPPH. Quercetin and its glycoside showed remarkable activity for scavenging DPPH radical and inhibiting peroxidation of liposome. Two new compounds also exhibited strong DPPH radical-scavenging activity and inhibitory effect on the peroxidation od liposome as myricetin.

Antioxidants↗

Antioxidant activity and protective effect on DNA strand scission of Rooibos tea (Aspalathus linearis).

Rooibos tea (Aspalathus linearis) was extracted by refluxing with water and 75% ethanol as a solvent. Antioxidant activity and protective effect on DNA strand scission were investigated by using different antioxidant assay systems and DNA strand nicking assay, respectively. 75% Ethanol extract has higher content of total soluble phenolics and flavonoid than water extract. Antioxidant activities such as hydrogen donating capacity and scavenging activity of hydrogen peroxide were higher in 75% ethanol extract than in water extract except the rate constant with hydroxyl radical. Peroxyl radical induced DNA strand scission was prevented by both 75% ethanol and water extract and hydroxyl radical induced DNA strand scission was not. This result indicates that total soluble phenolics, specially flavonoid, of Rooibos tea are responsible for several kinds of antioxidant activities and preventive activity on peroxyl radical induced DNA strand scission.

Antioxidants↗

Radical scavenging mechanism of phenol carboxylic acids: reaction of protocatechuic esters.

Solvent-dependency of the antiradical reaction of protocatechuic acid (3,4-dihydroxybenzoic acid, PA) and its esters has been investigated. In aprotic solvents, methyl protocatechuate (PAMe) was readily oxidized by two molar equivalents of DPPH radical and converted to protocatechuquinone methyl ester (PQMe). On the other hand, in alcoholic solvents such as methanol, PAMe rapidly consumed five radicals in 30 min and changed to complex oxidized mixtures. A 1H-NMR analysis of the reaction mixture of PAMe and DPPH radical in methanol showed that PAMe was rapidly converted to PQMe and its 3-hemiacetal. In addition, a signal of 2-methoxy-PQMe 3-hemiacetal was also detected in the reaction mixture. The results suggested that PQMe undergoes a nucleophilic attack by the solvent alcohol molecule at the C-2 of the ring in methanol, leading to a regeneration of catechol structure, which accounts well for the higher DPPH radical scavenging activity of PAMe in alcohols than in aprotic solvents.

Biphenyl Compounds↗

DPPH radical scavenging activity of ten natural p-terphenyl derivatives obtained from three edible mushrooms indigenous to China.

The antioxidant activities of ten natural p-terphenyl derivatives, 1-10, obtained from the fruiting bodies of three edible mushrooms (Thelephora ganbajun, Thelephora aurantiotincta, Boletopsis grisea) indigenous to China were evaluated in comparison with BHA ('butylated hydroxyanisole'=(1,1-dimethylethyl)-4-methoxyphenol) and alpha-tocopherol by the DPPH ('1,1-diphenyl-2-picrylhydrazyl'=2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl) radical-scavenging method. The compounds 1-3 showed significant antioxidant activity. The antioxidant activities of compounds 1-10 and reference compounds followed the order: 2>BHA>1>3>alpha-tocopherol>10>9>6 > 5>8>7>4. The compound 2 exhibited the strongest radical-scavenging activity with an EC(50) value of 0.07 (EC(50)(BHA) 0.09; EC(50)(alpha-tocopherol) 0.25).

Agaricales↗

New phenolic constituents from Balanophora polyandra with radical-scavenging activity.

The 80% acetone extract of Balanophora polyandra Griff. (Balanophoraceae) was found to exhibit high radical-scavenging activity (SC(50)=14.48 mug/ml) towards 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. Further chemical investigation led the isolation of two new hydrolysable tannins, balapolyphorins A (1) and B (2), together with 20 known phenolic compounds (3-22). Their structures were established by detailed spectroscopic analysis, and the radical-scavenging properties of all isolated compounds were determined by DPPH assay.

Acetone↗

Insight into the free-radical-scavenging mechanism of hydroxyl-substituent Schiff bases in the free-radical-induced hemolysis of erythrocytes.

This work aimed to explore the mechanism by which hydroxyl-substituent Schiff bases scavenge free-radicals. Thus, four Schiff bases, that is benzylidene aniline (BAN), 2-(phenyliminomethyl)phenol (BAH), 4-benzimidoylphenol (PBH) and 2-benzimidoylphenol (OBH), were applied to protect human erythrocytes against 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced hemolysis. The results revealed that the --OH attached to the ortho-position of methylene in Schiff base scavenges 1.46 radicals per molecule, the --OH attached to the para-position of the N atom scavenges 2.94 radicals and the --OH attached to the ortho-position of the N atom scavenges 3.63 radicals. In addition, four Schiff bases were used together with some familiar antioxidants, such as 6-hydroxyl-2,5,7,8-tetramethyl chroman-2-carboxylic acid (Trolox), L-ascorbic acid (VC), alpha-tocopherol (TOH) and L-ascorbyl-6-laurate (VC-12) in AAPH-induced hemolysis of erythrocytes. It was found that, except for BAN+VC-12, BAH + VC-12, OBH + VC-12 and PBH+TOH, all the other combinations protected erythrocytes more perfectly than when used individually. This result demonstrated that a promotive protection existed between Schiff base and other antioxidants and this improved their ability to scavenge free-radicals. Finally, IC(50) values of the aforementioned Schiff bases together with 2-((o-hydroxylphenylimino) methyl)phenol (OSAP) and 2-((p-hydroxylphenylimino)methyl)phenol (PSAP) were determined by reaction with two radical species, that is, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical (ABTS(+.)) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH). The results implied that the molecular framework of a Schiff base and an --OH attached to the ortho-position of methylene were apt to reduce radicals, but the --OH attached to the aniline ring in a Schiff base was prone to scavenge radicals directly.

Amidines↗

Separated and aligned molecular fibres in solid state self-assemblies of cyclodextrin [2]rotaxanes.

The conformations of two [2]rotaxanes, each comprising alpha-cyclodextrin as the rotor, a stilbene as the axle and 2,4,6-trinitrophenyl substituents as the capping groups, have been examined in solution and in the solid state, using (1)H NMR spectroscopy and X-ray crystallography, respectively. In solution, introducing substituents onto the stilbene prevents the cyclodextrin from being localized over one end of the axle. Instead the cyclodextrin moves back and forth along the substituted stilbene. In the solid state, the axles of the rotaxanes form extended molecular fibres that are separated from each other and aligned along a single axis. The molecular fibres are strikingly similar to those formed by the axle component of one of the rotaxanes in the absence of the cyclodextrin, but in the latter case they are neither separated nor all aligned.

Crystallography, X-Ray↗

Split unresponsiveness to the trinitrophenyl determinant. I. Manoeuvers which suppress either humoral or cell-mediated immune responses.

2,4,6-Trinitrobenzene sulfonic acid (TNBS) injected intravenously (i.v.) makes mice fully tolerant to the trinitrophenyl (TNP) determinant. Administration of in vitro TNP-labeled syngeneic erythrocytes or thymocytes renders mice unable to develop contact sensitivity to picryl chloride, while the humoral anti-TNP responses seem to be unaffected. The reverse was found after pretreatment of mice with TNP-labeled isologous IgG (MGG) since only anti-TNP antibody responses, but not contact sensitivity to picryl chloride, were significantly reduced. TNP-coupled macrophages given to animals suppressed both the cell-mediated and humoral responses, and this might be due to the presence on their surface of TNP-labeled cytophilic antibody. TNBS administered i.v. binds to circulating proteins and formed blood elements. Thus the split unresponsiveness affecting either humoral or cell-mediated compartments after the injection of TNP-MGG or of haptenated cells respectively, is presumably due to dissecting events which in vivo after the injection of TNBS, occur simultaneously. These results may be interpreted to indicate that split unresponsive states to TNP determinants are mediated two independent mechanisms which require different tolerogen presentations to be triggered.

Animals↗

Cross-reactivity of IgG anti-DNA-secreting B cells in patients with systemic lupus erythematosus.

This study is the first to analyze the cross-reactivity of in vivo activated B cells from patients with systemic lupus erythematosus. A chamber ELIspot assay was used to determine whether lymphocytes secreting antibodies that bound to DNA or 2,4,6-trinitrophenol (TNP)-keyhole limpet-hemocyanin (KLH) could simultaneously bind to the unrelated antigens actin or ovalbumin. IgM anti-DNA-, IgM anti-TNP-KLH- and IgG anti-TNP-KLH-secreting B cells from patients and controls showed similar levels of cross-reactivity (ranging from 6% to 23%, depending upon the antibody isotype and antigen pair examined). In general, IgG-producing cells were less cross-reactive than IgM producers from the same individual (on the average threefold, p < 0.001). In contrast, IgG anti-DNA-secreting B cells from lupus patients (i) showed no decrease in cross-reactivity when compared to IgM anti-DNA-secreting cells and (ii) were significantly more cross-reactive than control IgG anti-DNA-secreting cells and IgG anti-TNP-KLH secreting cells from patients (p < 0.001). The degree of IgG anti-DNA cross-reactivity correlated with disease activity (r = 0.52, p < 0.02). The implications of these findings with respect to repertoire expression and disease pathogenesis are discussed.

Adult↗

Step change of counterion--a new option in capillary zone electrophoresis.

In contrast to common capillary zone electrophoresis, with a constant composition of the background electrolyte, a new method is described, based on a change of the ionic matrix composition during the migration of the sample components. The proposed method expands the scale of pK's of analyzed compounds in a single run and affords new potential for optimizing the separation. The step change of the ionic matrix composition was achieved by displacing the primary matrix by the modified ionic matrix (electrolyte) which migrated against the movement of the sample components. The separation thus proceeded in the primary ionic matrix (electrolyte) while the detection was performed in the modified matrix with resultant shortening of the analysis time and convenient quantitation. A model mixture of five compounds, with a difference of four units in pK could be well resolved.

Dinitrophenols↗

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation.

A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.

Acrylamides↗

The silver staining procedure of sodium dodecyl sulfate-gels may be accelerated by shortening fixation time.

In most silver staining methods the first step in the staining of proteins separated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis is a rather protracted fixation of the gels. Optimum fixation should be short, cause no background staining and effectively immobilize the proteins in the gel without masking the proteins for reaction with the staining solution. Further, the concentration of the fixing compounds should be as low as possible due to the potential toxicity of fixatives. Fixation for only 5 min with mixtures of very low concentrations of formaldehyde and glutaraldehyde in ethanol, or a solution of formaldehyde or glutaraldehyde in picric acid and ethanol, fulfill these demands, provided that the gels were prefixed in ethanol-acetic acid for 10 min. As a consequence of these results a fast and sensitive silver staining procedure is proposed.

Amino Acids↗

Evidence of intra- and extracellular modifications of monoclonal IgG polypeptide chains generating charge heterogeneity.

The heavy and light chains of IgG monoclonal antibodies (mAbs) can be shown to be heterogeneous, with respect to isoelectric points, when analyzed by two-dimensional electrophoresis (2-DE). The molecular basis for this charge heterogeneity has not been clearly defined but it has been suggested that it could be due, in part, to differences in glycosylation. To investigate this possibility we have compared the 2-DE pattern of glycosylated and aglycosylated forms of the mouse IgG1 mAb (1B7-11), produced in vitro in the presence and absence of tunicamycin. Charge heterogeneity was shown not to be a consequence of glycosylation status. Intracellular and secreted IgG mAbs were also analyzed to investigate the time course of change in charge properties of the heavy and light chains. The charge heterogeneity was found to be generated intracellularly, and alterations in charge properties could be induced during incubation under physiological conditions. Semilogarithmic plots of the density of the principal heavy and light chain spots against incubation time showed linear relationships, suggesting that the charge shifts result from a first-order reaction. The semilogarithmic plot for the light chain correlated well with the time after IgG synthesis. These results suggest that the charge heterogeneity of an IgG mAb is due to intra- and extracellular modifications of the polypeptide chains which reflect "aging" of antibody molecules.

Animals↗

A new type of migrating zone boundary in electrophoresis: 3. The hybrid boundary and stacking criteria.

This article investigates the principles of stacking of analytes by a zone of a bulk sample component that possesses one hybrid boundary. The model system investigated comprises a transient stacking zone in a BGE where the rear boundary of this zone is a hybrid one. A theoretical description of such a system is given, and general rules and mathematical criteria for quantitative stacking are presented. These criteria are based on comparison of migration velocities of analytes to be stacked and velocities of the boundaries of the stacking zone. It is shown that the presence of a hybrid boundary brings about additional constraints for stacking of analytes due to the presence of the sharp part of the hybrid boundary that migrates faster than regular sharp boundary of this zone. An experimental example related to the described theory is also shown.

Computer Simulation↗

Protection from radiation-induced mitochondrial and genomic DNA damage by an extract of Hippophae rhamnoides.

Hippophae rhamnoides or seabuckthorn is used extensively in Indian and Tibetan traditional medicine for the treatment of circulatory disorders, ischemic heart disease, hepatic injury, and neoplasia. In the present study, we have evaluated the radioprotective potential of REC-1001, a fraction isolated from the berries of H. rhamnoides. Chemical analysis of the extract indicated that REC-1001 was approximately 68% by weight polyphenols, and contained kaempferol, isorhamnetin, and quercetin. The effect of REC-1001 on modulating radiation-induced DNA damage was determined in murine thymocytes by measuring nonspecific nuclear DNA damage at the whole genome level using the alkaline halo assay and by measuring sequence/gene-specific DNA damage both in nuclear DNA (beta-globin gene) and in mitochondrial DNA using a quantitative polymerase chain reaction. Treatment with 10 Gy resulted in a significant amount of DNA damage in the halo assay and reductions in the amplification of both the beta-globin gene and mitochondrial DNA. REC-1001 dose-dependently reduced the amount of damage detected in each assay, with the maximum protective effects observed at the highest REC-1001 dose evaluated (250 micro g/ml). Studies measuring the nicking of naked plasmid DNA further established the radioprotective effect of REC-1001. To elucidate possible mechanisms of action, the antioxidant properties and the free-radical scavenging activities of REC-1001 were evaluated. REC-1001 dose-dependently scavenged radiation-induced hydroxyl radicals, chemically-generated superoxide anions, stabilized DPPH radicals, and reduced Fe(3+) to Fe(2+). The results of the study indicate that the REC-1001 extract of H. rhamnoides protects mitochondrial and genomic DNA from radiation-induced damage. The polyphenols/flavonoids present in the extract might be responsible for the free radical scavenging and DNA protection afforded by REC-1001.

Animals↗

[Comparative investigations on the precipitation behavior of various protein precipitates in organs and tissues].

The differences of the non-precipitable N-quota in blood plasma, liver, intestine, and muscles after treatment with various precipitants reveal data on the height of protein precipitation in the corresponding body fraction. Organs of hens treated with picric acid (1%), trichloroacetic acid (10%), and sulphosalicylic acid were used for the protein precipitation. Because of contradictory literature data as to the most suitable concentration of sulphosalicylic acid a preliminary determination of the most favourable acid concentration was necessary. The application of a 5% solution of sulphosalicylic acid gave the highest precipitation rate depending on the analyzed organs. In the succession picric acid, trichloroacetic acid, and sulphosalicylic acid nitrogen increases in the soluble supernatant. Furthermore, dependences of the protein precipitation on the kind of the analyzed organs were indicated.

Animals↗

Antimutagenic and antiradical properties of flavones from the roots of Scutellaria baicalensis georgi.

The four flavonoids baicalin, baicalein, wogonoside, and wogonin have been isolated from Scutellaria baicalensis (Lamiaceae) root extract as main bioactive components. We evaluated their antimutagenic activity as potential chemopreventive natural substances using the Ames test. Free radical scavenging properties have also been measured with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Both crude extract and isolated flavone glycosides and aglycones have antimutagenic and free radical scavenging properties. The lipophilic flavone baicalein has been confirmed to be the most powerful antimutagen among the compounds tested.

Antimutagenic Agents↗

Effects of drying process on antioxidant activity of purple carrots.

Purple carrot was analysed for its antioxidant activity by measuring its scavenging effect on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. The amount of purple carrot necessary to decrease by 50% the initial DPPH concentration (EC50) was found 30.23 +/- 5.17 (mg sample/mg DPPH). Effects of the dehydration process on the antioxidant activity of purple carrots were also determined after dehydration by using tray drier and microwave+tray drier combination. Purple carrots were cleaned, washed, sorted, sliced, and blanched in water at 98 +/- 1 degree C for 2 min. Then half of the samples were dehydrated in microwave (45 min) and tray drier (105 min) combination, and others were dehydrated in tray drier (150 min) until reaching the constant weight. Raw, blanched and dehydrated purple carrots were compared for their antioxidant activity and total anthocyanin contents, color values (L*, a*, b*, C*, H degrees) and dry matter contents. The antioxidant activity as measured by DPPH, EC50 value ranged from 7.80 to 30.23 of processed and raw carrots where the H degrees values were changed from -3.68 to -23.96 corresponding to a bluish hue.

Air↗