In vitro and in vivo anti-arthritic evaluation of Polystichum braunii to validate its folkloric claim.

Medicinal plants are playing an imperative role in the therapy for treating various chronic ailments including arthritis. The present study was focused on finding in-vitro and in-vivo anti-arthritic potential of P. braunii roots. In vitro protein denaturation, membrane stabilization and anti-trypsinase assays were carried out to demonstrate anti-arthritic activity of the extracts. Furthermore, the extracts exerting promising in vitro anti-arthritic potential were tested orally at 150, 300 and 600mg/kg/day against formaldehyde induced arthritis in Wistar rats. The methanolic, aqueous and ethyl acetate extracts of the plant revealed noteworthy in vitro anti-arthritic activities while mitigating formaldehyde induced paw edema in dose dependent manner. Methanolic and aqueous extracts showed the highest inhibition (p<0.05) of paw edema, arthritic indices, reduced elevated level of platelets and leukocytes while increasing hemoglobin and body weight of arthritic rats. Anti-arthritic activity of the plant extracts may be due to inhibition of protein denaturation and lysosomal membrane stabilization. The plant exhibited good anti-arthritic potential.

Policosanol composition, antioxidant and anti-arthritic activities of milk thistle (Silybium marianum L.) oil at different seed maturity stages.

BACKGROUND: Several anti-arthritic drugs and synthetic antioxidants have wide pharmaceutical uses and are often associated with various side effects on the human health. Dietary seed oils and their minor components like policosanol may offer an effective alternative treatment for arthritic and oxidative-stress related diseases. The biological effects of seed oils were affected by different parameters such as the stage of seed maturity. Hence, this study seeks to determine the policosanol content, antioxidant and anti-arthritic activities of milk thistle (Silybium marianum L.) oil extracted at various stages of seed maturation. METHODS: Milk thistle oil samples were extracted from seeds collected at three maturation stages (immature, intermediate, and mature). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) radical scavenging assays were used to determine the antioxidant activity of the extracted oils. The anti-arthritic activity of oil samples was evaluated with bovine serum protein denaturation and egg albumin denaturation methods. Gas chromatography coupled to mass spectrometry (GC-MS) was employed to determine the policosanol profile. RESULTS: Policosanol profile, antioxidant and anti-arthritic activities of milk thistle oil were influenced by the seed maturity stages. The oil extracted from the immature seeds had the highest total policosanol content (987.68 mg/kg of oil) and displayed the maximum antiradical activity (96.42% and 90.35% for DPPH test and ABTS assay, respectively). Nine aliphatic alcohols were identified in the milk thistle oil. The dominant poliosanol in the mature seed oil was octacosanol (75.44%), while triacontanol was the major compound (40.25%) in the immature seed oil. Additionally, the maximum inhibition of bovine serum protein denaturation (92.53%) and egg albumin denaturation (86.36%) were observed in immature seed oil as compared to mature seed oil. A high correlation was found between the total policosanol content, anti-arthritic activity and antioxidant capacity of oil. CONCLUSIONS: The milk thistle oil exhibited a potential anti-arthritic and antioxidant activities and that it might contribute to the protection of humans from a variety of diseases like rheumatoid arthritis. Also, it could serve as natural antioxidant and anti-arthritic agents for application in the food industries and pharmaceutic. Policosanol level in the seed oils might contribute to their anti-arthritic and antioxidant activities.

Synthesis and structure elucidation of novel salophen-based dioxo-uranium(VI) complexes: In-vitro and in-silico studies of their DNA/BSA-binding properties and anticancer activity.

The synthesis and characterization of three dioxo U(VI) complexes, [UO2(L1)(OH2)], [UO2(L2)DMF], and [UO2(L2)DMSO], [L1]2- = 1,1'-(4-methyl-1,2-phenylenebis (nitrilomethylidyne))di-2-naphtholate: [L2]2- = 1,1'-(o-phenylenebis (nitrilomethylidyne)) di-2-naphtholate, are reported. Elemental analysis, FT-IR, 1HNMR, UV-Vis spectroscopy, molar conductivity and single crystal X-ray diffraction were used to characterize the complexes. It was found that the complexes adopt a distorted pentagonal bipyramidal coordination geometry. The interaction of the synthesized complexes with DNA and bovine serum albumin was thoroughly investigated using both experimental and theoretical studies. UV-Vis absorption and fluorescence quenching techniques were applied to determine the binding parameters as well as the mechanism of the interaction of each complex with DNA and the protein. The results obtained suggested that interaction of the complexes with DNA occurred through partial intercalation into the minor grooves of DNA with binding constants in the range of 0.661 × 105-1.56 × 105 M-1. In addition, interaction of the complexes with bovine serum albumin quenched the fluorescence emission of the tryptophan residues of the protein binding constants and thermodynamic parameters were obtained from the fluorescence quenching experiments at different temperatures. The values of binding constants revealed moderate interactions between the synthesized complexes and the protein suggesting that this protein could act as a suitable vehicle for transportation of the compounds. The results of molecular docking confirmed those of the experimental studies. The anticancer properties of the title complexes were also evaluated through a study of the in vitro cytotoxicity of the compounds against the HT-29 and MCF-7 cancer cell lines and the DPSC normal cell line using an MTT assay.

Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential.

Glucitol-core containing gallotannins (GCGs) are polyphenols containing galloyl groups attached to a 1,5-anhydro-d-glucitol core, which is uncommon among naturally occurring plant gallotannins. GCGs have only been isolated from maple (Acer) species, including the red maple (Acer rubrum), a medicinal plant which along with the sugar maple (Acer saccharum), are the major sources of the natural sweetener, maple syrup. GCGs are reported to show antioxidant, α-glucosidase inhibitory, and antidiabetic effects, but their antiglycating potential is unknown. Herein, the inhibitory effects of five GCGs (containing 1-4 galloyls) on the formation of advanced glycation end-products (AGEs) were evaluated by MALDI-TOF mass spectroscopy, and BSA-fructose, and G.K. peptide-ribose assays. The GCGs showed superior activities compared to the synthetic antiglycating agent, aminoguanidine (IC50 15.8-151.3 vs. >300 µM) at the early, middle, and late stages of glycation. Circular dichroism data revealed that the GCGs were able to protect the secondary structure of BSA protein from glycation. The GCGs did not inhibit AGE formation by the trapping of reactive carbonyl species, namely, methylglyoxal, but showed free radical scavenging activities in the DPPH assay. The free radical quenching properties of the GCGs were further confirmed by electron paramagnetic resonance spectroscopy using ginnalin A (contains 2 galloyls) as a representative GCG. In addition, this GCG chelated ferrous iron, an oxidative catalyst of AGE formation, supported a potential antioxidant mechanism of antiglycating activity for these polyphenols. Therefore, GCGs should be further investigated for their antidiabetic potential given their antioxidant, α-glucosidase inhibitory, and antiglycating properties.

STUDIES ON THE INTERACTION BETWEEN TRIPTOLIDE AND BOVINE SERUM ALBUMIN (BSA) BY SPECTROSCOPIC AND MOLECULAR MODELING METHODS.

BACKGROUND: Triptolide is a major active constituent isolated from Tripterygiumwilfordii Hook F, a Chinese herbal medicine. This study investigated the intermolecular interaction between triptolide and bovine serum albumin (BSA). MATERIALS AND METHODS: The fluorescence, circular dichroism (CD) and molecular docking methods were used to investigate the intermolecular interaction between triptolide and BSA. The binding constant, the number of binding sites, binding subdomain and the thermodynamic parameters were measured. RESULTS: The results of this experiment revealed that the intrinsic fluorescence of BSA was effectively quenched by triptolide via static quenching. The experimental results of synchronous fluorescence and CD spectra showed that the conformation of BSA was changed in the presence of triptolide. CONCLUSION: It indicated that triptolide could spontaneously bind on site II (subdomain IIIA) of BSA mainly via hydrogen bonding interactions and Van der Waals force.

The inhibitory effect of selenium nanoparticles on protein glycation in vitro.

Selenium nanoparticles (Se NPs) possess well-known excellent biological activities and low toxicity, and have been employed for numerous applications except as inhibitors to protein glycation. Herein, the present study is carried out to investigate the inhibitory effect of Se NPs on protein glycation in a bovine serum albumin (BSA)/glucose system. By measuring the amount of glucose covalently bound onto BSA, the formation of fructosamine and fluorescent products, it is found that Se NPs can hinder the development of protein glycation in a dose-dependent but time-independent manner under the selected reaction conditions (55 °C, 40 h). And after comparing the increase of inhibitory rate in different stages, it is observed that Se NPs show the greatest inhibitory effect in the early stage, then in the advanced stage, but no effect in the intermediate stage. Fourier transform infrared spectroscopy characterization of Se NPs collected after glycation and determination of ·OH influence and glyoxal formation show that the mechanism for the inhibitory efficacy of Se NPs is related to their strong competitive activity against available amino groups in proteins, their great scavenging activity on reactive oxygen species and their inhibitory effect on α-dicarbonyl compounds' formation. In addition, it is proved that Se NPs protect proteins from structural modifications in the system and they do not exhibit significant cytotoxicity towards BV-2 and BRL-3A cells at low concentrations (10 and 50 µg mL(-1)). Consequently, Se NPs may be suitable for further in vivo studies as novel anti-glycation agents.

Design, synthesis and destructive dynamic effects of BODIPY-containing and curcuminoid boron tracedrugs for neutron dynamic therapy.

BACKGROUND: We previously designed the boron tracedrugs UTX-42, UTX-43, and UTX-44, which possess antioxidant potency. In order to explore their destructive dynamic effects when bombarded by weak thermal neutrons, we performed thermal neutron irradiation of bovine serum albumin (BSA) treated with the boron tracedrugs. MATERIALS AND METHODS: Boron tracedrugs, including the boron dipyrromethene (BODIPY)-containing compounds UTX-42, UTX-44, and UTX-47 and the curcuminoid compounds UTX-50 and UTX-51, were designed for neutron dynamic therapy based on their molecular orbital calculation. Newly designed UTX-47, UTX-50, and UTX-51 were synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to detect decomposition by thermal neutron irradiation of BSA treated with these boron tracedrugs. RESULTS: The combination of 1.0 µM BSA with 100 µM of each of the boron tracedrugs showed a decrease in band intensity after irradiation. CONCLUSION: All boron tracedrugs tested caused destructive dynamic damage of BSA during thermal neutron irradiation, suggesting that boron tracedrugs could be used as dynamic drugs for neutron dynamic therapy.

Confident identification of 3-nitrotyrosine modifications in mass spectral data across multiple mass spectrometry platforms.

3-nitrotyrosine (3NT) is an oxidative posttranslational modification associated with many diseases. Determining the specific sites of this modification remains a challenge due to the low stoichiometry of 3NT modifications in biological samples. Mass spectrometry-based proteomics is a powerful tool for identifying 3NT modifications, however several reports identifying 3NT sites were later demonstrated to be incorrect, highlighting that both the accuracy and efficiency of these workflows need improvement. To advance our understanding of the chromatographic and spectral properties of 3NT-containing peptides we have adapted a straightforward, reproducible procedure to generate a large set of 3NT peptides by chemical nitration of a defined, commercially available 48 protein mixture. Using two complementary LC-MS/MS platforms, a QTOF (QSTAR Elite) and dual pressure ion trap mass spectrometer (LTQ Velos), we detected over 200 validated 3NT-containing peptides with significant overlap in the peptides detected by both systems. We investigated the LC-MS/MS properties for each peptide manually using defined criteria and then assessed their utility to confirm that the peptide was 3NT modified. This broad set of validated 3NT-containing peptides can be utilized to optimize mass spectrometric instrumentation and data mining strategies or further develop 3NT peptide enrichment strategies for this biologically important, oxidative posttranslational modification.

Skin cell protection against UVA by Sideroxyl, a new antioxidant complementary to sunscreens.

Oxidative stress resulting from photosensitized ROS production in skin is widely accepted as the main contributor to the deleterious effects of UVA exposure. Among the mechanisms known to be involved in UVA-induced oxidative damage, iron plays a central role. UVA radiation of skin cells induces an immediate release of iron, which can then act as a catalyst for uncontrolled oxidation reactions of cell components. Such site-specific damage can scarcely be counteracted by classical antioxidants. In contrast, iron chelators potentially offer an effective way to protect skin against UVA insults. However, iron chelation is very difficult to achieve without disturbing iron homeostasis or inducing iron depletion. A novel compound was developed to avoid these potentially harmful side effects. Sideroxyl was designed to acquire its strong chelating capability only during oxidative stress according to an original process of intramolecular hydroxylation. Herein, we describe in vitro results demonstrating the protective efficiency of Sideroxyl against deleterious effects of UVA at the molecular, cellular, and tissular levels. First, the Sideroxyl diacid form protects a model protein against UVA-induced photosensitized carbonylation. Second, intracellular ROS are dose-dependently decreased in the presence of Sideroxyl in both human cultured fibroblasts and human keratinocytes. Third, Sideroxyl protects normal human fibroblasts against UVA-induced DNA damage as measured by the comet assay and MMP-1 production. Finally, Sideroxyl provides protection against UVA-induced alterations in human reconstructed skin. These results suggest that Sideroxyl may prevent UVA-induced damage in human skin as a complement to sunscreens, especially in the long-wavelength UVA range.

Quercus infectoria galls possess antioxidant activity and abrogates oxidative stress-induced functional alterations in murine macrophages.

The present study reports the antioxidant activity of ethanolic extract of Quercus infectoria galls. The antioxidant potency of galls was investigated employing several established in vitro model systems. Their protective efficacy on oxidative modulation of murine macrophages was also explored. Gall extract was found to contain a large amount of polyphenols and possess a potent reducing power. HPTLC analysis of the extract suggested it to contain 19.925% tannic acid (TA) and 8.75% gallic acid (GA). The extract potently scavenged free radicals including DPPH (IC(50)~0.5 microg/ml), ABTS (IC(50)~1 microg/ml), hydrogen peroxide (H(2)O(2)) (IC(50)~2.6 microg/ml) and hydroxyl (*OH) radicals (IC(50)~6 microg/ml). Gall extract also chelated metal ions and inhibited Fe(3+) -ascorbate-induced oxidation of protein and peroxidation of lipids. Exposure of rat peritoneal macrophages to tertiary butyl hydroperoxide (tBOOH) induced oxidative stress in them and altered their phagocytic functions. These macrophages showed elevated secretion of lysosomal hydrolases, and attenuated phagocytosis and respiratory burst. Activity of macrophage mannose receptor (MR) also diminished following oxidant exposure. Pretreatment of macrophages with gall extract preserved antioxidant armory near to control values and significantly protected against all the investigated functional mutilations. MTT assay revealed gall extract to enhance percent survival of tBOOH exposed macrophages. These results indicate that Q. infectoria galls possess potent antioxidant activity, when tested both in chemical as well as biological models.

3-O-beta-D-Galactopyranoside of quercetin as an active principle from high altitude Podophyllum hexandrum and evaluation of its radioprotective properties.

The aqueous-ethanolic extract (AEE) of high altitude Podophyllum hexandrum has earlier been reported to render a radioprotective effect against lethal gamma radiation in in vitro model. AEE has also been reported to possess metal chelating and DNA protecting properties. The present study was undertaken to isolate and characterize the bioactive principle present in AEE and investigate its role in radiation protection. A novel molecule was found to be present in AEE and was assigned as 3-O-beta-D-galactoside of quercetin by acid hydrolysis, LC-MS, LC-APCI-MS/MS and 13C NMR spectra. Various biological activities were investigated at in vitro level. The antioxidant potential of AEE in lipid and aqueous phase was determined against numerous stresses. AEE was found to be significantly (p < 0.05) protective, i.e., against Fe2+ and Cu2+-induced linoleic acid degradation, respectively. Radiation-induced lipid oxidation studies revealed that AEE maximally works at a [lignan]/0.25 kGy ratio 400 (ratio of concentration of AEE divided by the radiation dose, i.e., 0.25 kGy) and no drug-induced lipid oxidation at all concentrations tested was found. In a time-dependent study, total antioxidant activity was maximally exhibited at 1 mg/ml. The site-specific and non-site-specific deoxyribose degradation assay exhibited a dose-dependant hydroxyl scavenging potential of AEE (0.05-500 microg/ml). The anti-lipid peroxidation ability of AEE against radiation (0.25 kGy)-induced lipid peroxidation was higher in case of neural tissue homogenate as compared to kidney homogenate [activity ratio: 0.039 (brain) < 0.24 (kidney)]. The protein protection study using bovine serum albumin was also done for two time intervals (2 h and 4 h) and significant (p < 0.05) protection was observed at 500 microg/ml (> 97%). This study implies that 3-O-beta-D-galactoside present in AEE renders radioprotection by protecting lipids, proteins in renal and neural model system against supra-lethal (0.25 kGy) gamma radiation.

Antioxidative effects of polyphenols in leaves of Houttuynia cordata on protein fragmentation by copper-hydrogen peroxide in vitro.

The leaves of Houttuynia cordata have been traditionally used as medicinal foods in East Asia. However, there have been few data about the biological effects. The antioxidative effects of polyphenols in the leaves of H. cordata were investigated on protein fragmentation by copper-hydrogen peroxide in vitro. The total polyphenol content in the leaves of H. cordata was 1.14%. The condensed tannin content was 2.46% by vanillin assay and 0.54% by proanthrocyanidin assay. The polyphenols in the leaves of H. cordata inhibited bovine serum albumin fragmentation by copper-hydrogen peroxide. These results demonstrated that the leaves of H. cordata have antioxidative effects on biological damage such as protein fragmentation.

Inhibitory effects of polyphenols in leaves of Artemisia princeps PAMP on protein fragmentation by Cu(II)-H2O2 in vitro.

The leaves of Artemisia princeps PAMP have traditionally been used as teas and foods in Japan. Polyphenols in Artemisia plants have been shown to have inhibitory effects against biological damages. The inhibitory effects of polyphenols in the leaves of A. princeps PAMP were investigated on protein fragmentation induced by Cu(II)-H(2)O(2) in vitro. The total polyphenol content in the leaves of A. princeps PAMP was 4.58%. The condensed tannin content was 0.62% by vanillin assay and 0.14% by proanthrocyanidin assay. The polyphenols in the leaves of A. princeps PAMP inhibited bovine albumin fragmentation by Cu(II)-H(2)O(2). The effects of polyphenols in the leaves of A. princeps PAMP were similar to those of tannic acid, studied as a related polyphenol. These results demonstrated that the leaves of A. princeps PAMP have inhibitory effects on protein fragmentation damage.

Development of a screening assay to measure the loss of antibacterial activity in the presence of proteins: its use in optimizing compound structure.

An assay quantifying the loss of antibacterial potency of compounds, originally identified via target-based screening, in the presence of increasing albumin concentration was developed and used as a technique to measure potential association of compounds with proteins unrelated to their molecular target. Minimum inhibitory concentrations (MICs) of test compounds were measured against Staphylococcus aureus strain ATCC 6538 in the presence of 0-12 muM bovine serum albumin (BSA). The linear regression coefficient r(2) for the correlation between MIC and BSA concentration was >/= 0.9 for 49 and > 0.5 for 62 out of a total of 69 compounds tested. The slope of these correlations varied widely from < 1 to 99, suggesting that the loss of potency due to a given concentration of BSA could vary from compound to compound due to wide variation in the apparent stoichiometry for protein-ligand association. Follow-up experiments using additional proteins and a fatty acid, oleic acid, showed that this compound:BSA association was not protein specific, but was likely driven by hydrophobicity. The method described in this report can be used to optimize compound design and minimize this undesirable effect.

Effect of Mangifera indica L. extract (QF808) on protein and hepatic microsome peroxidation.

The antioxidant activities of QF808, a steam bark extract of Mangifera indica L., were studied on hydroxyl-mediated oxidation of bovine serum albumin (BSA) and in a hepatic microsome system. The extract was effective in reducing the oxidation of BSA, since its half- maximal inhibition concentration (IC(50)) was 0.0049% w/v in the inhibition of carbonyl group formation and lower than 0.0025% w/v in the inhibition of sulfhydryl group loss. QF808 inhibited lipid peroxidation which was initiated enzymatically by reduced nicotinamide adenine dinucleotide phosphate (NADPH), IC(50)= 0.00075% w/v, or non-enzymatically by ascorbic acid, IC(50) = 0.0126% w/v. The extract tested did not inhibit NADPH-dependent cytochrome P-450 reductase activity, since it had no effect on the oxidation rate of NADPH. These results suggest that QF808 has an antioxidant activity, probably due to its ability to scavenge free radicals involved in microsome lipid peroxidation. In addition, QF808 antioxidant profile in vitro is probably similar to its principal polyphenolic component, mangiferin, a glycosylated xanthone.

Ferric nitrilotriacetate induced DNA and protein damage: inhibitory effect of a fermented papaya preparation.

The carcinogen Fe-NTA catalyzes the hydrogen peroxide-derived production of free radicals and possibly acts through a mechanism involving oxidative stress. Fermented papaya preparation (FPP) has been reported as a natural antioxidant able to prevent lipid peroxidation in vitro and in vivo. However, little is known about the antioxidant properties of FPP regarding iron-mediated oxidative damage to DNA and proteins. In the present study FPP protected supercoiled plasmid DNA against Fe-NTA plus H2O2 induced single and double strand breaks. Similar protective effects of FPP were evident when human T-lymphocytes were challenged with Fe-NTA/H2O2 and DNA damage was determined using the Comet assay. Fe-NTA/H2O2 also induced fragmentation of bovine serum albumin (BSA) in vitro and depleted cellular GSH levels in lymphocytes. BSA fragmentation and GSH depletion were dose-dependently counteracted by FPP. EPR spin trapping studies demonstrated that antioxidant properties of FPP are related to both hydroxyl scavenging as well as iron chelating properties.

[The effects of antitumor agents of croton alkaloids and cisplatin on human red blood cell membranes].

The effects of antitumor drugs of croton alkaloids (CA) extracted from croton, and cisplatin on the structure of human erythrocyte membrane and bovine serum albumin (BSA) were studied with the techniques of circular dichroism, fluorescence spectroscopy and ultraviolet absorption. From measuring circular dichroism between 200-240nm, it was found that the alpha helix of membrane and BSA increased after CA addition. Cisplatin was also found effective in altering membrane alpha helical content. BSA showed a loss of fluorescence intensity and a blue-shift of emission peak after CA addition. The florescence quenching and peak shift may reflect the movement of tryptophan to a more nonpolar environment (the hydrophobic portions of proteins). This is consistent with the increase in helicity of BSA in the presence of CA. From measuring fluorescence polarization of membrane, it was found that membrane fluidity decreased in the presence of CA, but did not change in the presence of cisplatin. The results indicate that both CA and cisplatin lead to alterations in the secondary structure of membrane protein. The conformation change in membrane protein may account for the pharmocological effect of CA and cisplatin.