In vitro antioxidant activity of Hedyotis corymbosa (L.) Lam. aerial parts

The methanolic extract of the aerial part of Hedyotis corymbosa (L.) Lam. (Rubiaceae) was screened for antioxidant activity using 1,1-diphenyl-2-picryl hydroxyl (DPPH) quenching assay, 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS) cation decolorization test, ferric reducing power (FRP), scavenging capacity towards hydroxyl ion (OH*) radicals and nitric oxide (NO) radical inhibition activity using established assay procedures. Total phenolics and total flavonoid contents were, also determined. The plant yielded 210 mg gallic acid equivalent/100 g phenolic content and 55 mg quercetin equivalent/100 g flavonoid content. The extract exhibited high antiradical activity against DPPH, ABTS, nitric oxide and hydroxyl radicals with EC50 value of 82, 150, 130, and 170 microg/ml, respectively. The FRP increased with increasing concentration of the sample. The antioxidant activity of the extract was comparable with that of the standard butylated hydroxyl toluene (BHT). High correlation between total phenolic/flavonoid contents and scavenging potential of different reactive oxygen species (R2 = 0.785-0.998) indicated the polyphenols as the main antioxidants.     

In vitro antioxidant and cytotoxic activities of polyherbal extracts from Vetiveria zizanioides,Trichosanthes cucumerina,and Mollugo cerviana on HeLa and MCF-7 cell lines

Various metabolites exist in the medicinal plants have lot of potential to cure various diseases and disorders. Plants such as, Vetiveria zizanioides, Trichosanthes cucumerina, and Mollugo cerviana were collected from Western Ghats, Tamilnadu, India. Phytochemicals were extracted from these plants using various organic solvents and tested against Gram-positive and Gram-negative bacteria. The phytochemicals such as, carbohydrate, alkaloids, steroids, saponins, flavonoids and tannin were detected from these medicinal plants. Among the extracts, methanol showed potent activity and this solvent was used to extract polyherbal medicinal plants. Methanol extract of V. zizanioides was found to be highly active against E. coli (27 ± 2 mm), P. mirabilis (19 ± 3 mm) and B. subtilis (18 ± 2 mm). Ethyl acetate extract showed high activity against E. coli (24 ± 2 mm), P. mirabilis (22 ± 3 mm) and B. subtilis (20 ± 1 mm). These three plants were taken at 1:1:1 ratio and extracted with methanol at 1:10 ratio and synergistic activity was tested against bacterial pathogens. Synergistic activity of polyherbal extract was analyzed. The extracted crude herbal medicine was found to be effective against Staphylococcus aureus, E. coli, Enterbacter sp., Pseudomonas aeruginosa, Bacillus subtilis and Proteus mirabilis. The zone of inhibition was 33 ± 3 mm, 17 ± 2 mm, 22 ± 2 mm, 40 ± 2 mm, 33 ± 1 mm and 38 ± 2 mm zone of inhibition against E. coli, S. aureus, P. aeruginosa, P. mirabilis, B. subtilis and Enterobacter sp. Polyherbal extract was found to be highly effective against P. mirabilis and Enterobacter sp. MIC values of polyherbal extract ranged from 29 ± 2.5 µg/ml to 34 ± 2.5 µg/ml. MIC value was found to be less against P. mirabilis and was high against S. aureus. Antioxidant property varied between 49 ± 3% and 95.3 ± 2%. At 20 µg/ml antioxidant activity was reported as 49 ± 3% and it was increased at higher concentrations of polyherbal extract. Two cell lines (HeLa and MCF cell lines) were selected to analyze cytotoxic activity of polyherbal extract. The methanol extract of polyherbal fraction showed cytotoxicity against these two cell lines. The LC50 value was 467 ± 2.9 µg/ml against HeLa cell line and >800 µg/ml against MCF-7 cell lines. The polyherbal extract showed antibacterial, antioxidant and anticancer activities.     

In vitro evaluation of the antioxidant activity of calcium fructoborate

Although increasing evidence shows the nutritional benefits of calcium fructoborate (CF) on animals and humans, its action mechanism has not been clearly identified. The present study aims to investigate the possible antioxidant function of CF. Based on its efficiency in skin wound healing, the authors tested whether CF possesses antioxidant properties on human keratinocytes cultures, in a complete serum-free medium (KMK-2; Sigma). The cells treated with CF (0–450 nmol/culture medium) were exposed to exogenous 100 μmol of hydrogen peroxide to mimic the oxidative stress. The changes in general cell oxidant production evaluated with dihydrorhodamine-123 showed that the intracellular reactive oxygen species (ROS) were markedly reduced by preincubation with CF. The maximum antioxidant activity was notice at 90 nmol CF. To assess the reactivity of CF on ROS, we analyzed its ability to inhibit the superoxide-dependent auto-oxidation of pyrogallol. The CF inhibited the pyrogallol auto-oxidation depending on time and concentration, which suggests its possible role as a superoxide radical scavenger. Taken together, our results indicate that CF has antioxidant activity, which could have clinical significance in protecting cells from oxidant-induced injury. A hypothetic mechanism for the antioxidant activity of CF is proposed.     

Deoxypodophyllotoxin content and antioxidant activity of aerial parts of Anthriscus sylvestris Hoffm

Deoxypodophyllotoxin content of the aerial parts of Anthriscus sylvestris Hoffm. growing at different altitudes was evaluated in comparison to the roots. The lignan accumulation in ground parts was at least double compared to aerial ones. In addition antioxidant-guided fractionation of the crude methanol extract of aerial parts was performed with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test. Active fractions contained mainly luteolin-7-O-glucoside and chlorogenic acid. Antioxidant properties of both crude extract and isolated compounds were also investigated with the Briggs-Rauscher (BR) oscillating reaction. A satisfactory agreement between the results obtained with the two methods was observed.     

In vivo and in vitro antioxidant properties of furosemide

The aim of this study was to investigate in vivo and in vitro antioxidant properties of furosemide. In vitro, human red blood cells were submitted to oxidative stress (AAPH), in absence or in presence of different concentrations of furosemide. Potassium efflux was measured in order to quantify the oxidative stress after the action of AAPH on red blood cells. Allophycocyanin assay was also used to investigate antioxidant capacities of furosemide. For the in vivo experiment, male Wistar rats were used. A control group (n = 5) was treated by a daily intraperitoneal injection of saline solution (0.2 ml); 2 other groups (J0 and J+) were treated for 7 days by one daily intraperitoneal injection of furosemide (0.10 mg/kg/day). In the J+group, the injection of furosemide was done one hour before the experiment, while in the J0 group the last injection of furosemide was done on the 6th day and an injection of saline was performed one hour before the experiment. On the day of experiment, a laparotomy was performed under general anesthesia and blood was collected from abdominal aorta. Oxidative stress and antioxidant capacities were evaluated on Wistar rat red blood cells and plasma. In vitro results (oxidative challenge with AAPH) showed that oxidative stress was decreased in presence of furosemide. This was due to a potent free radical scavenging effect of furosemide. In vivo studies confirmed that furosemide had antioxidant properties. These data may be of great relevance in clinical practice, considering the use of large doses of furosemide in patients presenting pathology involving the production of free radicals.     

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.     

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.     

体内和体外评价法分析内生真菌粗提取物的抗氧化活性

对几株经初筛具较高抗氧化活性的疏花水柏枝的内生真菌展开研究,在体内和体外两种方法下分析了其抗氧化能力。本研究选择1,1-二苯基-2-三硝基苯肼(DPPH)法、总抗氧化能力(T-AOC)试剂盒和铁氰化钾还原力测定法来评价内生真菌的体外抗氧化活性,并对不同方法进行了比较分析;建立并优化了大肠杆菌的氧化损伤模型,并将内生真菌对其保护作用与对人神经母瘤细胞SH-SY5Y的氧化损伤保护作用进行比较,分析了不同内生真菌在体内的抗氧化活性。结果表明体内和体外的抗氧化分析方法存在一定偏差,体外分析中活性最高的菌株是SJ-12和QY-1,体内分析中对大肠杆菌抗氧化保护效果最好的菌株也是QY-1和SJ-12,但对神经细胞保护效果最好的菌株是QY-1和MG-9。综合几种方法,本实验证明了内生真菌QY-1及其发酵产物具较强的抗氧化活性,可以作为潜在的新型抗氧化剂开发利用。     

In vitro evaluation of antioxidant activity by electrophoresis and high performance liquid chromatography

Two methods for the analysis of antioxidants, based on polyacrylamide gel electrophoresis (PAGE) and gel permeation high performance liquid chromatography (HPLC) were developed. Both of them exploit the variations of the signal (band or peak) given by human serum albumin (0.2% w/v in 100 mM sodium phosphate pH 7) upon oxidation with hypochlorite (1% of a solution containing 4% active Cl), quantitatively determined by densitometric analysis or peak integration. Based on such changes, two formulas were defined which allowed the determination of the antioxidant activity of ascorbic acid (EC(50,PAGE)=4.8x10(-4) M, EC(50,HPLC)=3.6x10(-4) M), glutathione (EC(50,PAGE)=1.5x10(-4) M, EC(50,HPLC)=2.0x10(-4) M) and melatonin (EC(50,PAGE)=5.2x10(-4) M, EC(50,HPLC)=3.2x10(-4) M), chosen as reference compounds. A good correlation was found between the activities of these substances in the two assays, which are also in good agreement with literature data, indicating that the two methods are essentially equivalent. These assays could be useful for the screening of new antioxidant drugs for pathological conditions such as cataract, rheumatic diseases, atherosclerosis and Alzheimer's disease.     

In vitro and in vivo assessment of the antioxidant activity of melatonin and related indole derivatives

Effects of melatonin and some structurally related indole compounds were studied by in vitro methods such as (i) an inhibition of the hyaluronic acid degradation and (ii) a standard lipid peroxidation assay. In vivo approach was based on the alloxan model of hyperglycaemia. Reduction of the viscosity of a hyaluronic acid solution in the reaction mixture was inhibited by tryptamine (91% inhibition), as well as by indole-3-carboxylic acid and indomethacin (80% and 77% inhibition, respectively). Lipid peroxidation with tert-butyl hydroperoxide as a source of radicals was followed by the formation of thiobarbituric acid reactive substances. Tested drugs inhibited lipid peroxidation in the order: tryptamine (59%) > indole-2-carboxylic acid (38%) > indomethacin (26%) > melatonin and indole-3-carboxylic acid (13%). In vivo, alloxan-induced hyperglycaemia was reduced in mice pretreated with drugs tested. The highest protective effect was observed with indomethacin (52% inhibition), followed by tryptamine and melatonin (18% and 16% inhibition, respectively).     

In vitro and in vivo evaluation of antioxidant and neuroprotective properties of antipsychotic D2AAK1

Neurochemical Research - The susceptibility of neurons to free radical toxicity partially underlies the pathomechanism of neurodegenerative diseases. On the other hand, excitotoxicity also...     

In vitro and in vivo evaluation of the prebiotic activity of water-soluble blueberry extracts

The prebiotic effects of water extracts of two blueberry (BBE) cultivars (‘Centurion’ and ‘Maru’) were studied using pure and mixed cultures of human faecal bacteria. The results demonstrated for the first time that addition of BBE from both cultivars to broth media containing pure cultures of Lactobacillus rhamnosus and Bifidobacterium breve resulted in a significant increase (P < 0.05–0.0001) in the population size of these strains. Batch fermentation system was used to monitor the effect of BBE addition on the mixed faecal bacterial populations (obtained from healthy human donors). Addition of BBE from both cultivars to batch cultures inoculated with mixed human faecal cultures resulted in a significant increase in the number of lactobacilli (P < 0.01–0.0001) and bifidobacteria (P < 0.05–0.0001). Furthermore, a significant influence on the population size of lactobacilli and bifidobacteria was observed after administration of extracts from both cultivars to rats daily for 6 days in comparison with the control group. In rats gavaged orally with 4 ml kg⁻¹ day⁻¹ of BBE for 6 days, the population size of lactobacilli (P < 0.05) and bifidobacteria (P < 0.05–0.01) was increased significantly. We hypothesize that BBE could modify the bacterial profile by increasing the numbers of beneficial bacteria and thereby improving gut health.     

In vivo and in vitro genotoxic evaluation of indorenate

5-Methoxytryptamine, beta-methylcarboxylate hydrochloride (indorenate) is a new antihypertensive serotonin derivative. We evaluated its genotoxic activity using the mouse bone marrow and cytogenetic test and the human lymphocyte culture cytogenetic assay. As endpoints we measured chromosomal aberrations, sister-chromatid exchanges and cellular proliferation kinetics. Our results agree in both systems showing that indorenate is a non-genotoxic agent in these assays.     

In vitro and in vivo determination of antioxidant activity and mode of action of isoquercitrin and Hyptis fasciculata

Reactive oxygen species (ROS) are thought to underline the process of ageing and the pathogenicity of various diseases, such as neurodegenerative disorders and cancer. The use of traditional medicine is widespread and plants still present a large source of natural antioxidants that might serve as leads for the development of novel drugs. In this paper, the alcoholic extract from leaves of Hyptis fasciculata, a Brazilian medicinal plant, and isoquercitrin, a flavonoid identified in this species, showed to be active as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavengers. The extract of Hyptis fasciculata and isoquercitrin were also able to increase tolerance of the eukaryotic microorganism Saccharomyces cerevisiae to both hydrogen peroxide and menadione, a source of superoxide. Cellular protection was correlated with a decrease in oxidative stress markers, such as levels of ROS, protein carbonylation and lipid peroxidation, confirming the antioxidant potential of Hyptis fasciculata and isoquercitrin.     

In vitro evaluation of the antioxidant activity and biomembrane interaction of the lazaroid U-74389G

The aim of this paper was to clarify whether the interaction of the lazaroid U-74389G with phospholipid membranes might be relevant as to its antioxidant activity. Thus we evaluated the "in vitro" antioxidant activity of U-74389G in two experimental models: 1) bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical; 2) peroxidation, induced by the water-soluble radical initiator 2,2'-azobis(2-amidino-propane) hydrochloride, on mixed dipalmitoyl-phosphatidylcholine/linoleic acid unilamellar vesicles. Moreover, given that biophysical techniques may help in explaining the role of a drug in its interaction with the microenvironment of the model lipid membranes, we used a classical approach to investigate the U-74389G/model membrane interaction: the differential scanning calorimetry technique on dimyristoylphosphatidylcholine multilamellar and unilamellar vesicles and the Langmuir-Blodgett technique on dimyristoylphosphatidylcholine monolayers. The results evidenced the strong antioxidant activity of U-74389G (especially in a membranous system) and its capability to interact with and be transported across model membranes. Thus one can speculate that U-74389G can act as scavenger of chain-propagating lipid peroxyl radicals within the membranes and may be able to protect not only cell membranes, but also intracellular components against peroxidative attack. Furthermore, also if there is no certain proof that the effect on the lipid packing order may play a key role in its antioxidant activity, the fluidifying effect on phospholipid bilayers of U-74389G favourably complements its free radical scavenging characteristics.     

Thermodynamic and kinetic analysis of porphyrin binding to Trichosanthes cucumerina seed lectin.

The interaction of several metallo-porphyrins with the galactose-specific lectin from Trichosanthes cucumeirna (TCSL) has been investigated. Difference absorption spectroscopy revealed that significant changes occur in the Soret band region of the porphyrins upon binding to TCSL and these changes have been monitored to obtain association constants (Ka) and stoichiometry of binding (n). The dimeric lectin binds two porphyrin molecules and the presence of the specific saccharide lactose did not affect porphyrin binding significantly, indicating that the sugar and the porphyrin bind at different sites. The Ka values obtained for the binding of different porphyrins with TCSL at 25 degrees C were in the range of 2 x 10(3)-5 x 10(5) m(-1). Association constants for meso-tetra(4-sulphonatophenyl)porphyrinato copper(II) (CuTPPS), a porphyrin bearing four negative charges and meso-tetra(4-methylpyridinium)porphyrinato copper(II) (CuTMPyP), a porphyrin with four positive charges, were determined at several temperatures; from the temperature dependence of the association constants, the thermodynamic parameters change in enthalpy (DeltaH degrees ) and change in entropy (DeltaS degrees ) associated with the binding process were estimated. The thermodynamic data indicate that porphyrin binding to TCSL is driven largely by a favourable entropic contribution; the enthalpic contribution is very small, suggesting that the binding process is governed primarily by hydrophobic forces. Stopped-flow spectroscopic measurements show that binding of CuTMPyP to TCSL takes place by a single-step process and at 20 degrees C, the association and dissociation rate constants were 1.89 x 10(4) m(-1).s(-1) and 0.29 s(-1), respectively.     

Physicochemical and saccharide-binding studies on the galactose-specific seed lectin from Trichosanthes cucumerina

Physicochemical and saccharide-binding studies have been performed on Trichosanthes cucumerina seed lectin (TCSL). The agglutination activity of TCSL is highest in the pH range 8.0-11.0, whereas below pH 7.0 it decreases quite rapidly, which is consistent with the involvement of imidazole side chains of His residues, which titrate in this pH range, in the sugar-binding activity of the lectin. The lectin activity is unaffected between 0 and 60 degrees C, but a sharp decline occurs at higher temperatures. Isoelectric focusing studies show that TCSL has three isoforms with pI values of 5.3, 6.2, and 7.1, with the isoform of pI 6.2 being the most abundant. Circular dichroism spectroscopic studies reveal that TCSL contains about 28.4% beta-sheet, 10.6% beta-turns, 7% polyproline type 2 structure, with the remainder comprising unordered structure; the alpha-helix content is negligible. Binding of 4-methylumbelliferyl-beta-D-galactopyranoside (MeUmbbetaGal) to TCSL results in a significant increase in the fluorescence intensity of the ligand, and this change has been used to obtain the association constant for the interaction. At 25 degrees C, the association constant, K(a), for the TCSL-MeUmbbetaGal interaction was determined as 6.9 x 10(4)M(-1). Binding of nonfluorescent, inhibitory sugars was studied by monitoring their ability to reverse the fluorescence changes observed when MeUmbbetaGal was titrated with TCSL.     

In vitro and in vivo osteogenic activity of licochalcone A

We investigated the in vitro and in vivo osteogenic activity of licochalcone A. At low concentrations, licochalcone A stimulated the differentiation of mouse pre-osteoblastic MC3T3-E1 subclone 4 (MC4) cells and enhanced the bone morphogenetic protein (BMP)-2-induced stimulation of mouse bi-potential mesenchymal precursor C2C12 cells to commit to the osteoblast differentiation pathway. This osteogenic activity of licochalcone A was accompanied by the activation of extracellular-signal regulated kinase (ERK). The involvement of ERK was confirmed in a pharmacologic inhibition study. Additionally, noggin (a BMP antagonist) inhibited the osteogenic activity of licochalcone A in C2C12 cells. Licochalcone A also enhanced the BMP-2-stimulated expression of various BMP mRNAs. This suggested that the osteogenic action of licochalcone A in C2C12 cells could be dependent on BMP signaling and/or expression. We then tested the in vivo osteogenic activity of licochalcone A in two independent animal models. Licochalcone A accelerated the rate of skeletal development in zebrafish and enhanced woven bone formation over the periosteum of mouse calvarial bones. In summary, licochalcone A induced osteoblast differentiation with ERK activation in both MC4 and C2C12 cells and it exhibited in vivo osteogenic activity in zebrafish skeletal development and mouse calvarial bone formation. The dual action of licochalcone A in stimulating bone formation and inhibiting bone resorption, as described in a previous study, might be beneficial in treating bone-related disorders.     

In vitro antioxidant activity of Diospyros malabarica Kostel bark

Antioxidant activity of defatted methanol extract of D. malabarica bark was studied for its free radical scavenging property on different in vitro models e.g. 1,1-diphenyl-2-picryl hydrazyl (DPPH), nitric oxide, superoxide, hydroxyl radical and lipid peroxide radical model. The extract showed good dose-dependent free radical scavenging property in all the models except in hydroxyl radical inhibition assay. IC50 values were found to be 9.16, 13.21, 25.27 and 17.33 microg/ml respectively in DPPH, nitric oxide, superoxide and lipid peroxidation inhibition assays. In hydroxyl radical inhibition assay 1000 microg/ml extract showed only 10% inhibition with respect to the control. Measurement of total phenolic compounds by Folin-Ciocalteu's phenol reagent indicated that 1 mg of the extract contained 120.7 microg equivalent of pyrocatechol. The results indicate that the antioxidant property of the extract may be due to the high content of phenolic compounds. However, the underlying mechanism may not involve hydroxyl radical scavenging property.