In vitro antioxidant capacity and free radical scavenging evaluation of active metabolite constituents of Newbouldia laevis ethanolic leaf extract

Background

The aim of the present study was to evaluate the in vitro antioxidant and free radical scavenging capacity of bioactive metabolites present in Newbouldia laevis leaf extract.

Results

Chromatographic and spectrophotometric methods were used in the study and modified where necessary in the study. Bioactivity of the extract was determined at 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 400 μg/ml concentrations expressed in % inhibition. The yield of the ethanolic leaf extract of N.laevis was 30.3 g (9.93%). Evaluation of bioactive metabolic constituents gave high levels of ascorbic acid (515.53 ± 12 IU/100 g [25.7 mg/100 g]), vitamin E (26.46 ± 1.08 IU/100 g), saponins (6.2 ± 0.10), alkaloids (2.20 ± 0.03), cardiac glycosides(1.48 ± 0.22), amino acids and steroids (8.01 ± 0.04) measured in mg/100 g dry weight; moderate levels of vitamin A (188.28 ± 6.19 IU/100 g), tannins (0.09 ± 0.30), terpenoids (3.42 ± 0.67); low level of flavonoids (1.01 ± 0.34 mg/100 g) and absence of cyanogenic glycosides, carboxylic acids and aldehydes/ketones. The extracts percentage inhibition of DPPH, hydroxyl radical (OH^.), superoxide anion (O₂^.-), iron chelating, nitric oxide radical (NO), peroxynitrite (ONOO⁻), singlet oxygen (¹O₂), hypochlorous acid (HOCl), lipid peroxidation (LPO) and FRAP showed a concentration-dependent antioxidant activity with no significant difference with the controls. Though, IC₅₀ of the extract showed significant difference only in singlet oxygen (¹O₂) and iron chelating activity when compared with the controls.

Conclusions

The extract is a potential source of antioxidants/free radical scavengers having important metabolites which maybe linked to its ethno-medicinal use.     

Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA

Heat-induced formation of 8-oxoguanine was demonstrated in DNA solutions in 10^–3 M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chemical yield of 3.7 × 10^–2 µmol J^–1 for 8-oxoguanine production in DNA upon γ-irradiation was used as an adequate standard for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate constants for 8-oxoguanine formation were determined at elevated temperatures; the activation energy was found to be 27 ± 2 kcal/mol. Extrapolation to 37°C gave a value of k₃₇ = 4.7 × 10^–10 s^–1. Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced production of H₂O₂ in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H₂O₂. Gas saturation (O₂, N₂ and Ar), D₂O, scavengers of ¹O₂, O₂^–• and OH^• radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.     

Influence of Prior Exercise on VO2 Kinetics Subsequent Exhaustive Rowing Performance

Prior exercise has the potential to enhance subsequent performance by accelerating the oxygen uptake (VO₂) kinetics. The present study investigated the effects of two different intensities of prior exercise on pulmonary VO₂ kinetics and exercise time during subsequent exhaustive rowing exercise. It was hypothesized that in prior heavy, but not prior moderate exercise condition, overall VO₂ kinetics would be faster and the VO₂ primary amplitude would be higher, leading to longer exercise time at VO_2max. Six subjects (mean ± SD; age: 22.9±4.5 yr; height: 181.2±7.1 cm and body mass: 75.5±3.4 kg) completed square-wave transitions to 100% of VO_2max from three different conditions: without prior exercise, with prior moderate and heavy exercise. VO₂ was measured using a telemetric portable gas analyser (K4b², Cosmed, Rome, Italy) and the data were modelled using either mono or double exponential fittings. The use of prior moderate exercise resulted in a faster VO₂ pulmonary kinetics response (τ₁ = 13.41±3.96 s), an improved performance in the time to exhaustion (238.8±50.2 s) and similar blood lactate concentrations ([La⁻]) values (11.8±1.7 mmol.L⁻¹) compared to the condition without prior exercise (16.0±5.56 s, 215.3±60.1 s and 10.7±1.2 mmol.L⁻¹, for τ₁, time sustained at VO_2max and [La⁻], respectively). Performance of prior heavy exercise, although useful in accelerating the VO₂ pulmonary kinetics response during a subsequent time to exhaustion exercise (τ₁ = 9.18±1.60 s), resulted in a shorter time sustained at VO_2max (155.5±46.0 s), while [La⁻] was similar (13.5±1.7 mmol.L⁻¹) compared to the other two conditions. Although both prior moderate and heavy exercise resulted in a faster pulmonary VO₂ kinetics response, only prior moderate exercise lead to improved rowing performance.     

Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O₂^·− and H₂O₂. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H₂O₂ but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (^·OH) as a consequence of the reaction of H₂O₂ with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H₂O₂ in the presence of an α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN–^·CH(CH₃)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating ^·OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H₂O₂ in a similar manner, the magnitude of ^·OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of ^·OH spin trapped. Exogenous SOD failed to inhibit ^·OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in ^·OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H₂O₂-mediated killing seen in these mutants lacking SOD.     

Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO₂ ^– to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are k _app1 = 9.6±0.2 M^–1 s^–1 and k _app2 = 1.2±0.1 M^–1 s^–1 (at pH 7.4 and 20°C). The k _app1 and k _app2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are h _app = 3.8×10⁴ M^–1 s^–1 and h ₀ = 2.8×10^–1 s^–1 (at pH 7.4 and 20°C). The pH-dependence of h _on and h ₀ values reflects the acid-base equilibrium of peroxynitrite (pK _a = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.     

Early Picosecond Events in the Photocycle of Bacteriorhodopsin

The primary processes of the photochemical cycle of light-adapted bacteriorhodopsin (BR) were studied by various experimental techniques with a time resolution of 5 × 10^-13 s. The following results were obtained. (a) After optical excitation the first excited singlet state S₁ of bacteriorhodopsin is observed via its fluorescence and absorption properties. The population of the excited singlet state decays with a lifetime τ₁ of ~0.7 ps (430 ± 50 fs) (52). (b) With the same time constant the first ground-state intermediate J builds up. Its absorption spectrum is red-shifted relative to the spectrum of BR by ~30 nm. (c) The second photoproduct K, which appears with a time constant of τ₂ = 5 ps shows a red-shift of 20 nm, relative to the peak of BR. Its absorption remains constant for the observation time of 300 ps. (d) Upon suspending bacteriorhodopsin in D₂O and deuterating the retinal Schiff base at its nitrogen (lysine 216), the same photoproducts J and K are observed. The relaxation time constants τ₁ and τ₂ remain unchanged upon deuteration within the experimental accuracy of 20%.     

Structural Evidence for Inter-Residue Hydrogen Bonding Observed for Cellobiose in Aqueous Solution

The structure of the disaccharide cellulose subunit cellobiose (4-O-β-D-glucopyranosyl-D-glucose) in solution has been determined via neutron diffraction with isotopic substitution (NDIS), computer modeling and nuclear magnetic resonance (NMR) spectroscopic studies. This study shows direct evidence for an intramolecular hydrogen bond between the reducing ring HO3 hydroxyl group and the non-reducing ring oxygen (O5′) that has been previously predicted by computation and NMR analysis. Moreover, this work shows that hydrogen bonding to the non-reducing ring O5′ oxygen is shared between water and the HO3 hydroxyl group with an average of 50% occupancy by each hydrogen-bond donor. The glycosidic torsion angles φ_H and ψ_H from the neutron diffraction-based model show a fairly tight distribution of angles around approximately 22^° and −40^°, respectively, in solution, consistent with the NMR measurements. Similarly, the hydroxymethyl torsional angles for both reducing and non-reducing rings are broadly consistent with the NMR measurements in this study, as well as with those from previous measurements for cellobiose in solution.     

Different Inactivation Behaviors of MS-2 Phage and Escherichia coli in TiO2 Photocatalytic Disinfection

Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO₂ photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (·OH), which is generated on the surface of UV-illuminated TiO₂, plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H₂O₂ and O₂·⁻, etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and Escherichia coli as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction, para-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers, tert-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe²⁺, were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that E. coli is inactivated by both the free and the surface-bound hydroxyl radicals. E. coli might also be inactivated by other ROS, such as O₂·⁻ and H₂O₂, according to the present results.     

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 10⁶ m⁻¹ s⁻¹ (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s⁻¹. This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s⁻¹. 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent k_cat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s⁻¹. Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s⁻¹) and hydroxylation (36 ± 2 s⁻¹) partially control the overall turnover.     

Relationship Between Lignin Degradation and Production of Reduced Oxygen Species by Phanerochaete chrysosporium

The relationship between the production of reduced oxygen species, hydrogen peroxide (H₂O₂), superoxide (O₂), and hydroxyl radical (·OH), and the oxidation of synthetic lignin to CO₂ was studied in whole cultures of the white-rot fungus Phanerochaete chrysosporium Burds. The kinetics of the synthesis of H₂O₂ coincided with the appearance of the ligninolytic system; also, H₂O₂ production was markedly enhanced by growth under 100% O₂, mimicking the increase in ligninolytic activity characteristic of cultures grown under elevated oxygen tension. Lignin degradation by whole cultures was inhibited by a specific H₂O₂ scavenger, catalase, implying a role for H₂O₂ in the degradative process. Superoxide dismutase also inhibited lignin degradation, suggesting that O₂ is also involved in the breakdown of lignin. The production of ·OH was assayed in whole cultures by a benzoate decarboxylation assay. Neither the kinetics of ·OH synthesis nor the final activity of its producing system obtained under 100% O₂ correlated with that of the lignin-degrading system. However, lignin degradation was inhibited by compounds which react with ·OH. It is concluded that H₂O₂, and perhaps O₂, are involved in lignin degradation; because these species are relatively unreactive per se, their role must be indirect. Conclusions about a role for ·OH in ligninolysis could not be reached.     

Cytoprotective Effects of Grape Seed Extract on Human Gingival Fibroblasts in Relation to Its Antioxidant Potential

Cytoprotective effects of short-term treatment with grape seed extract (GSE) upon human gingival fibroblasts (hGFs) were evaluated in relation to its antioxidant properties and compared with those of a water-soluble analog of vitamin E: trolox (Tx). GSE and Tx showed comparable antioxidant potential in vitro against di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH; a stable radical), hydroxyl radical (^•OH), singlet oxygen (¹O₂), and hydrogen peroxide (H₂O₂). Pretreatment or concomitant treatment with GSE for 1 min protected hGFs from oxidative stressors, including H₂O₂, acid-electrolyzed water (AEW), and ¹O₂, and attenuated the intracellular formation of reactive oxygen species induced by H₂O₂ and AEW. Tx also reduced the H₂O₂- and AEW-induced intracellular formation of reactive oxygen species, but showed no cytoprotective effects on hGFs exposed to H₂O₂, AEW, or ¹O₂. These results suggest that the cytoprotective effects of GSE are likely exerted independently of its antioxidant potential.     

Diminution of singlet oxygen-induced DNA damage by curcmin and related antioxidants

Curcumin, the natural antioxidant from turmeric, an Indian spice, and its derivatives have significant abilities to protect plasmid pBR322 against single-strand breaks induced by singlet oxygen (¹O₂), a reactive oxygen species with potential genotoxic/mutagenic properties. ¹O₂ was generated at 37°C in an aqueous buffer system by the thermal dissociation of the endoperoxide of 3,3′-(1,4-naphthylene)dipropionate (NDPO₂). Among the compounds, tested, curcumin was the most effective inhibitor of DNA damage followed by desmethoxycurcumin, bisdesmethoxycurcumin and other derivatives. The observed antioxidant activity was both time-and concentration-dependent. The protectice ability of curcumin was higher than that of the well-known biological antioxidants lipoate, α-tocopherol and β-carotene. However, the highest protective ability with saturating concentrations of curcumin did not exceed 50%. The ability of curcumin and its derivatives to protect DNA against ¹O₂ seems to be related to their structures and may at least partly explain the therapeutic and other beneficial effects of these compounds including anticarcinogenic and antimutagenic properties.     

Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate

The H⁺-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl⁻ and NO₃⁻. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K_m of the enzyme for ATP but caused relatively little alteration of the V_max. Nitrate increased K_m only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl⁻. Other monovalent anions that specifically increased the K_m of the ATPase for ATP were, in order of increasing K_i, SCN⁻, ClO₄⁻, and ClO₃⁻. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO₃⁻, was a competitive inhibitor of the ATPase but ADP and NO₃⁻ did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO₃⁻ does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO₃⁻ exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl⁻ nor NO₃⁻ are involved in substrate binding.     

Nature of photooxidative events in leaves treated with chlorosis-inducing herbicides

Leaves of rye seedlings (Secale cereale L.) grown in the presence of four chlorosis-inducing herbicides under a low light intensity of 10 lux formed chlorophyll. When segments of such dim-light-grown leaves were exposed to 30,000 lux at either 0°C or 30°C, treatments with aminotriazole or haloxidine (group 1) showed no or only minor changes of their chlorophyll contents. In treatments with San 6706 or difunon (group 2), however, rapid photodestruction of chlorophyll occurred both at 0°C and at 30°C and was accompanied by an increase of malondialdehyde that was not seen in the presence of group 1 herbicides. Unlike the in vivo behavior, virtually equal rates of chlorophyll breakdown were observed for aminotriazole and San 6706 treatments in suspensions of isolated chloroplasts from 10 lux-grown leaves after exposure to strong light. The free radical scavengers p-benzoquinone and hydroquinone and the d-penicillamine copper complex exerting superoxide dismutating activity effectively prevented photooxidation of chlorophyll in 10 lux-grown herbicide-treated leaf segments or even restored an accumulation of chlorophyll at 30,000 lux. Ascorbate and several singlet oxygen or hydroxyl radical scavengers had no protective effects. Deuterium oxide and H₂O₂ did not enhance the degradation of chlorophyll. Superoxide dismutase activity was decreased in leaves bleached in the presence of group 2 herbicides.     

Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a hydroxymethyl group transfer from l-serine to tetrahydrofolate (H₄folate) to yield glycine and 5,10-methylenetetrahydrofolate (CH₂-H₄folate). SHMT is crucial for deoxythymidylate biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary complex mechanism. To define the kinetic mechanism of this catalysis, we explored the PvSHMT reaction by employing various methodologies including ligand binding, transient, and steady-state kinetics as well as product analysis by rapid-quench and HPLC/MS techniques. The results indicate that PvSHMT can bind first to either l-serine or H₄folate. The dissociation constants for the enzyme·l-serine and enzyme·H₄folate complexes were determined as 0.18 ± 0.08 and 0.35 ± 0.06 mm, respectively. The amounts of glycine formed after single turnovers of different preformed binary complexes were similar, indicating that the reaction proceeds via a random-order binding mechanism. In addition, the rate constant of glycine formation measured by rapid-quench and HPLC/MS analysis is similar to the k_cat value (1.09 ± 0.05 s⁻¹) obtained from the steady-state kinetics, indicating that glycine formation is the rate-limiting step of SHMT catalysis. This information will serve as a basis for future investigation on species-specific inhibition of SHMT for antimalarial drug development.     

Kinetics of Leptin Binding to the Q223R Leptin Receptor

Studies in human populations and mouse models of disease have linked the common leptin receptor Q223R mutation to obesity, multiple forms of cancer, adverse drug reactions, and susceptibility to enteric and respiratory infections. Contradictory results cast doubt on the phenotypic consequences of this variant. We set out to determine whether the Q223R substitution affects leptin binding kinetics using surface plasmon resonance (SPR), a technique that allows sensitive real-time monitoring of protein-protein interactions. We measured the binding and dissociation rate constants for leptin to the extracellular domain of WT and Q223R murine leptin receptors expressed as Fc-fusion proteins and found that the mutant receptor does not significantly differ in kinetics of leptin binding from the WT leptin receptor. (WT: k_a 1.76×10⁶±0.193×10⁶ M⁻¹ s⁻¹, k_d 1.21×10⁻⁴±0.707×10⁻⁴ s⁻¹, K_D 6.47×10⁻¹¹±3.30×10⁻¹¹ M; Q223R: k_a 1.75×10⁶±0.0245×10⁶ M⁻¹ s⁻¹, k_d 1.47×10⁻⁴±0.0505×10⁻⁴ s⁻¹, K_D 8.43×10⁻¹¹±0.407×10⁻¹¹ M). Our results support earlier findings that differences in affinity and kinetics of leptin binding are unlikely to explain mechanistically the phenotypes that have been linked to this common genetic variant. Future studies will seek to elucidate the mechanism by which this mutation influences susceptibility to metabolic, infectious, and malignant pathologies.     

Assessment of the phytochemical constituents and antioxidant activity of a bloom forming microalgae Euglena tuba

Background

Unstable generation of free radicals in the body are responsible for many degenerative diseases. A bloom forming algae Euglena tuba growing abundantly in the aquatic habitats of Cachar district in the state of Assam in North-East India was analysed for its phytochemical contents, antioxidant activity as well as free radical scavenging potentials.

Results

Based on the ability of the extract in ABTS^•+ radical cation inhibition and Fe³⁺ reducing power, the obtained results revealed the prominent antioxidant activity of the algae, with high correlation coefficient of its TEAC values to the respective phenolic and flavonoid contents. The extract had shown its scavenging activity for different free radicals and 41.89 ± 0.41 μg/ml, 5.83 ± 0.07 μg/ml, 278.46 ± 15.02 μg/ml and 223.25 ± 4.19 μg/ml were determined as the IC₅₀ values for hydroxyl, superoxide, nitric oxide and hypochlorous acid respectively, which are lower than that of the corresponding reference standards. The phytochemical analysis also revealed that the phenolics, flavonoids, alkaloids, tannins and carbohydrates are present in adequate amount in the extract which was confirmed by HPLC analysis.

Conclusions

The results showed that 70% methanol extract of the algae possesses excellent antioxidant and free radical scavenging properties.     

Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by Mycobacterium sp. Strain RJGII-135

The mechanism of uptake of phenanthrene by Mycobacterium sp. strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-¹⁴C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state ¹⁴C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (K_t) of 26 ± 3 nM (mean ± standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated ¹⁴C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (K_d) of 41 ± 21 nM (mean ± standard deviation). Given the low values of K_t and K_d, Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.     

Pronghorn (Antilocapra americana) enamel phosphate δ18O values reflect climate seasonality: Implications for paleoclimate reconstruction

Stable oxygen isotope (δ¹⁸O) compositions from vertebrate tooth enamel are widely used as biogeochemical proxies for paleoclimate. However, the utility of enamel oxygen isotope values for environmental reconstruction varies among species. Herein, we evaluate the use of stable oxygen isotope compositions from pronghorn (Antilocapra americana Gray, 1866) enamel for reconstructing paleoclimate seasonality, an elusive but important parameter for understanding past ecosystems. We serially sampled the lower third molars of recent adult pronghorn from Wyoming for δ¹⁸O in phosphate (δ¹⁸O_PO4) and compared patterns to interpolated and measured yearly variation in environmental waters as well as from sagebrush leaves, lakes, and rivers (δ¹⁸O_w). As expected, the oxygen isotope compositions of phosphate from pronghorn enamel are enriched in ¹⁸O relative to environmental waters. For a more direct comparison, we converted δ¹⁸O_w values into expected δ¹⁸O_PO4* values (δ¹⁸O_W‐_PO4*). Pronghorn δ¹⁸O_PO4 values from tooth enamel record nearly the full amplitude of seasonal variation from Wyoming δ¹⁸O_W‐PO4* values. Furthermore, pronghorn enamel δ¹⁸O_PO4 values are more similar to modeled δ¹⁸O_W‐PO4* values from plant leaf waters than meteoric waters, suggesting that they obtain much of their water from evaporated plant waters. Collectively, our findings establish that seasonality in source water is reliably reflected in pronghorn enamel, providing the basis for exploring changes in the amplitude of seasonality of ancient climates. As a preliminary test, we sampled historical pronghorn specimens (1720 ± 100 AD), which show a mean decrease (a shift to lower values) of 1–2‰ in δ¹⁸O_PO4 compared to the modern specimens. They also exhibit an increase in the δ¹⁸O amplitude, representing an increase in seasonality. We suggest that the cooler mean annual and summer temperatures typical of the 18th century, as well as enhanced periods of drought, drove differences among the modern and historical pronghorn, further establishing pronghorn enamel as excellent sources of paleoclimate proxy data.