Rat Brain Homogenates Research Articles

A quality by design paradigm, novel, and fast LC-MS/MS method to quantify vortioxetine in rat brain homogenate and its assessment of greenness, whiteness, and blueness tools

Vortioxetine (VOR) is an antidepressant drug with multimodal mechanism of action. In this work a sensitive and simple bioanalytical LC-MS/MS method for analysis of VOR in spiked rat brain tissue was developed and validated. Chromatographic analyses were performed in isocratic mode, and Citalopram (CT) was found as a suitable internal standard. Supelco Ascentis® Express Phenyl-Hexyl column was used as stationary phase, and the mobile phase was 0.15 % formic acid in acetonitrile:water (55:45, v/v). A Box-Behnken Design − based method optimization was applied to explore the impact of analytical conditions on chromatographic separation of VOR and CT. Detection was realized via a triple quadrupole LC-MS/MS system; the mass transition ion-pair has been followed as m/z 299.05 → 149.90 for VOR, m/z 325.20 → 109.05 for CT. Analytical method validation was performed according to International Council for Harmonisation (ICH) guideline M10 on bioanalytical method validation. The calibration curve was within the range of 30 to 450 ng/mL. LOD and LLOQ were calculated at 10 and 30 ng/mL, respectively. Recovery was 90 % for the lower limit of quantification and between 98 % to 105 % for the other concentration levels. Total run time was 5 min, retention times of citalopram and vortioxetine were 2.4 and 3.1 min, respectively, and a good chromatographic separation was obtained under optimal conditions. The solvents used to make the method greener were evaluated with the Green Solvent Selection Tool and Green Index Spider chart. The AGREE score for the greenness of the developed method was calculated as 0.61 and was also evaluated with the ComplexGAPI pictogram. The BAGI score for blueness was calculated as 72.5, and the RGBfast score for whiteness was calculated as 50. The method was determined to be most green, white, and blue.

Experimental and Computational Evaluation of Lipidomic In-Source Fragmentation as a Result of Postionization with Matrix-Assisted Laser Desorption/Ionization.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can provide spatially resolved molecular information about a sample. Recently, a postionization approach (MALDI-2) has been commercially integrated with MALDI-MSI, allowing for bettered sensitivity and consequent improved spatial resolution. While advantages of MALDI-2 have previously been established, we demonstrate here statistically increased in-source fragmentation (ISF) results from postionization with a commercial instrument. Via lipid standard analyses, known MALDI ISF pathways (e.g., loss of trimethylamine) were statistically increased in MALDI-2 compared to MALDI-1 (65-172% increase in fragmentation). Gas phase molecular modeling with density functional theory estimated that the most-weighted virtual orbitals to excite within lipids involve ester and phosphate bonds. Protonated lipid excitation energies are furthermore red-shifted compared to those of other adduct types [e.g., 254 nm for protonated PC(16:0/18:1)] and approach the MALDI-2 laser energy (266 nm). Analysis of rat brain homogenate detected statistically more positive-ion mode peaks with MALDI-2 (1090) than that with MALDI-1 (719), where Kernel density estimations showed that the majority of this enhancement occurs with low m/z ions (i.e., m/z 75-500). Taken together with the lipid standard data, these observations may indicate ISF due to postionization. While artifact contributions from matrix blanks were also noted, both experimental and computational data sets suggest that the overall extent of ISF is statistically increased in MALDI-2 compared to MALDI-1.

Characterization and Hydrolysis Studies of a Prodrug Obtained as Ester Conjugate of Geraniol and Ferulic Acid by Enzymatic Way.

Ferulic acid (Fer) and geraniol (Ger) are natural compounds whose antioxidant and anti-inflammatory activity confer beneficial properties, such as antibacterial, anticancer, and neuroprotective effects. However, the short half-lives of these compounds impair their therapeutic activities after conventional administration. We propose, therefore, a new prodrug (Fer-Ger) obtained by a bio-catalyzed ester conjugation of Fer and Ger to enhance the loading of solid lipid microparticles (SLMs) designed as Fer-Ger delivery and targeting systems. SLMs were obtained by hot emulsion techniques without organic solvents. HPLC-UV analysis evidenced that Fer-Ger is hydrolyzed in human or rat whole blood and rat liver homogenates, with half-lives of 193.64 ± 20.93, 20.15 ± 0.75, and 3.94 ± 0.33 min, respectively, but not in rat brain homogenates. Studies on neuronal-differentiated mouse neuroblastoma N2a cells incubated with the reactive oxygen species (ROS) inductor H2O2 evidenced the Fer-Ger ability to prevent oxidative injury, despite the fact that it appears ROS-promoting. The amounts of Fer-Ger encapsulated in tristearin SLMs, obtained in the absence or presence of glucose, were 1.5 ± 0.1%, allowing the control of the prodrug release (glucose absence) or to sensibly enhance its water dissolution rate (glucose presence). These new "green" carriers can potentially prolong the beneficial effects of Fer and Ger or induce neuroprotection as nasal formulations.

Potential for in vivo visualization of intracellular pH gradient in the brain using PET imaging.

Intracellular pH is a valuable index for predicting neuronal damage and injury. However, no PET probe is currently available for monitoring intracellular pH in vivo. In this study, we developed a new approach for visualizing the hydrolysis rate of monoacylglycerol lipase, which is widely distributed in neurons and astrocytes throughout the brain. This approach uses PET with the new radioprobe [11C]QST-0837 (1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-phenyl-1H-pyrazol-3-yl)azetidine-1-[11C]carboxylate), a covalent inhibitor containing an azetidine carbamate skeleton for monoacylglycerol lipase. The uptake and residence of this new radioprobe depends on the intracellular pH gradient, and we evaluated this with in silico, in vitro and in vivo assessments. Molecular dynamics simulations predicted that because the azetidine carbamate moiety is close to that of water molecules, the compound containing azetidine carbamate would be more easily hydrolyzed following binding to monoacylglycerol lipase than would its analogue containing a piperidine carbamate skeleton. Interestingly, it was difficult for monoacylglycerol lipase to hydrolyze the azetidine carbamate compound under weakly acidic (pH 6) conditions because of a change in the interactions with water molecules on the carbamate moiety of their complex. Subsequently, an in vitro assessment using rat brain homogenate to confirm the molecular dynamics simulation-predicted behaviour of the azetidine carbamate compound showed that [11C]QST-0837 reacted with monoacylglycerol lipase to yield an [11C]complex, which was hydrolyzed to liberate 11CO2 as a final product. Additionally, the 11CO2 liberation rate was slower at lower pH. Finally, to indicate the feasibility of estimating how the hydrolysis rate depends on intracellular pH in vivo, we performed a PET study with [11C]QST-0837 using ischaemic rats. In our proposed in vivo compartment model, the clearance rate of radioactivity from the brain reflected the rate of [11C]QST-0837 hydrolysis (clearance through the production of 11CO2) in the brain, which was lower in a remarkably hypoxic area than in the contralateral region. In conclusion, we indicated the potential for visualization of the intracellular pH gradient in the brain using PET imaging, although some limitations remain. This approach should permit further elucidation of the pathological mechanisms involved under acidic conditions in multiple CNS disorders.

Disposable paper electrodes for detection of changes in dopamine concentrations in rat brain homogenates

Dopamine, the main catecholamine neurotransmitter plays an important role in renal, cardiovascular, central nervous systems, and pathophysiological processes. The abnormal dopamine levels can result in neurological disorders such as Parkinson's, Alzheimer's, schizophrenia, acute anxiety, neuroblastoma and also contribute to cognitive dysfunctions. Given the widespread importance of dopamine concentration levels, it is imperative to develop sensors that are able to monitor dopamine. Herein, we have developed pre-anodized disposable paper electrode modified with 1-pyrenebutyric acid, for the selective and sensitive determination of dopamine. The sensor was characterized with Fourier transform infrared spectroscopy, Energy dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and electrochemical techniques for addressing the robust formation and electrochemical activity. The modified electrode exhibited excellent electrocatalytic activity towards dopamine without the common interference from ascorbic acid. The calibration plot for the dopamine sensor resulted linear range from 0.003 μM to 0.5 μM with a detection limit of 0.11 nM. The sensor's potential utility was tested by monitoring dopamine concentration changes in rat brain homogenates when subjected to neurotoxicity. The developed sensor was validated with gold-standard UV–Vis spectroscopy studies and computational studies were performed to understand the interaction between 1-pyrenebutyric acid and dopamine.

A novel PET probe to selectively image heat shock protein 90α/β isoforms in the brain

BackgroundHeat shock proteins (HSPs) are present throughout the brain. They function as molecular chaperones, meaning they help with the folding and unfolding of large protein complexes. These chaperones are vital in the development of neuropathological conditions such as Alzheimer’s disease and Lewy body disease, with HSP90, a specific subtype of HSP, playing a key role. Many studies have shown that drugs that inhibit HSP90 activity have beneficial effects in the neurodegenerative diseases. Therefore, HSP90 PET imaging ligand can be used effectively to study HSP90 in neurodegenerative diseases. Among four HSP90 isoforms, two cytosolic isoforms (HSP90α and HSP90β) thought to be involved in the structural homeostasis of the proteins related to the neurodegenerative diseases. Currently, no useful PET imaging ligands selectively targeting the two cytosolic isoforms of HSP90 have been available yet.ResultsIn this study, we developed a novel positron emission tomography (PET) imaging ligand, [11C]BIIB021, by 11C-radiolabeling (a positron emitter with a half-life of 20.4 min) 6-Chloro-9-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-9H-purin-2-amine (BIIB021), an inhibitor with a high affinity for and selectivity to HSP90α and HSP90β. [11C]BIIB021 was synthesized with a high yield, molar activity and radiochemical purity. [11C]BIIB021 showed a high binding affinity for rat brain homogenate as well as human recombinant HSP90α and HSP90β proteins. Radioactivity was well detected in the rat brain (SUV 1.4). It showed clear specific binding in PET imaging of healthy rats and autoradiography of healthy rat and human brain sections. Radiometabolite was detected in the brain, however, total distribution volume was well quantified using dual-input graphical model. Inhibition of p-glycoprotein increased brain radioactivity concentrations. However, total distribution volume values with and without p-glycoprotein inhibition were nearly the same.ConclusionsWe have developed a new PET imaging agent, [11C]BIIB021, specifically targeting HSP90α/β. We have been successful in synthesizing [11C]BIIB021 and in vitro and in vivo imaging HSP90α/β. However, the quantification of HSP90α/β is complicated by the presence of radiometabolites in the brain and the potential to be a substrate for p-glycoprotein. Further efforts are needed to develop radioligand suitable for imaging of HSP90α/β.

Extraction Techniques Optimization, Suitable Solvents Selection and In Vitro Evaluation of Lipid Peroxidation Inhibition of Five Traditional Medicinal Plants using Egg Yolk, Rat Liver and Rat Brain Homogenates

Extraction Techniques Optimization, Suitable Solvents Selection and In Vitro Evaluation of Lipid Peroxidation Inhibition of Five Traditional Medicinal Plants using Egg Yolk, Rat Liver and Rat Brain Homogenates

Targeting inflammation and oxidative stress for protection against ischemic brain injury in rats using cupressuflavone

Inflammatory responses and oxidative stress contribute to the pathogenesis of brain ischemia/reperfusion (IR) injury. Naturally occurring bioflavonoids possess antioxidant and anti-inflammatory properties. The phytochemicals of Juniperus sabina L., known as “Abhal” in Saudi Arabia, have been studied and cupressuflavone (CUP) has been isolated as the major bioflavonoid. This study aimed to investigate the neuroprotective potential of CUP in reducing brain IR damage in rats and to understand probable mechanisms. After 60 min of inducing cerebral ischemia by closing the left common carotid artery (CCA), blood flow was restored to allow reperfusion. The same surgical procedure was performed on sham-operated control rats, excluding cerebral IR. CUP or vehicle was given orally to rats for 3 days prior to ischemia induction and for a further 3 days following reperfusion. Based on the findings of this study, compared to the IR control group, CUP-administered group demonstrated reduced neurological deficits, improved motor coordination, balance, and locomotor activity. Additionally, brain homogenates of IR rats showed a decrease in malondialdehyde (MDA) level, an increase in reduced glutathione (GSH) content, and an increase in catalase (CAT) enzyme activity following CUP treatment. CUP suppressed neuro-inflammation via reducing serum inflammatory cytokine levels, particularly those of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) and enhancing the inflammatory cytokine levels, such as Nuclear factor kappa- B (NF-κB), TANK-binding kinase-1 (TBK1), and interferon beta (IFN-β) in brain tissues. Furthermore, CUP ameliorated the histological alterations in the brain tissues of IR rats. CUP significantly suppressed caspase-3 expression and downregulated the Toll-like receptor 4 (TLR4)/NF-κB signaling pathway as a result of suppressing High mobility group box 1 (HMGB1). To our knowledge, this is the first study to document the neuroprotective properties of CUP. Thus, the study findings revealed that CUP ameliorates IR–induced cerebral injury possibly by enhancing brain antioxidant contents, reducing serum inflammatory cytokine levels, potentiating the brain contents of TBK1 and IFN-β and suppressing the HMGB1/TLR-4 signaling pathway. Hence, CUP may serve as a potential preventive and therapeutic alternative for cerebral stroke.

Bioassays guided isolation of berberine from Berberis lycium and its neuroprotective role in aluminium chloride induced rat model of Alzheimer's disease combined with insilico molecular docking.

Berberis lycium is an indigenous plant of Pakistan that is known for its medicinal properties. In the current study, we investigated the anti-Alzheimer's effect of berberine isolated from Berberis lycium. Root extract of B. lycium was subjected to acetylcholinesterase inhibition assay and column chromatography for bioassays guided isolation of a compound. The neuroprotective and memory improving effects of isolated compound were evaluated by aluminium chloride induced Alzheimer's disease rat model, elevated plus maze (EPM) and Morris water maze (MWM) tests., Levels of dopamine and serotonin in rats brains were determined using HPLC. Moreover, western blot and docking were performed to determine interaction between berberine and β-secretase. During fractionation, ethyl acetate and methanol (3:7) fraction was collected from solvent mixture of ethyl acetate and methanol. This fraction showed the highest anti-acetylcholinesterase activity and was alkaloid positive. The results of TLC and HPLC analysis indicated the presence of the isolated compound as berberine. Additionally, the confirmation of isolated compound as berberine was carried out using FTIR and NMR analysis. In vivo EPM and MWM tests showed improved memory patterns after berberine treatment in Alzheimer's disease model. The levels of dopamine, serotonin and activity of antioxidant enzymes were significantly (p<0.05) enhanced in brain tissue homogenates of berberine treated group. This was supported by decreased expression of β-secretase in berberine treated rat brain homogenates and good binding affinity of berberine with β-secretase in docking studies. Binding energies for interaction of β-secretase with berberine and drug Rivastigmine is -7.0 kcal/mol and -5.8 kcal/mol respectively representing the strong interactions. The results of docked complex of secretase with berberine and Rivastigmine was carried out using Gromacs which showed significant stability of complex in terms of RMSD and radius of gyration. Overall, the study presents berberine as a potential drug against Alzheimer's disease by providing evidence of its effects in improving memory, neurotransmitter levels and reducing β-secretase expression in the Alzheimer's disease model.

Resveratrol-loaded copolymer nanoparticles with anti-neurological impairment, antioxidant and anti-inflammatory activities against cerebral ischemia–reperfusion injury

During cerebral ischemia/reperfusion (I/R) injury, oxidative stress and inflammation are major contributors to disability. Finding compounds that reduce oxidative stress, inflammation and strengthen the antioxidant defense system properties is one of the vital areas of research. Therefore, the present research aimed to evaluate the effects of resveratrol loaded in copolymer nanoparticles (RES.CP.NPs) on cerebral I/R injury in rats. After preparing and characterization RES.CP.NPs, these platforms were administered to cerebral I/R injured rats at a dose of 20 mg/kg after 2 h occlusion and the neurological disability scores were measured 24 h later. The content of malondialdehyde (MDA), glutathione (GSH), and catalase (CAT) activity as well as the expression levels of TNF-α, IL-10 and NF-κB-p65 in rat brain homogenates were measured. The RES.CP.NPs synthesized in the present study had a spherical structure with a zeta potential of −38 mV and a stable RES release for 72 h. Administration of RES.CP.NPs to I/R rats resulted in significant improvement in neurological disability score, decreased MDA levels, increased GSH content, increased CAT activity, decreased TNF-α expression, increased IL-10 expression, and decreased NF-κB-p65 expression (P < 0.0001). RES.CP.NPs can have neuroprotective effects in cerebral I/R conditions as a result of their strong antioxidant and anti-inflammatory activities.

Ex vivo antiacetylcholinesterase studies on silver nanoparticles synthesized using green approach

For the green fabrication of silver nanoparticles (AgNPs), scientists are favoring herbal sources to avoid the toxic effects of synthetic sources. Thus, in this study, the AgNPs were generated using the herbal extract of the whole plant Hippeastrum hybridum L. (HH) and 1 mM AgNO3 aqueous solution. These HH‐AgNPs were characterized via UV–Vis Spectroscopy, which showed a maximum absorbance of 1.61 at a 432 nm sharp peak; FT‐R analysis, which confirmed the functional groups in HH extract and their AgNPs; XRD analysis, which gives 4‐Bragg's reflections at 2θ and confirmed the HH‐AgNPs crystal structure with 13.3 nm average size; SEM analysis, which confirmed the irregular‐shaped morphology with 40 nm mean size; and EDX analysis, which confirmed the elemental composition and reported that Ag was present in 22.75%. After characterization, these AgNPs were tested as anti‐Alzheimer agents against acetylcholinesterase (AChE) in ex vivo activity using rat brain homogenate as a source of AChE enzyme. AChE showed 48 ± 0.03 and 42 ± 0.05% activity at 150 μg concentration of HH plant and HH‐AgNPs, respectively, with 145 ± 0.24 and 124.2 ± 0.14 μg IC50 concentration. According to enzyme kinetics results, the plant extract inhibits AChE in competitive mode (Km increased from 166.2% to 1,379.2% and Vmax not affected), while HH‐AgNPs showed the mixed mode of inhibition (Km increased from 0.029 to 0.048, and Vmax decreased from 9 to 5.4). KIapp and Vmaxiapp were also calculated (KIapp increased from 39 to 95.1 μg, and Vmaxiapp remained constant for the plant), while for HH‐AgNPs, both Kmaxipp and Vmaxiapp increased from 122.32 to 325 μg and 8.15 to 9.8 μg, respectively. The Km, Ki, and KI were also calculated and were found to be 0.017 mM (HH‐plant) and 0.2 mM (HH‐AgNPs); 98 μg (HH‐plant); and 129 μg (HH‐AgNPs), 53 μg (HH‐plant), and 179 μg (HH‐AgNPs), respectively. γKm (18 mM) was calculated for HH‐AgNPs, while for HH‐plant, it is not applicable. In conclusion, it could be said that HH‐plant plays a significant role in the generation of small‐size HH‐AgNPs. These AgNPs exhibited significant anti‐AChE potential as compared to HH‐plant extract and proved to be an herbal source for the treatment of AD.

Study of fingolimod, nitric oxide inhibitor, and P-glycoprotein inhibitor in modulating the P-glycoprotein expression via an endothelin-sphingolipid pathway in an animal model of pharmacoresistant epilepsy.

The overexpression of P-glycoprotein (P-gp) contributes to drug resistance in patients with epilepsy, and the change of P-gp expression located at the blood-brain barrier alienates the anti-seizure effects of P-gp substrates. Thus, the present study explored the effect of fingolimod (FTY720) acting through an endothelin-sphingolipid pathway on P-gp-induced pentylenetetrazol (PTZ)-kindled phenobarbital (PB)-resistant rats. PTZ kindling (30 mg/kg; i.p.) and PB (40 mg/kg; orally) were used to develop an animal model of refractory epilepsy. The effect of Fingolimod on seizure score (Racine scale), plasma and brain levels of PB (high-performance liquid chromatography), and blood-brain barrier permeability (Evans blue dye) was determined. Further, Fingolimod's neuroprotective effect was determined by measuring the levels of various inflammatory cytokines, oxidative stress parameters, and neurotrophic factors in rat brain homogenate. The Fingolimod's effect on P-gp expression was estimated by reverse transcriptase-polymerase chain reaction and immunohistochemistry in rat brain. The H and E staining was done to determine the neuronal injury. Fingolimod significantly (P < 0.001) reduced the seizure score in a dose-dependent manner and alleviated the blood-brain barrier permeability. It decreased the P-gp expression, which further increased the brain PB concentration. Fingolimod significantly (P < 0.01) reduced oxidative stress as well as inflammation. Moreover, it attenuated the raised neuronal injury score in a resistant model of epilepsy. The modulation of the P-gp expression by Fingolimod improved drug delivery to the brain in an animal model of refractory epilepsy. Therefore, S1P signaling could serve as an additional therapeutic target to overcome refractoriness.

Evaluation of Eugenia uniflora L. leaf extracts obtained by pressurized liquid extraction: Identification of chemical composition, antioxidant, antibacterial, and allelopathic activity

Pitangueira (Eugenia uniflora L.) leaves are rich in phenolic compounds. To evaluate these compounds, pitangueira leaves were submitted to extraction by aqueous infusion (AI) and by pressurized liquid extraction (PLE). PLE extracts were obtained with water (PLE-W), water:ethanol (PLE-W:E), and ethanol (PLE-E). Fifty-four extracted compounds were tentatively identified by LC-MS. The PLE-W:E extraction resulted in a higher concentration of phenolic acids and tannin compounds, while PLE-E showed a higher concentration of flavonoids. All extracts tested in vitro showed good antioxidant activity, as evaluated by thiobarbituric acid reactive substances (TBARS) and reactive oxygen species (ROS) assays in rat brain homogenates model systems and in protecting yeasts under oxidation. Among the eight bacterial strains tested with the pitangueira leaf extracts, antibacterial activity was observed mainly against Staphylococcus aureus with the extracts of AI, PLE-W, and PLE-W:E. And for Pseudomonas aeruginosa, only the AI extract showed antibacterial activity. The other strains were not affected by the extracts. All extracts exhibit an allelopathic effect against lettuce seeds. Even though 72.2% of the putatively identified compounds were common to all extracts, PLE extracts provide more regular antioxidant, antibacterial, and allelopathic activities.

OXIDATIVE MECHANISMS UNDERLYING BRAIN CELL DAMAGE IN RATS UNDER CHANGES IN DURATION OF THE LIGHTDARK CYCLES

One of the main mechanisms underlying cell damage is lipid peroxidation, which is characterized by increased levels of free radical, products of lipid peroxidation chain reactions, and alterations in the activity of the antioxidant system in tissues. Therefore, the objective of this study is to investigate the proand antioxidant balance in rat brain homogenate under conditions of disrupted "light-dark" cycles. The study involved 28 white Wistar rats weighing 150-200 gm, divided into two groups: control (15) and the group of animals exposed to acute desynchronosis simulation (13). To simulate desynchronosis, a normal "light-dark" cycle of 12 hours of light and 12 hours of darkness was initially established for three weeks, followed by a shift of the "light-dark" phases backward by six hours over the next three days. The animals were kept in standard conditions with a balanced diet in the vivarium, and the study adhered to the principles of biomedical ethics.&#x0D; The results obtained indicate a significant increase in the activity of superoxide anion radical production in the brain tissues of the rats with modelled desynchronosis by 75.8% compared to the control group. This increase is attributed to the NADPH-oxidase mechanism by 20.2% and to xanthine oxidase mechanisms by 28%. The concentration of TBA-active products increased by 36.8%, the growth of TBA-active products elevated by 30%, and the activity of the key antioxidant enzymes, superoxide dismutase and catalase, decreased by 53% and 14%, respectively, in the second group of animals. Furthermore, in the brain tissues of rats with disrupted "light-dark" cycles, there were found elevated levels of nitrites by 23.8%, nitrosothiols by 19%, and peroxynitrites by 69%, along with a reduction in the activity of total NO-synthase by 59.1%, inducible NO-synthase by 61.1%, and constitutive NO-synthase by 9% compared to the control group. The activity of ornithine decarboxylase increased by 26.5% in the brain tissues of the rats with simulated desynchronosis compared to the control group.&#x0D; In conclusion, the disruption of "light-dark" cycles leads to the development of oxidative stress in brain tissues of animals and is characterized by increased production of reactive oxygen species and diminished antioxidant protection. Acute desynchronosis decreases nitric oxide production through the NO-synthase pathway, while increasing the accumulation of toxic nitric oxide metabolites in the brain tissues of rats, thereby posing a potential risk of nitrosative stress. Furthermore, alterations in the normal light regimen of the day activate the arginase pathway of L-arginine metabolism and result in increased glutamate production in the brain tissues of rats, potentially leading to oxidative stress.

Synthesis, optimization, and characterization of silver/gold allied bimetallic from Hippeastrum hybridum (L.) and their ex vivo anti‐acetylcholinesterase activity in rat brain

Nanotechnology, a modern technique, is apprehensive for small‐size nanoparticle (NP) generation via superficial, straightforward, and pharmaceutically modes. The main aim of the current research was to project the development of a green justifiable method to synthesize Ag/Au bimetallic NPs (BNPs) via Hippeastrum hybridum (HH) extract and then to use these BNPs as rat brain anti‐acetylcholinesterase (anti‐AChE) in ex vivo mode. According to the current results, BNPs exhibited a sharp peak of 542 nm when characterized by ultravioet (UV)–visible spectroscopy; Fourier transform infrared (FT‐IR) analysis exhibited peaks, which confirmed the existence of various phytochemicals in HH plant extract; X‐ray diffraction (XRD) analysis confirmed the crystal BNPs 8.34 nm mean size; scanning electron microscopy (SEM) showed that BNPs had a square shape with 20‐nm nanomeasures calculated sizes; and energy dispersive X‐ray (EDX) analysis confirmed Ag and Au composition, which was reported to be 12% and 38.26%, respectively. Moreover, the anti‐AChE potential of BNPs was also tested to cure Alzheimer's disease (AD). An excellent anti‐AChE activity was exhibited by BNPs in which AChE exhibited 31 ± 0.22% activity at 150‐μg concentration of BNPs with 103 ± 0.42 μg IC50. Statistical analysis exhibited that BNPs inhibit AChE noncompetitively, Km remain constant, whereas Vmax decreased from 7.24 to 6.1 (18.23% to 31.11%). Ag/Au BNPs do not affect KIapp, while increasing Vmaxiapp from 11.07 to 16 (17.39% to 67.4%). The Km, Ki, and KI were described to be 0.024 mM, 169 μg, and 91 μg, respectively. So, it is concluded that small‐size, stable, potent Ag/Au BNPs were synthesized from HH extract, and consequently, these BNPs significantly inhibit AChE activity in rat brain homogenate and thus prove to be a significant remedy in the treatment of AD.

A Comparative Study on GC-MS Profiles and Effect of Ethanolic Extracts of Stem-Bark and Leaves of Daniellia oliveri on Cerebral Sodium Pump and Thiols Status in Fenton Reaction Treated Rat Organs In-vitro

Daniellia oliveri is one of the most extensively utilized medicinal plants in Nigeria and some West African countries for the treatment of various ailments. In the present study, the GC-MS profiles and effect of ethanolic extracts of stem bark and leaves of Daniellia oliveri on lipid peroxidation, Na+/K+-ATPase activity and thiols status in H2O2 and Fe2+ (fenton reaction) assaulted cerebral and hepatic tissue homogenates was investigated. The stem bark and leaves were extracted with absolute ethanol for 72hrs and concentrated in a rotary evaporator. Wistar rats were euthanized and the brains and livers were removed, homogenized, centrifuged and the supernatants were used for lipid peroxidation, Na+/K+-ATPase activity, and thiol assays in the presence of prooxidants (1mM H2O2 or 10μM Fe2+ or H2O2 and Fe2+).The results revealed that ethanol extract of both the leaf and stem-bark inhibited lipid peroxidation induced by H2O2, Fe2+ and combination of H2O2 and Fe2+ and this was evident in the reduction of lipid peroxidation adducts formation in rat liver and brain homogenates with the stem-bark possessing more efficacy. Furthermore, the results of Na+/K+-ATPase and thiol assays revealed that H2O2 and Fe2+ inhibited the activity of cerebral Na+/K+-ATPase, while H2O2/Fe2+ caused a marked reduction in the levels of both protein and non-protein thiols in the tissues homogenates. However, the ethanolic extract of Daniellia oliveri extirpated this anomaly by increasing the activity of cerebral Na+/K+-ATPase as well as the thiol level in the rat hepatic and cerebral tissues. Finally, GC-MS analysis was carried out on the ethanolic extracts of the stem bark and leaves of Daniellia oliveri and results revealed the presence of 21 compounds including 1,2-Benzisothiazol-3-amine, 1H-Indole-2-carboxylic acid, 2-Ethylacridine, Ethyl 2-(2-chloroacetamido)-3,3,3-trifluoro-2-(3-fluoroanilino) propionate and trans-1-Butyl-2-methylcyclopropane in the stem bark and 10 compounds including Methylthio-acetonitrile, 2-Methyl-Z,Z-3,13-octadecadienol, Desulphosinigrin and 4H-1,2,4-triazole-3,5-diamine in the leaves, justifying the observed biological activities of Daniellia oliveri and the higher potency of the stem bark may be attributed to the presence of more bioactive constituents found in it. This study therefore justifies the medicinal usage of leaves and stem-bark of Daniellia oliveri and suggests its consideration in the treatment and management of degeneration diseases with etiology associated with oxidative stress.

Pharmacological Characterization of [18F]-FNM and Evaluation of NMDA Receptors Activation in a Rat Brain Injury Model

PurposeNMDA receptors (NMDARs) dysfunction plays a central role in the physiopathology of psychiatric and neurodegenerative disorders whose mechanisms are still poorly understood. The development of a PET (positron emission tomography) tracer able to selectively bind to the NMDARs intra-channel PCP site may make it possible to visualize NMDARs in an open and active state. We describe the in vitro pharmacological characterization of [18F]-fluoroethylnormemantine ([18F]-FNM) and evaluate its ability to localize activated NMDA receptors in a rat preclinical model of excitotoxicity.ProceduresThe affinity of the non-radioactive analog for the intra-channel PCP site was determined in a radioligand competition assay using [3H]TCP ([3H]N-(1-[thienyl]cyclohexyl)piperidine) on rat brain homogenates. Selectivity was also investigated by the displacement of specific radioligands targeting various cerebral receptors. In vivo brain lesions were performed using stereotaxic quinolinic acid (QA) injections in the left motor area (M1) of seven Sprague Dawley rats. Each rat was imaged with a microPET/CT camera, 40 min after receiving a dose of 30 MBq + / − 20 of [18F]-FNM, 24 and 72 h after injury. Nine non-injured rats were also imaged using the same protocol.ResultsFNM displayed IC50 value of 13.0 ± 8.9 µM in rat forebrain homogenates but also showed significant bindings on opioid receptors. In the frontal and left somatosensory areas, [18F]FNM PET detected a mean of 37% and 41% increase in [18F]FNM uptake (p < 0,0001) 24 and 72 h after QA stereotaxic injection, respectively, compared to the control group.ConclusionsIn spite of FNM’s poor affinity for NMDAR PCP site, this study supports the ability of this tracer to track massive activation of NMDARs in neurological diseases.

Quantitative determination of monoamine neurotransmitters in rat brain homogenates using HPLC-MS/MS

Relevance. Evaluation of the effect of drugs on neurotransmitter processes is an important component of pharmacodynamic studies. The quantitative determination of monoamine neurotransmitters in the brain structures of laboratory animals is an urgent task of pharmacology and physiology.Purpose of the study. Development of a method for the quantitative determination of serotonin, dopamine, norepinephrine, histamine and epinephrine in rat brain homogenates using HPLC-MS/MS.Methods. The isolation of neurotransmitters from the brain of rats was carried out by homogenizing the biomaterial with acetonitrile and hydrochloric acid. The extraction was purified by liquid-liquid extraction with chloroform and isopropanol. Monoamines were detected using an AB Sciex QTrap 3200MD mass spectrometer, chromatography was performed using an Agilent Technologies 1260 Infinity II HPLC. Methanol and deionized water were used as eluent.Results. Sample preparation consisted of centrifugation of the resulting homogenate, drying of the supernatant in a stream of nitrogen, dissolution of the precipitate in the mobile phase, and purification of the solution using a mixture of chloroform and isopropanol. An Agilent InfinityLab Poroshell 120 EC-C18 4.6×100 mm, 2.7 μm analytical column was used to separate monoamine neurotransmitters. The total time of the chromatographic analysis was 12 minutes, the retention time of norepinephrine, epinephrine, dopamine, serotonin, histamine was 2.8; 3.2; 5.4; 7.9; and 2.2 minutes, respectively. The analytical range of the technique was 25.0–5000.0 ng/g for epinephrine, histamine, and dopamine; 5.0–5000.0 ng/g for serotonin and 50.0–5000.0 for norepinephrine. To test the technique, we analyzed monoamine neurotransmitters in the striatum of intact Wistar rats.Conclusion. The developed bioanalytical HPLC-MS/MS method for the quantitative determination of monoamine neurotransmitters in the rat brain fully complies with the validation requirements. The metrological characteristics of the technique make it possible to estimate the content of norepinephrine, epinephrine, dopamine, serotonin, and histamine in the brain structures of rats with high accuracy.

Synthesis and evaluation of small organic molecule as reactivator of organophosphorus inhibited acetylcholinesterase

A series of uncharged salicylaldehyde oximes were synthesized and evaluated for the reactivation of organophosphorus (OP) nerve agents simulants Diethylchlorophosphonate (DCP) & Diethylcyanophosphonate (DCNP) and pesticides (paraoxon & malaoxon) inhibited electric eel Acetylcholinesterase (AChE). The computational software Swiss ADME and molinspiration were used to unfold the probability of drug-likeness properties of the oximes derivatives. Substituted aromatic oximes with diethylamino or bromo group with free hydroxyl group ortho to oxime moiety were found efficient to regenerate the enzymatic activity in in-vitro AChE assay. The alkylation of the ortho hydroxyl group of derivatives led to the loss of reactivation potential. The derivatives with a hydroxyl group and without oxime group and vice versa did not show significant reactivation potency against tested OP toxicants. Further, we also evaluated the reactivation potential of these selected molecules on the rat brain homogenate against different OPs inhibited ChE and found maximum reactivation potency of oxime 2e. The in-vitro results were further validated by molecular docking and dynamic studies which showed that the hydroxyl group interacted with serine amino acids in the catalytic anionic site of AChE enzyme and was stable up to 200 ns consequently providing proper orientation to oxime moiety for reactivating the OP inhibited enzyme. It has thus been proved by the structure-activity relationship of oximes derivatives that hydroxyl group ortho to oxime is essential for reactivating OP inhibited electric eel AChE. Amongst the twenty-one oximes derivatives, 2e was found to be most active in regenerating the paraoxon, malaoxon, DCP and DCNP inhibited AChE enzyme.

An HPLC-based assay for improved measurement of glutamate decarboxylase inhibition/activation

L-Glutamic acid decarboxylase (GAD) is an enzyme that ensures the balance between the levels of two neurotransmitters, γ-aminobutyric acid (GABA) and L-glutamic acid (L-Glu), necessary for proper brain functioning. A reduction in the concentrations of GABA and/or GAD activity has been implicated in the symptoms associated with epilepsy, which could be plausibly alleviated by the application of GAD activators. As any unnecessary interference in GAD catalytic activity could be detrimental, it is important to study whether CNS (or other) drug candidates act on GAD or not. The ability to identify and reduce this risk early could significantly improve the process of drug development. Although many methods for measuring GAD activity in various biological samples have been described, only few (such as manometric and radiometric) were adopted as in vitro assays for the screening of potential GAD inhibitors/activators. However, these methods require specialized equipment and/or an expensive radiolabeled substrate, and may have sensitivity and/or reliability issues. Therefore, this study aimed to develop an HPLC-DAD-based assay that would allow a simple and more accurate measurement of GAD inhibition or activation using unpurified mice or rat brain homogenates. This assay is based on the quantification of GABA, formed during the enzymatic reaction, after its derivatization with dansyl chloride. Various parameters were evaluated to optimize the assay procedure (e.g. homogenate volume, incubation time, DMSO content, GAD, GABA, and dansyl-GABA stabilities). This assay was validated for pharmacological screenings using 3-mercaptopropionic acid and gallic acid and GAD obtained from different experimental animals.