Asparagus recemosus enhances memory and protects against amnesia in rodent models.

Asparagus Racemosus (AR) is an Ayurvedic rasayana possessing multiple neuropharmacological activities. The adpatogenic and antidepressant activity of AR is well documented. The present study was undertaken to assess nootropic and anti-amnesic activities of MAR in rats. The Morris water maze (MWM) and elevated plus maze (EPM) models were employed to evaluate learning and memory activity. Subsequently, the anti-amnestic activity was evaluated in scopolamine and sodium nitrite (NaNO(2))-induced amnestic models in rats. Rats pre-treated with MAR (50, 100 and 200mg/kg, p.o) for 7 days showed significant decrease in escape latency in the MWM test indicating nootropic activity. MAR also significantly reversed scopolamine and sodium nitrite-induced increase in transfer latency on EPM indicating anti-amnesic activity. Further, MAR dose-dependently inhibited acetylcholinesterase enzyme in specific brain regions (prefrontal cortex, hippocampus and hypothalamus). Thus, MAR showed nootropic and anti-amnesic activities in the models tested and these effects may probably be mediated through augmentation of cholinergic system due to its anti-cholinesterase activity.

Antidepressant activity of Asparagus racemosus in rodent models.

Asparagus racemosus Linn. (AR) is an Ayurvedic rasayana used as an adaptogen. Adaptogenic drugs are those which are useful as anti-stress agents by promoting non-specific resistance of the body. Although, the adaptogenic effect of AR is well documented, its use in psychological disorders like depression is not scientifically evaluated. Hence, the present investigation evaluates the antidepressant effect of methanolic extract of roots of AR (MAR) standardized to saponins (62.2% w/w). Rats were given MAR in the doses of 100, 200 and 400 mg/kg daily for 7 days and then subjected to forced swim test (FST) and learned helplessness test (LH). The results show that MAR decreases immobility in FST and increases avoidance response in LH indicating antidepressant activity. In behavioral experiments, MAR increased the number of head twitches produced by 5-HTP and increased clonidine-induced aggressive behavior indicating facilitatory effect on both serotonergic and adrenergic systems respectively. However, MAR had insignificant effect on l-DOPA-induced aggressive behavior indicating absence of activity on dopaminergic system. MAR also reversed changes to the endogenous antioxidant system induced by FST. Thus, MAR has significant antidepressant activity and this effect is probably mediated through the serotonergic and the noradrenergic systems and augmentation of antioxidant defenses.

Isolation of nitric oxide synthase from human platelets.

We are reporting a distinct constitutive isoform of nitric oxide synthase that has been purified to homogeneity from human platelet cytosolic fractions. Purification involved ultra centrifugation at 100,000 x g followed by two sequential affinity chromatography procedures: adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin Sepharose 4B. Purified enzyme appeared as a single band (approximately 80 kDa) under denaturing condition (SDS-PAGE). The native enzyme appears to be dimeric, since its molecular weight estimated by gel filtration was approximately 150 kDa. Enzyme activity was dependent on L-arginine, NADPH and (6R)-5,6,7,8-tetrahydro-L-biopterine. Partially purified platelet NOS (100,000 x g supernatant) activity was sensitive to calmodulin antagonists and to the N omega-Monomethyl-L-arginine, a substrate analog of L-arginine.

Identification of the site of non-enzymatic glycation of glutathione peroxidase: rationalization of the glycation-related catalytic alterations on the basis of three-dimensional protein structure.

Bovine erythrocyte glutathione peroxidase has been glycated in vitro by incubation in 0.05 M glucose at pH 7.4. Upon glycation the estimated KM for t-butylhydroperoxide reduction increased by approx. 3-fold in comparison to non-glycated glutathione peroxidase. The glycated protein fraction was stabilized by NaBH4 reduction and subjected to tryptic cleavage. Affinity chromatography of the tryptic digest on m-aminophenylboronate-Agarose resulted in the isolation of a single glycated peptide. The peptide was identified as T94-K117 by amino-acid composition comparison to the published amino-acid sequence for this enzyme. The glycation site has been identified as the epsilon-NH2 group of K110. Examination of the three-dimensional structure of bovine erythrocyte glutathione peroxidase indicates that K110 lies on the surface of the protein approximately 15 A away from the active site selenocysteine (SEC 45). Modeling studies indicate that K110 can communicate via H-bonded interactions with the alpha-helix containing the active site residues (SEC-45 and R50). The observed elevation of KM upon glycation of bovine glutathione peroxidase is discussed in terms of the disruption of the long range H-bonded interaction.

A distinct strategy to generate high-affinity peptide binders to receptor tyrosine kinases.

We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocyte cultures differentiated in vitro.

Glutamate plays an important role in brain development, physiological function, and neurodegeneration. Astrocytes control synaptic concentration of glutamate via the high affinity glutamate transporters, GLT-1 and GLAST, and the glutamate catabolizing enzyme, glutamine synthetase. In this study we show that astrocytes cultured from rat brain in various stages of development including embryonic (E18), postnatal (P1-P21) and mature (P50), show distinct patterns of GLT-1 and GLAST expression, glutamine synthetase activity, and phenotypic changes induced by dibutyryl-cyclic adenosine monophosphate. The transcripts for GLT-1 message were detectable in embryonic astrocytes only, whereas the GLAST message was highly expressed in E18 and P1-P4 astrocyte cultures, declined in P10-P21, and was undetectable in P50 astrocytes. Uptake of 3H-glutamate correlated well with GLAST expression in astrocyte cultures of all developmental stages. Glutamine synthetase activity significantly declined from high embryonic levels in P4 astrocytes and remained low throughout postnatal maturation. Exposure of astrocyte cultures to the differentiating agent, db-cAMP (250-500 microM; 6 days), resulted in a pronounced stellation, up-regulation of GLT-1 and GLAST in E18, and GLAST in P4 cultures, while it was ineffective in P10 astrocytes. By contrast, db-cAMP induced a more pronounced stimulation of glutamine synthetase activity (up to 10-fold above basal) in P10 than in E18 cultures (up to 2 times above basal). The differences in expression/inducibility of glutamate transporters and glutamine synthetase observed in astrocyte cultures derived from various stages of fetal and postnatal development suggest that astrocytes in vivo might also respond differently to environmental or injurious stimuli during development and maturation.

ELISA for in vivo assessment of nonenzymatically glycated platelet glutathione peroxidase.

Using a combination of boronate affinity chromatography and ELISA methodology, a simple procedure was devised to selectively determine the in vivo glycated state of the platelet glutathione peroxidase (GSH-Px) from normal and diabetic subjects. The mean total GSH-Px levels in the normal (n = 14) and diabetic (n = 18) platelets were 1167 +/- 97 and 1007 +/- 73 ng/mg protein, respectively. The mean percentage glycated GSH-Px in the normal and diabetic platelets were 5.79 +/- 0.72% and 11.68 +/- 0.95%, respectively. When the percentage glycated GSH-Px was compared with the fructosamine values, a correlation coefficient of 0.71 was obtained. This indicates that the glycation status of platelet GSH-Px can be utilized as a sensitive, short-term index of plasma glucose levels.

Glutathione metabolic enzyme activities in diabetic platelets as a function of glycemic control.

Type 1 diabetic subjects categorized on the basis of the glycated haemoglobin content of their blood (low less than 7%; medium, greater than 7% and less than 11%; high, greater than 11%) were analyzed for total intraplatelet GSH as well as for the steady-state kinetic parameters (apparent KM and apparent Vmax) of some glutathione metabolic enzymes including glutathione reductase, glutathione peroxidase, gamma-glutamyltrans-peptidase and glutathione-S-transferase. This study indicates that intraplatelet GSH content of subjects with low glycated-haemoglobin is approximately 2-fold higher than those with medium glycated-haemoglobin. There was no further decrease in intraplatelet-GSH in subjects with high glycated-haemoglobin. The kinetic parameters of the platelet-enzymes studied (glutathione reductase, gamma-glutamyltranspeptidase and glutathione-S-transferase) were essentially independent of the glycation state of the subject. However, the apparent KM of glutathione peroxidase was approximately 4-fold higher in the subjects with high glycated-haemoglobin, in comparison to low subjects. This decrease in affinity could possibly result from the susceptibility of this enzyme to non-enzymatic glucosylation as purified samples of glutathione peroxidase incubated in vitro with glucose showed similar increases in apparent KM. These results are discussed in terms of the potential contribution of glutathione peroxidase impairment, to the hyperaggregability of the diabetic platelet.

Visible light photochemical release of nitric oxide from S-nitrosoglutathione: potential photochemotherapeutic applications.

Some aspects of the physiological role of NO may be mediated by stable NO-carriers such as S-nitrosoglutathione and related S-nitrosothiols. In this report we show that irradiation of S-nitrosoglutathione at either absorption band (lambda max = 340 nm or 545 nm) results in the release of nitric oxide. Photolysis of S-nitrosoglutathione at 545 nm exhibited a quantum yield of 0.056 +/- 0.002 and was best approximated by a first-order process with kobs = 4.9 x 10(-7) +/- 0.3 x 10(-7) s-1. The photolytic release of NO from S-nitrosoglutathione resulted in an enhanced cytotoxic effect of S-nitrosoglutathione on HL-60 leukemia cells. That the cytotoxic effect of S-nitrosoglutathione was diminished by the addition of oxyhemoglobin strongly suggests that NO is the cytotoxic species. The finding that NO can be readily liberated from S-nitrosoglutathione by visible radiation indicates that the photochemical properties of this compound in the visible spectrum must be considered in order to obtain meaningful data as to its physiological role and the S-nitrosoglutathione and related compounds may find use as photochemotherapeutic agents.

Adaptogenic activity of Withania somnifera: an experimental study using a rat model of chronic stress.

Withania somnifera (WS) Dunal is classified in Ayurveda, the ancient Hindu system of medicine, as a rasayana, a group of plant-derived drugs reputed to promote physical and mental health, augment resistance of the body against disease and diverse adverse environmental factors, revitalise the body in debilitated conditions and increase longevity. These attributes are remarkably similar to the properties ascribed to adaptogens like Panax ginseng (PG) in contemporary medicine. As such, the adaptogenic activity of a standardised extract of WS roots was investigated against a rat model of chronic stress (CS). The stress procedure was mild, unpredictable footshock, administered once daily for 21 days to adult male Wistar rats. CS induced significant hyperglycaemia, glucose intolerance, increase in plasma corticosterone levels, gastric ulcerations, male sexual dysfunction, cognitive deficits, immunosuppression and mental depression. These CS induced perturbations were attenuated by WS (25 and 50 mg/kg po) and by PG (100 mg/kg po), administered 1 h before footshock for 21 days. The results indicate that WS, like PG, has significant antistress adaptogenic activity, confirming the clinical use of the plant in Ayurveda.

Glutathione homeostasis and leukotriene-induced permeability in human blood-brain barrier endothelial cells subjected to in vitro ischemia.

Ischemic alterations in the glutathione (GSH) redox system of the blood-brain barrier (BBB) may facilitate oxidative injury and formation of vasogenic brain edema. In this study, both the intra- and extracellular GSH contents of human cerebromicrovascular endothelial cells (HCEC) were reduced by 35% after exposing the cells to 4 h in vitro ischemia and 24 h-recovery. The intracellular/extracellular GSH ratio was not affected, indicating a constant rate of GSH efflux. The activities of the peroxide detoxifying enzymes, glutathione peroxidase and glutathione S-transferase, increased by 35%-50%, whereas the GSH regenerating enzyme, glutathione reductase, remained unchanged in ischemic HCEC. gamma-glutamyl transpeptidase (GGTP), a GSH catabolizing enzyme enriched in brain capillaries, was reduced by 30-50% in ischemic HCEC. The effect of in vitro ischemia on HCEC permeability was assessed by measuring sodium fluorescein clearance across a compartmentalized in vitro BBB model. Sodium fluorescein clearance across HCEC monolayers exposed to leukotriene C4 in the presence of the GGTP inhibitor, acivicin (1 microM), or after in vitro ischemia was increased by 60% and 30%, respectively, suggesting that oxidative stress and loss of GGTP may 'unmask' BBB permeabilizing actions of leukotrienes. These results indicate that oxidative stress and loss of GGTP activity in HCEC contribute to ischemic BBB disruption and vasogenic brain edema.

Effect of Indian Hypericum perforatum Linn on animal models of cognitive dysfunction.

The effect of a standardised 50% ethanolic extract of Indian Hypericum perforatum (IHp) was investigated for its putative nootropic activity on various experimental paradigms of learning and memory, viz. transfer latency (TL) on elevated plus-maze, passive avoidance (PA), active avoidance (AA), scopolamine and sodium nitrite induced amnesia (SIA & NIA) in albino rats. Pilot studies indicated that single dose administration of IHp had little or no acute behavioural effects, hence the extract of IHp was administered orally at two dose levels (100 and 200 mg/kg, p.o.), once in daily for three consecutive days, while piracetam (500/kg, i.p.), a clinically used nootropic agent, was administered acutely to rats as the standard drug. Control rats were treated with equal volume of vehicle (0.3% carboxymethyl cellulose (CMC)). IHp and piracetam when given alone shortened the TL on day 1, 2, 9 and also antagonised the amnesic effects of scopolamine and sodium nitrite on the TL significantly. IHp had no significant per se effect on the retention of the PA in rats. Only the higher dose (200 mg/kg, p.o.) produced a significant reversal of scopolamine induced PA retention deficit but no significant reversal was observed with sodium nitrite. Piracetam showed significant per se facilitatory effect on PA retention and also reversed the scopolamine and sodium nitrite induced impaired PA retention. In the AA test, IHp in both the doses, and piracetam, facilitated the acquisition and retention of AA in rats and the IHp effects were found to be dose dependent. Both the doses of IHp and piracetam significantly attenuated the scopolamine and sodium nitrite induced impaired retention of AA. These results indicate a possible nootropic action of IHp, which was qualitatively comparable with that induced by piracetam.

Antidepressant activity of hyperforin conjugates of the St. John's wort, Hypericum perforatum Linn.: an experimental study.

Nine extracts of H. perforatum, containing hyperforin in conjugated forms, but devoid of free hyperforin and adhyperforin, were subjected to antidepressant screening using the forced swim test (FST). The observed activity was compared with that of SJW extracts containing hyperforin and adhyperforin (in free form). Results indicate that hyperforin conjugates exhibit significant antidepressant activity as evidenced by the reduced immobility period in the FST in rats.

Effect of Emblica officinalis tannoids on a rat model of tardive dyskinesia.

Effect of active tannoid principles of E. officinalis, comprising of emblicanin A (37%), emblicanin B (33%), punigluconin (12%) and pedunculagin (14%), was investigated on a rat model of tardive dyskinesia (TD) induced by once daily administration of haloperidol (1.5 mg/kg, ip) for 28 days. Involuntary orofacial movements (chewing movements, buccal tremors and tongue protusion) were assessed as TD parameters. The tannoid principles of E. officinalis (EOT) were administered concomitantly with haloperidol in the doses of 10, 20 and 50 mg/kg, po, for 28 days. Sodium valproate (200 mg/kg, po), a Gaba-mimetic agent, and vitamin E (400 mg/kg, po), an antioxidant, were used as the standard drugs and administered for the same period. EOT induced a dose-related inhibition of all the three TD parameters assessed, as did vitamin E. The effect of sodium valproate remained statistically insignificant. The results suggest that EOT exerts a prophylactive effect against neuroleptic-induced TD which is likely to be due to its earlier reported antioxidant effects in rat brain areas, including striatum.

Effect of poly herbal formulation, EuMil, on chronic stress-induced homeostatic perturbations in rats.

EuMil, is a herbal formulation comprising the standardised extracts of Withania somnifera (L) Dunal, Ocimum sanctum L, Asparagus racemosus Willd and Emblica officinalis Gaertn., all of which are classified in Ayurveda as rasayanas to promote physical and mental health, improve defense mechanisms of the body and enhance longevity. These attributes are similar to the modern concept of adaptogenic agents, which are, known to afford protection to the human physiological system against diverse stressors. The present study was undertaken to investigate the adaptogenic and antistress activity of EuMil against chronic unpredictable, but mild, footshock stress-induced perturbations in behaviour (depression), glucose metabolism, suppressed male sexual behaviour, immunosuppression and cognitive dysfunction in CF strain albino rats. Panex ginseng (PG) was used for comparison. Gastric ulceration, plasma corticosterone levels, serum lipid, hepatic and renal functions were used as the stress indices. These effects were attenuated by EuMil (dose 100 mg/kg, p.o.) and PG (100 mg/kg. p.o.), administered once daily over a period of 14 days, the period of stress induction period. Further, chronic stress also induced glucose intolerance, suppressed male sexual behaviour, induced behavioural despair and cognitive dysfunction and immunosuppression. All these chronic stress-induced perturbations were attenuated, in a dose dependent manner by EuMil and PG. Thus, the results indicate that EuMil has significant adaptogenic and anti-stress, activity, qualitatively comparable to PG, against a variety of behavioural, biochemical and physiological perturbations, induced by unpredictable stress, which has been proposed to be a better indicator of clinical stress than acute stress. The likely contribution of the individual constituents of EuMil in the observed adaptogenic action of the polyherbal formulation, has been discussed.

Hypericum perforatum: nature's mood stabilizer.

Hypericum perforatum (HP), better known as St. John's Wort, has been used clinically for centuries. Modern usage is still quite diverse and includes kidney and lung ailments, insomnia and depression. Standardised extracts of HP are widely used in the treatment of psychovegetative disorders and especially for mild forms of depression. Several bioactive constituents of this plant may play important role in its well-known antidepressant activity, which are discussed in the present article. Furthermore, emphasis is also given on its botany, chemistry, pharmacology and clinical efficacy.

Effect of poly herbal formulation, EuMil, on neurochemical perturbations induced by chronic stress.

EuMil, a polyherbal formulation consisting of standardised extracts of Withania somnifera (L) Dunal, Ocimum sanctum L, Asparagus racemosus Wilid and Emblica officinalis Gaertn., is used as an anti-stress agent to attenuate the various aspects of stress related disorders. In the present study, the neurochemical mechanisms underlying the anti-stress activity of EuMil were evaluated by measuring the rat brain monoamine neurotransmitter levels and tribulin activity. Chronic electroshock stress (14 days) significantly decreased the nor-adrenaline (NA) and dopamine (DA) levels in frontal Cortex, pons-medulla, hypothalamus, hippocampus and striatal, hypothalamal region, respectively, and increased the 5-hydroxytryptamine (5HT) level in frontal cortex, pons medulla, hypothalamus and hippocampus. Chronic stress, also increased the rat brain tribulin activity. EuMil (100 mg/kg, p.o., 14 days) treatment normalized the perturbed regional NA, DA, 5HT concentrations, induced by chronic stress. EuMil also significantly attenuated the stress-induced increase in the rat brain tribulin activity. The amelioration of chronic stress-induced neurochemical perturbations by EuMil explains the neurochemical mechanisms underlying the observed putative anti-stress activity of the product.

Asparagus racemosus competitively inhibits in vitro the acetylcholine and monoamine metabolizing enzymes.

Asparagus racemosus (AR) has earlier been reported to possess antidepressant activity possibly mediated through the monoaminergic system, and nootropic and anti amnestic activities possibly through the cholinergic system. In the present study to further understand the mechanism of action, we evaluated the kinetics of acetyl (AChE) and butyryl (BuChE) cholinesterases, and monoamine oxidase (MAO-A and B) enzyme inhibitory activities of different fractions of AR. The results showed that methanolic extract of AR (MAR) significantly inhibited cholinesterase and MAO activities as compared to hexane (HAR) and chloroform (CAR) extracts of AR as evident from the IC(50) values. The kinetic analysis of enzyme inhibition of MAR shows that the V(max) does not change with different concentrations of MAR but the K(m) value increases. This indicates that MAR is a non-selective competitive inhibitor for both cholinesterase and monoamine oxidase enzymes. Evaluation of K(i) values show that MAR inhibited these enzymes less potently compared to the respective standard drugs. There seems to be a positive correlation between the saponin content and, cholinesterase and monoamine inhibitory activities as MAR had 62.20% of saponins, whereas HAR and CAR had no measurable saponin content. The non-selective competitive inhibitory activity on cholinesterase and monoamine oxidase enzymes can explain many reported neuropharmacological activities of AR. AR apart being used as a drug is also used as a food. As such AR may have potential drug-drug, drug-food and food-food interactions with drugs and foods sharing the cholinergic and monoaminergic pathways.

Glycosphingolipid composition of a new immortalized human cerebromicrovascular endothelial cell line.

Previous studies have demonstrated the involvement of glycosphingolipid (GSL) antigens in the pathogenesis of immune-mediated neurological disorders such as peripheral neuropathies and multiple sclerosis. To study the role of the blood-brain barrier (BBB) in these disorders, we used a new human cerebromicrovascular endothelial cell (HCEC) line that has been immortalized through transfection with the plasmid pSV3-neo encoding for the SV40 large T-antigen and the neomycin gene. The immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates but maintained phenotypic properties of early-passage control cells. Therefore, this human cell line may serve as a useful in vitro model for studying the properties of the human BBB. We first investigated the GSL composition of cultured SV-HCECs. The major gangliosides were GM3 (62% of total gangliosides), GM2 (18%), GM1 (3%), and GD1a (15%). The major neutral GSLs were glucosylceramide (15% of the total neutral glycolipids), lactosylceramide (36%), globotriaosylceramide (3%), and globoside (43%). Trace amounts of paragloboside, lactosaminyl paragloboside, and sulfoglucuronyl paragloboside could also be detected by TLC-immunostaining. These results provide the basis for further investigations of the expression of these cell surface antigens in cultured SV-HCECs on activation with inflammatory cytokines such as interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma, which have been implicated as playing an important role in the pathogenesis of many nervous system disorders.

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier.

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3-neo coding for the SV40 large T antigen and the neomycin gene. The neomycin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent cell colonies. Single-cell clones of near-diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV-HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII-related antigen, uptake of acetylated low-density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor-mediated endocytosis, and high activities of the BBB-specific enzymes alkaline phosphatase and gamma-glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV-HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.