Anticonvulsant activity of methanolic extract of Withania cogulans in mice.

Mental and neurological diseases including depression, Parkinson's disease, dementia, epilepsy, anxiety disorders and bipolar disorders account for a considerable amount of the world's disease burden. Unfortunately, drugs used in the treatment of neurological diseases are expensive, symptomatic and they produce undesirable side effects. People from different cultures prefer to use medicinal plants for the treatment of various ailments ranging from plain to perplex disorders because they are most affordable, cost effective and easily accessible source of treatment in the primary healthcare system throughout the world. Withania coagulans, an erect grayish under-shrub belongs to family Solanaceae. It is common in Pakistan, East India, Iran and Afghanistan. The objective of this study was to analyze the anti-seizure activity of crude methanolic extract of Withania coagulans fruits (MeWc). For screening of this activity, maximal electroshock seizures model (MES) and chemically-induced seizures models were used. In maximal electroshock seizures test MeWc showed significant dose dependent percent protection against hind-limb tonic extension; significant and dose-dependent increase in latency to myoclonic jerks and tonic clonic convulsions and decrease in seizures duration were observed in PTZ-induced seizures. In strychnine-induced convulsions MeWc significantly increased latency to hind-limb tonic extension and percent protection from death in a dose-dependent manner. Thus, it was inferred from the experiments that extract of Withania coagulans showed anticonvulsant activity.

Comparison of acute and chronic effects of Bacopa monnieri, Ginkgo biloba, and Lavandula angustifolia and their mixture on learning and memory in mice.

Forty-seven million people are living with memory-related disorders worldwide. Phytomedicines are gaining extensive interest in the treatment of these ailments. Memory-enhancing (acute and chronic) potentials of commercial grade extracts of Bacopa monnieri (200 mg/kg, po), Ginkgo biloba (150 mg/kg, po), and Lavandula angustifolia (200 mg/kg, po) and their mixture (B. monnieri 100 mg/kg, G. biloba 75 mg/kg, and L. angustifolia 100 mg/kg, po) were compared for their synergistic/additive effects on the Morris water maze (MWM) test and elevated plus maze (EPM) test in scopolamine-induced amnesia in mice. Escape latency and accumulative path length were significantly reduced both in acute (up to day 6) and chronic trials (days 8-14) in B. monnieri-, G. biloba-, and L. angustifolia-treated animals and their mixtures (n = 8, p < .05) in MWM. Furthermore, in probe trials (acute on day 7 and chronic on day 15), the number of crossing-overs at platform position and time spent in platform quadrant were significantly increased, while transfer latency in EPM was decreased in treated animals as compared to the saline group (n = 8, p < .05). The mixture showed synergistic effects on memory enhancement as compared to each extract individually in mice. Further studies may be carried out on the active compounds of B. monnieri at the cellular and molecular levels.

Evaluation of safety profile and stress suppressant activity of Rosa moschata in mice.

Stress is a state that seriously disturbs psychological or physiological homeostasis of the body and subsequently affects the morphology and function of the hippocampus. Currently available anti-stress medications provide limited benefits with cost of severe adverse effects. In the present study, effect of Rosa moschata extract was evaluated using acute restraint model in mice. The stress suppressant activity of Rosa moschata was evaluated by using elevated plus maze test (EPM), dark light box test and open field test (OFT) following restraint stress protocol. Results showed that the Rosa moschata extract significantly enhanced the number of transitions and the time spent in the open arm in the EPM, increased the number of transitions and time spent in the light compartment of the dark light box, and also enhanced the locomotor activity in OFT, as compared to the stress group. In addition, LD50 of the plant extract is greater than 5000mg/Kg. Thus the findings of our studies show that Rosa moschata significantly alleviates stress following the acute restraint stress in mice. Further studies dealing with underlying mechanism and characterization of active fraction/compound may provide an alternative therapy for stress and related neurological conditions.

Glutamine augments neuronal network activity in Rat hippocampal slices.

BACKGROUND: In recent past, a huge number of in vitro electrophysiological techniques have been developed to explore underlying mechanisms of most complicated functions of brain. Neurophysiologist and neuroscientist use different compositions of artificial cerebrospinal fluid (aCSF) usually based on ionic and energy demands of neurons but these compositions lack amino acids such as aspartic acid, taurine and glutamine. METHODS: We used in vitro electrophysiological recording technique to estimate the effects of glutamine, an amino acid and precursor of neurotransmitters glutamate and GABA, on hippocampal sharp wave ripple activity (SPW-R) in rats. We evoked SPW-Rs in hippocampal slices applying high frequency stimulation. RESULTS: We found that glutamine significantly enhanced the incidence and amplitude of sharp wave ripples. However, duration of sharp wave and ripples' frequency did not change significantly. It is interesting that glutamine neither prolonged sharp wave ripple activity nor transformed these into pathological events such as recurrent epileptiform discharges. CONCLUSIONS: Our data indicate that addition of glutamine in aCSF may optimize the experimental conditions for in vitro electrophysiology without disturbing excitatory - inhibitory balance. This study may provide a better experimental paradigm for exploring the underlying mechanisms of neurological disorders and for searching new therapeutic options to cure these neurological conditions.

Natural products from Cuscuta reflexa Roxb. with antiproliferation activities in HCT116 colorectal cell lines.

Parasitic Cuscuta reflexa Roxb. possesses many medicinal properties and is a rich source of a variety of biologically relevant natural products. Natural products are the prime source of leads, drugs, and drug templates, and many of the anticancer and antiviral drugs are either based on natural product or derived from them. Cancer is a devastating disease and one of the leading causes of death worldwide despite improvements in patient survival during the past 50 years; new and improved treatments for cancer are therefore actively sought. Colorectal cancer is the fourth most prevalent cancer worldwide and is responsible for nearly 9% of all cancer deaths. Our search for anticancer natural products from C. reflexa has yielded four natural products: Scoparone (1), p-coumaric acid (2), stigmasta-3,5-diene (3) and 1-O-p-hydroxycinnamoylglucose (4) and among them 1-O-p-hydroxycinnamoyldlucose (4) showed promising antiproliferative activities in HCT116 colorectal cell lines, whereas compounds 1-3 showed moderate activities.

Pretreatment with ß-adrenergic receptor agonists facilitates induction of LTP and sharp wave ripple complexes in rodent hippocampus.

Norepinephrine, is involved in the enhancement of learning and memory formation by regulating synaptic mechanisms through its ability to activate pre- and post-synaptic adrenergic receptors. Here we show that ß-agonists of norepinephrine facilitate the induction of both associational LTP and sharp wave ripples (SPW-Rs) in acute slices of rat hippocampus in area CA3. Surprisingly, this facilitating effect persists when slices are only pretreated with ß-receptor agonists followed by wash out and application of the unspecific ß-adrenoreceptor (ßAR) antagonist propranolol. During application of ßAR agonists repeated stimulation resulted in facilitated induction of SPW-Rs. Since SPW-Rs are thought to be involved in memory replay we studied the effects of ßAR-agonists on spontaneous SPW-Rs in murine hippocampus and found that amplitude and incidence of SPW-Rs increased. These effects involve cyclic-AMP and the activation of protein kinase A and suggest a supportive role in memory consolidation. © 2016 Wiley Periodicals, Inc.

Borneol inhibits TRPA1, a proinflammatory and noxious pain-sensing cation channel.

Borneol, a natural product isolated from several species of Artemisia, Blumea and Kaempferia, has a widespread use in traditional medicine. TRP ion channels are a class of nonselective cation channel proteins involved in a variety of physiological and pathological processes in mammals. TRPA1, a member of TRP family of cation channels, is involved in plethora of processes including noxious-cold, noxious-pain sensations, inflammation and the detection of irritant chemicals. Borneol is chemically related to camphor (a known inhibitor of TRPA1 ion channels); therefore, it is beneficial to investigate the effects of borneol on TRPA1. In the present investigation it was found that borneol inhibits TRPA1 mediated cationic currents in low millimolar range (IC50 0.3mM) in heterologous expression systems like Xenopus oocytes and in neurons cultured from trigeminal ganglia. Effects of nicotine, a known chemical irritant and agonist of TRPA1 are also inhibited by borneol in both systems. It is concluded that borneol, being an inhibitor of TRPA1, could be a safer therapeutic-combination in clinical situations where TRPA1 channelopathies like neuropathic-pain, trigeminal neuralgia or nicotine withdrawal treatments are involved.

Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues.

TRPV3 ion channels mediate thermo-transduction, nociception, inflammation and dermatitis in mammals. TRPV1-4 proteins have been shown to have conserved cysteine-residues in the pore-forming regions. These residues participate in channel activation via S-nitrosylation of channel proteins. Camphor is a commonly used ligand for TRPV3 channels. Thus the knowledge about the potential binding/interacting site(s) for camphor will help to design effective and potent analgesic compounds. In an overlap-extension PCR method, following primer-pairs were used to mutate conserved cysteine-residues in the pore-region of TRPV3 channels; GATTGAGAATcCTCCAAGGACAAAAAGGAC, TRPV3-C612S-Fw and GTCCTTGGAGgACTTCTCAATCAGTCAGTGAGG, TRPV3-C612S-Rv primers pair. And for TRPV3-C619S: GGACTCcAGTTCCTATGGCCAGC, TRPV3-C619S-Fw and GCTGGCCATAgGAACTGGAGTCC, TRPV3-C619S-Rv respectively. All cDNA constructs were confirmed by DNA-sequencing and used to make cRNAs. Oocytes expressing mTRPV3-C619S and mTRPV3-C612S mutant channels were challenged with 2-APB (1 mM), camphor (10 mM) and dihydrocarveol (10 mM) either at -40 mV or +40 mV holding potentials in voltage-clamp experiments. Responses of both mutants to 2-APB were similar to wild-type mTRPV3. Interestingly, responses to camphor were totally lost in mTRPV3-C619S mutant, while responses to dihydrocarveol remained intact. In contrast mTRPV3-C612S displayed slightly altered (16±2 % reduction) phenotype with respect to camphor sensitivity. It is concluded that pore-region cysteines play critical role in camphor sensitivity of TRPV3 ion channels.

Supercooling agent icilin blocks a warmth-sensing ion channel TRPV3.

Transient receptor potential vanilloid subtype 3 (TRPV3) is a thermosensitive ion channel expressed in a variety of neural cells and in keratinocytes. It is activated by warmth (33-39°C), and its responsiveness is dramatically increased at nociceptive temperatures greater than 40°C. Monoterpenoids and 2-APB are chemical activators of TRPV3 channels. We found that Icilin, a known cooling substance and putative ligand of TRPM8, reversibly inhibits TRPV3 activity at nanomolar concentrations in expression systems like Xenopus laeves oocytes, HEK-293 cells, and in cultured human keratinocytes. Our data show that icilin's antagonistic effects for the warm-sensitive TRPV3 ion channel occurs at very low concentrations. Therefore, the cooling effect evoked by icilin may at least in part be due to TRPV3 inhibition in addition to TRPM8 potentiation. Blockade of TRPV3 activity by icilin at such low concentrations might have important implications for overall cooling sensations detected by keratinocytes and free nerve endings in skin. We hypothesize that blockage of TRPV3 might be a signal for cool-sensing systems (like TRPM8) to beat up the basal activity resulting in increased cold perception when warmth sensors (like TRPV3) are shut off.

Characterization of selective TRPM8 ligands and their structure activity response (S.A.R) relationship.

PURPOSE: Transient receptor potential melastatin-8 (TRPM8) is an ion channel expressed extensively in sensory nerves, human prostate and overexpressed in a variety of cancers including prostate, breast, lung, colon and skin melanomas. It is activated by innoxious cooling and chemical stimuli. TRPM8 activation by cooling or chemical agonists is reported to induce profound analgesia in neuropathic pain conditions. Known TRPM8 agonists like menthol and icilin cross-activate other thermo-TRP channels like TRPV3 and TRPA1 and mutually inhibit TRPM8. This limits the usefulness of menthol and icilin as TRPM8 ligands. Consequently, the identification of selective and potent ligands for TRPM8 is of high relevance both in basic research and for therapeutic applications. In the present investigation, a group of menthol derivates was characterized. These ligands are selective and potent agonists of TRPM8. Interestingly they do not activate other thermo-TRPs like TRPA1, TRPV1, TRPV2, TRPV3 and TRPV4. These ion channels are also nociceptors and target of many inflammatory mediators. METHODS: Investigations were performed in a recombinant system: Xenopus oocytes microinjected with cRNA of gene of interest were superfused with the test substances after initial responses of known standard agonists. Evoked currents were measured by two-electrode voltage clamp technique. RESULTS: The newly characterized ligands possess an up to six-fold higher potency (EC50 in low microM) and an up to two-fold increase in efficacy compared to the parent compound menthol. In addition, it is found that chemical derivatives of menthol like CPS-368, CPS-369, CPS-125, WS-5 and WS-12 are the most selective ligands for TRPM8. The enhanced activity and selectivity seems to be conferred by hexacyclic ring structure present in all ligands as substances like WS-23 which lack this functional group activate TRPM8 with much lower potency (EC50 in mM) and those with pentacyclcic ring structure (furanone compounds) are totally inactive. CONCLUSION: The new substances activate TRPM8 with a higher potency, efficacy and specificity than menthol and will thus be of importance for the development of pharmacological agents suitable for treatment and diagnosis of certain cancers and as analgesics. STATEMENT OF NOVELTY: The new compounds have an unmatched specificity for TRPM8 ion channels with additional display of high potency and efficacy. Thus these substances are better pharmacological tools for TRPM8 characterization then known compounds and it is suggested that these menthol-derivates may serve as model substances for the development of TRPM8 ligands.

Monoterpenoids induce agonist-specific desensitization of transient receptor potential vanilloid-3 (TRPV3) ion channels.

PURPOSE: Transient receptor potential vanilloid-3 (TRPV3) is a thermo-sensitive ion channel expressed in skin keratinocytes and in a variety of neural cells. It is activated by warmth as well as monoterpenoids including camphor, menthol, dihydrocarveol and 1,8-cineol. TRPV3 is described as a putative nociceptor and previous studies revealed sensitization of the channel during repeated short-term stimulation with different agonists. In the present investigation TRPV3 was transiently expressed in either Xenopus oocytes or HEK293 cells. Whole-cell voltage-clamp techniques were used to characterize the behavior of TRPV3 when challenged with different agonists. METHODS: Similarly, a human keratinocyte-derived cell line (HaCaT cells) was used to monitor the behavior of native TRPV3 when challenged with different agonists. RESULTS: We report here that prolonged exposure (5-15 minutes) of monoterpenoids results in agonist-specific desensitization of TRPV3. Long-term exposure to camphor and 1,8-cineol elicits desensitizing currents in TRPV3 expressing oocytes, whereas the non-terpenoid agonist 2-APB induces sustained currents. Agonist-specific desensitization of endogenous TRPV3 was also found in HaCaT cells, which may be taken as a representative for the native system. Terpenoids have a long history of use in therapeutics, pharmaceuticals and cosmetics but knowledge about underpinning molecular mechanisms is incomplete. Our finding on agonist-induced desensitization of TRPV3 by some monoterpenoids displays a novel mechanism through which TRP channels could be functionally modulated. CONCLUSION: Desensitization of TRPV3 channels might be the molecular basis of action for some of the medicinal properties of camphor and 1,8-cineol.

Inhibition of tryptophan - pyrrolase activity and elevation of brain tryptophan concentration by fluoxetine in rats.

OBJECTIVE: To investigate in-vitro as well as in-vivo effects of various doses of fluoxetine (SSRI) on tryptophan metabolism in rats. DESIGN: A pre-clinical study. PLACE AND DURATION OF STUDY: Clinical Biochemistry Research Laboratory, Department of Biochemistry, University of Karachi. The investigation was carried out during 2000 to 2001. SUBJECTS AND METHODS: Male Wistar rats (150-200 g body wt) were selected and divided into control and test groups (n = 5) for comparison. RESULTS: In in-vitro (10 - 1000mM) as well in-vivo (0.5-30 mg/kg body wt.) studies, fluoxetine showed a statistically significant inhibition of rat liver tryptophan pyrrolase (tryptophan-2,3-dioxygenase; EC 1.13.11.11) activity. Significant increases were noted at 10 and 30 mg/kg doses in brain, serum (total and free) and liver L-tryptophan concentrations. Similarly, serum non-estrified free fatty acids showed a significant increase at both doses. There was no effect on serum glucose and albumin concentrations. CONCLUSION: It is suggested that major mechanism of action of fluoxetine is that of elevating brain tryptophan concentration and hence 5-HT synthesis by increasing the availability of circulating tryptophan to the brain secondarily to inhibition of major tryptophan degrading enzyme, hepatic tryptophan pyrrolase. It is assumed that fluoxetine inhibits the binding of apoenzyme form of tryptophan pyrrolase with its cofactor haem. The results are discussed in relation to possible involvement of disturbed hepatic tryptophan metabolism in depressive illness.

Similar pattern of inhibition of tryptophan pyrrolase activity by fluoxetine hydrochloride in both sexes of rats.

Compounds targeting the individual enzymes of kynurenine-nicotinamide pathway have led to new neuropharmacological concepts and provide novel opportunities for therapeutic intervention. Tryptophan pyrrolase (Tryptophan-2, 3-dioxygenase; EC 1.13.11.11), a metalloprotein, is the first and rate limiting enzyme of the most important pathway for tryptophan metabolism via kynurenine-nicotinamide pathway in the liver and therefore plays a key role in regulating the physiological flux of tryptophan in to relevant metabolic pathways like synthesis of neurotransmitter, serotonin in the brain. Fluoxetine, a clinically proven antidepressant, have shown statistically significant inhibition of hepatic tryptophan pyrrolase activities (holo, total and apo form) in both the sexes of rats at 10 & 30 mg/kg doses. Despite elevated holo-enzyme form of tryptophan pyrrolase in female rats, we have found similar inhibition of tryptophan pyrrolase activity by fluoxetine hydrochloride in both male and female rats. The results are discussed in relation to gender differences in the enzyme activity and its possible role in pathophysiology of depression in females.