Nasiara Karim

Anti-inflammatory activities of Sieboldogenin from Smilax china Linn.: Experimental and computational studies

ETHNOPHARMACOLOGICAL RELEVANCE Smilax china Linn. is extensively used in traditional Chinese medicine (TCM) as well as in Pakistan for several medicinal purposes including their use in inflammatory disorders. AIM OF THE STUDY The aims of the current study were to validate and assess the folk use of Smilax china Linn. on pharmacological grounds using the isolated compound at molecular, in vivo and computational levels. MATERIALS AND METHODS Seiboldogenin was isolated from ethyl acetate fraction of the plant crude extract. In vitro lipoxygenase and in vivo carrageenan-induced hind paw oedema models were used in experimental studies while molecular docking technique was used to conduct computational study. RESULTS Sieboldogenin showed significant lipoxygenase inhibition (IC50: 38 microM). It also exhibited significant inhibition (p<0.05) of carrageenan-induced hind paw oedema at the doses of 10 and 50mg/kg. Computational molecular docking showed its molecular interaction with important amino acid residues in the catalytic site of lipoxygenase, revealing its potential binding mode at molecular level. CONCLUSIONS Sieboldogenin seems to be a potential new anti-inflammatory compound responsible for anti-inflammatory activities of Smilax china Linn. Its in vitro and in vivo inflammatory activities are in good agreement with the folk medicinal use of Smilax china Linn. in inflammatory disorders.

α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABAA receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABAA receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC50 = 140 nM) over α4β(2/3)δ (EC50 = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABAA receptors and postulate a role for extrasynaptic α4δ-containing GABAA receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

2'-Methoxy-6-methylflavone: A novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA A receptors

BACKGROUND AND PURPOSE Flavonoids are known to have anxiolytic and sedative effects mediated via actions on ionotropic GABA receptors. We sought to investigate this further.

Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABAA receptor subtypes indicate a different binding mode for GABA at these receptors

Ionotropic GABA(A) receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant δ-containing extrasynaptic GABA(A) receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABA(A) receptors. α4/δ-Containing GABA(A) receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC₅₀=24 nM) and α4β3δ (EC₅₀=12 nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC₅₀(1)=16 nM; EC₅₀(2)=1.2 μM). At α4β2δ, GABA had low micromolar activity (EC₅₀=1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABA(A) receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA(A) receptors compared to their synaptic counterparts.

Structural insights to investigate Conypododiol as a dual cholinesterase inhibitor from Asparagus adscendens

The main aim of the current study was to explore molecular insights for potentially new dual cholinesterase inhibitor(s) from Asparagus adscendens via molecular docking. This medicinal plant is traditionally used as a nerve tonic and remedy for memory impairments. Conypododiol was isolated from the chloroform fraction of methanolic extract of A. adscendens, based on bioactivity guided isolation. Conypododiol exhibited significant inhibition of both acetylcholinesterase and butyrylcholinesterase, having the IC(50) values 2.17 ± 0.1 μM and 11.21 ± 0.1 μM, respectively. IC(50) values of the standard compound Galanthamine for both the enzymes were 0.537 ± 0.018 μM and 8.6 ± 0.27 μM, respectively. Based on MTT cytotoxicity assay, Conypododiol was found safe against LCMK-2 monkey kidney epithelial cells and mice hepatocytes. Molecular docking studies revealed the hydrogen bonding interactions of Conypododiol with His440 and Ser200 at esteratic site (ES), and also with Tyr334 at peripheral anionic site (PAS) of the aromatic gorge of acetylcholinesterase. Simultaneous contacts of Conypododiol with PAS and ES shows its significance as a bivalent ligand. This preliminary study highlighted the potential of Conypododiol to be further developed and modified as new lead compound identified by its folk use.

Terpenoid Content of Valeriana wallichii Extracts and Antidepressant-like Response Profiles

Three extracts of Valeriana wallichii DC (Valerianaceae) rhizome and fluoxetine were studied for antidepressant‐like activity in two behavioral models, namely the forced swim test (FST) and the tail suspension test (TST). Fluoxetine as well as methanolic and aqueous extracts of V. wallichii induced monophasic dose‐related decrements in immobility times in both tests. However, the aqueous‐ethanolic fraction induced a biphasic dose‐response profile since it produced a graded effect up to 200 mg/kg but the highest dose (250 mg/kg) was inactive in the FST. This extract also exhibited significantly reduced activity at 200 mg/kg compared to lower doses in the TST. The highest doses of aqueous‐ethanolic extract also reduced locomotor activity which will have led to a negative functional interaction with antidepressant‐like effects. Qualitative phytochemical analysis revealed that the aqueous‐ethanolic extract of V. wallichii was the only separated rhizome fraction containing terpenoids. Furthermore, since the methanolic and aqueous extracts were active in the tests, it is suggested that the antidepressant‐like action of this herbal plant is not contingent upon its terpenoid constituents. Copyright

Flavan‐3‐ol esters: new agents for exploring modulatory sites on GABAA receptors

BACKGROUND AND PURPOSE Enhancement of GABAergic function is the primary mechanism of important therapeutic agents such as benzodiazepines, barbiturates, neurosteroids, general anaesthetics and some anticonvulsants. Despite their chemical diversity, many studies have postulated that these agents may bind at a common or overlapping binding site, or share an activation domain. Similarly, we found that flavan‐3‐ol esters act as positive modulators of GABAA receptors, and noted that this action resembled the in vitro profile of general anaesthetics. In this study we further investigated the interactions between these agents.

Evaluation of antidiabetic and antihyperlipidemic activity of Artemisia indica linn (aeriel parts) in Streptozotocin induced diabetic rats

ETHNOPHARMACOLOGICAL RELEVANCE Diabetes mellitus is a major metabolic disorder affecting a huge population all over the world. Artemisia species have been extensively used for the management of diabetes in folkloric medicine. The present study is designed to investigate the antidiabetic and antihyperlipidemic effects of aeriel parts of Artemisia indica. MATERIALS AND METHODS Hydromethanolic crude extracts, chloroform, ethyl acetate and n-butanol fractions of aerial parts of Artemisia indica were tested for their antidiabetic potential in Streptozotocin (STZ) (50mg/kg, i.p.) induced diabetic Sprague-Dawley rats. Blood glucose level, body weight, serum lipid profile and activities of liver enzymes were determined. The extracts were further subjected to preliminary phytochemical analysis. RESULTS A daily oral dose of hydromethanolic crude extracts (200 and 400mg/kg b.w.) and chloroform fraction (200mg/kg b.w.) of Artemisia indica for 15 days showed a significant reduction in blood glucose level which was comparable to that of the standard antidiabetic drug, glibenclamide (500 μg/kg, p.o.). Artemisia indica extracts also showed reduction in total cholesterol, triglycerides and low density lipoproteins as well as serum creatinine level, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase (ALP) in diabetic rats. CONCLUSION According to the results Artemisia indica possesses hypoglycemic, antihyperlipidemic and valuable effects on liver and renal functions in diabetic rats, which seems to validate its traditional usage.

Covalent trapping of methyllycaconitine at the α4-α4 interface of the α4β2 nicotinic acetylcholine receptor: antagonist binding site and mode of receptor inhibition revealed.

Background: Methyllycaconitine is an antagonist at subtypes of the nicotinic acetylcholine receptor. Results: A reactive methyllycaconitine probe was covalently trapped by a cysteine introduced on the complementary face of the α4 subunit and only in the (α4)3(β2)2 nAChR stoichiometry. Conclusion: The α4-α4 interface on the α4β2 nAChR contains a methyllycaconitine binding site. Significance: Defining the molecular interactions of nAChR ligands at the α4-α4 interface may lead to superior therapeutics. The α4β2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and α4β2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4β2 receptor containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(β2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(β2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(β2)2 nAChRs.

GABAA receptor modulation and neuropharmacological activities of viscosine isolated from Dodonaea viscosa (Linn)

The objective of the present study was to evaluate the modulation of GABA-evoked currents by the flavonoid viscosine at recombinant GABA(A) receptors, and subsequently to study its anxiolytic, sedative and anticonvulsant activities. Viscosine (1-300μM) positively modulated GABA-evoked currents at human α1β2γ2L and α2β2γ2L GABA(A) receptors expressed in Xenopus oocytes in a flumazenil insensitive manner. In behavioral studies, viscosine at doses of 10-100mg/kg (i.p.) exerted significant anxiolytic effects in the elevated plus maze, light-dark and open field tests (*P<0.05, **P<0.01, ***P<0.001 n=6, One-way ANOVA post-Dunnetts test), and sedative effects at high doses (100mg/kg i.p.) in hole board and thiopental induced sleep time tests. The anxiolytic effect in the elevated plus maze test was not blocked by flumazenil whereas pentylenetetrazole (PTZ) completely attenuated the effect, indicating that the activity was mediated via the non-benzodiazepine sites of GABA(A) receptors. Furthermore, viscosine at doses of 10-100mg/kg (i.p.) exerted anticonvulsant effects in a dose-dependent manner in PTZ, picrotoxin and bicuculline induced seizure paradigms (*P<0.05, **P<0.01,***P<0.001 n=6, One-way ANOVA post-Dunnetts test). In conclusion, the results of the present study suggest that the anxiolytic and anticonvulsant actions of viscosine are likely mediated via its positive allosteric modulatory action of GABA at different GABA(A) receptor subtypes.