Aster tataricus alleviates constipation by antagonizing the binding of acetylcholine to muscarinic receptor and inhibiting Ca2+ influx.

BACKGROUND: The dried root and rhizome of Aster tataricus (RA), is a traditional Chinese medicine has been used for more than 2000 years with the function of antitussive, expectorant and antiasthmatic. Ancient books and modern pharmacological researches demonstrated that RA may have the function of moistening intestines and relieving constipation, but there was a lack of systematic evidence. The aim of this study was to comprehensively evaluate the efficacy and possible mechanisms of ethanol extract of Aster tataricus (ATE) in treating constipation from in vivo to in vitro. METHODS: In vivo, the ATE was studied in loperamide-induced constipation of mice. In vitro, different concentrations of ATE was tested separately or cumulatively on spontaneous and agonists-induced contractions of isolated rat duodenum strips. RESULTS: In vivo, at doses of 0.16, 0.8 g/mL, ATE showed significantly promotion of the small intestinal charcoal transit, decrease of the amount of remnant fecal, and increase of the content of fecal water in colon. In addition, ATE could effectively relieve colonic pathological damage caused by loperamide as well. In vitro, with the cumulative concentration increase of ATE from 0.8 to 6.4 mg/mL, it could significantly decrease the contraction caused by KCl or Ach, and gradually restore to near base tension value.Meanwhile, it could also partially but significantly inhibit the contractions induced by Ach and CaCl2 on rat duodenum in a concentration related manner. CONCLUSIONS: Taking all these findings together, it could be speculated that ATE may attenuate constipation mainly through antagonizing the binding of acetylcholine to muscarinic receptor, inhibiting Ca2+ influx and anti-inflammation.

Blood pressure lowering effects of Ranunculus scleratus Linn. in normal and fructose induced hypertensive rats and estimation of underlying mechanisms.

Ranunculus scleratus Linn. is used in folk medicine to treat hypertension. This study was aimed at providing validation to its traditional use and to explore underlying mechanisms of action. Effects of hydro-ethanolic crude extract of the plant and its fractions on blood pressure was evaluated using direct surgical method in normotensive and in fructose induced hypertensive rats. Various doses of crude extract, RSC, (5, 10, 20, 30mg/kg) and all fractions (3, 5, 10, 20mg/kg) were studied. Results suggested that aqueous fraction of R. scleratus (RSA) produced most pronounced effects at 10mg/kg in normotensive and at 20mg/kg in hypertensive animals. Underlying mechanisms, using various pharmacological antagonists were also elucidated. Results suggested the involvement of muscarinic receptor, angiotensin converting enzyme (ACE) inhibition, ganglionic block and nitric oxide (NO) release in presenting hypotensive response.

An evaluation of the pharmacokinetics of tiotropium following a single-dose inhalation in healthy subjects using an ultra-sensitive bioanalytical method.

Objective and significance: The systemic bioavailability of tiotropium following administration via inhalation is known to be very low. A validated ultra-sensitive bioanalytical method with the lowest lower limit of quantitation (LLOQ) was developed and used to evaluate the complete pharmacokinetic profile of tiotropium.Methods: This was a pharmacokinetic study performed in 18 healthy subjects. Each subject was administered a dose of 18 mcg of tiotropium from a dry powder inhaler (DPI). The subjects' plasma tiotropium concentrations were assayed with LLOQ of 0.1 pg/mL.Results: The results showed a mean Cmax of 4.98 ± 3.55 pg/mL, and a median (tmax) of 3.6 minutes (range: 1.8-12 minutes). The means for area under the concentration-time curve (AUC) from time zero hours to infinity (AUCinf) and AUC from time zero hours to the time of the last measurable tiotropium concentration (AUCt) were 51.11 ± 27.4 pg*h/mL and 37.37 ± 23.38 pg*h/mL, respectively. The mean apparent elimination half-life (t1/2) was 68.02 ± 24.55 hours. This calculated half-life is longer than what others have reported where a less sensitive LLOQ was used.Conclusion: The lower LLOQ enabled further insight into the pharmacokinetics of tiotropium that was not possible with other analytical methods. With this method, we were able to quantify tiotropium concentrations as early as one minute following drug administration and up to 144 hours after dosing. The application of this method will allow for studies to be designed properly and enable further investigations into the pharmacokinetics of tiotropium.

Spasmolytic activity of Aquilariae Lignum Resinatum extract on gastrointestinal motility involves muscarinic receptors, calcium channels and NO release.

CONTEXT: Aquilariae Lignum Resinatum (ALR), the dry rhizome of Aquilaria agallocha R. (Thymelaeaeeae), has been widely used to treat emesis, stomachache and gastrointestinal dysfunction. OBJECTIVE: This study evaluates the effects of ALR methanol extract on gastrointestinal motility (GIM) and possible mechanisms of the action involved. MATERIALS AND METHODS: In vivo, the study evaluated the effects of ALR (200-800 mg/kg) on gastric emptying and small intestinal motility in normal and neostigmine-induced adult KM mice. The in vitro effects of ALR (0.2-1.6 mg/mL) on GIM were performed on isolated jejunum of Wistar rats, pretreated with acetylcholine (ACh), KCl, CaCl2, and pre-incubation with l-NAME (a selective inhibitor of the nitric oxide synthase). RESULTS: In vivo, ALR (800 mg/kg) decreased gastric emptying (70.82 ± 9.81%, p < 0.01, compared with neostigmine group 91.40 ± 7.81%), small intestinal transit (42.82 ± 3.82%, p < 0.01, compared with neostigmine group 85.53 ± 5.57%). In vitro, ALR concentration dependently decreased the contractions induced by ACh (10-5 M) and KCl (60 mM) with respective EC50 values of 0.35 and 0.32 mg/mL. The Ca2+ concentration-response curves were shifted by ALR to the right, similar to that caused by verapamil (the positive). The spasmolytic activity of ALR was inhibited by pre-incubation with l-NAME. DISCUSSION AND CONCLUSIONS: ALR played a spasmolytic role in GIM, which is probably mediated through inhibition of muscarinic receptors, blockade of Ca2+ influx and NO release. This is the first study presenting a comprehensive description of the effects of ALR on GIM.

Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis.

Functional screening for compounds that promote remyelination represents a major hurdle in the development of rational therapeutics for multiple sclerosis. Screening for remyelination is problematic, as myelination requires the presence of axons. Standard methods do not resolve cell-autonomous effects and are not suited for high-throughput formats. Here we describe a binary indicant for myelination using micropillar arrays (BIMA). Engineered with conical dimensions, micropillars permit resolution of the extent and length of membrane wrapping from a single two-dimensional image. Confocal imaging acquired from the base to the tip of the pillars allows for detection of concentric wrapping observed as 'rings' of myelin. The platform is formatted in 96-well plates, amenable to semiautomated random acquisition and automated detection and quantification. Upon screening 1,000 bioactive molecules, we identified a cluster of antimuscarinic compounds that enhance oligodendrocyte differentiation and remyelination. Our findings demonstrate a new high-throughput screening platform for potential regenerative therapeutics in multiple sclerosis.

A simple and sensitive GC/MS method for the determination of atropine during therapy of anticholinesterase poisoning in serum samples.

Atropine is used in the daily clinical practice for the treatment of poisonings caused by anticholinesterase pesticides, due to its sympathomimetic action. The investigation of the cause of the adverse effects that appear during atropine administration showed the necessity for the development and validation of a simple, rapid, sensitive, and specific method for the determination of atropine levels in serum samples. The developed method includes liquid-liquid extraction with ethyl acetate: dichloromethane (3:1, v/v) and derivatization using N,O-bis(trimethylsilyl)-trifluoracetamide (BSTFA) with 1% trimethylchlorsilane (TMCS) in acetonitrile environment. The method was found to be selective, linear, accurate, and precise according to international guidelines. The recovery was higher than 85.9%, the limit of quantification was 2.00 ng/ml, and the calibration curve was linear within the range of 2.00-500 ng/ml (R(2) ≥ 0.992). Accuracy and precision were also calculated and were found to be less than 5.2 and 8.7%, respectively. The developed method was applied in a real case of accidental poisoning with chlorpyrifos in order to determine the atropine serum levels of the patient. The proposed method proved to be useful for the investigation of adverse effects that appear during atropine treatment of patients poisoned by anticholinesterase pesticides and it can also be used for the investigation of poisonings caused after consumption of atropine containing plants.

The antidiarrheal and spasmolytic activities of Phyllanthus emblica are mediated through dual blockade of muscarinic receptors and Ca2+ channels.

AIM OF THE STUDY: This study was aimed at providing the possible mechanisms for the medicinal use of Phyllanthus emblica in diarrhea. MATERIALS AND METHODS: The in vivo studies were conducted in mice, while isolated rabbit jejunum and guinea-pig ileum were used for the in vitro experiments. RESULTS: The crude extract of Phyllanthus emblica (Pe.Cr), which tested positive for alkaloids, tannins, terpenes, flavonoids, sterols and coumarins, caused inhibition of castor oil-induced diarrhea and intestinal fluid accumulation in mice at 500-700 mg/kg. In isolated rabbit jejunum, Pe.Cr relaxed carbachol (CCh) and K(+) (80 mM)-induced contractions, in a pattern similar to that of dicyclomine. The preincubation of guinea pig-ileum with Pe.Cr (0.3 mg/mL), caused a rightward parallel shift in the concentration-response curves (CRCs) of acetylcholine without suppression of the maximum response. While at the next higher concentration (1 mg/mL), it produced a non-parallel rightward shift with suppression of the maximum response, similar to that of dicyclomine, suggesting anticholinergic and Ca(2+) channel blocking (CCB)-like antispasmodic effect. The CCB-like activity was further confirmed when pretreatment of the tissue with Pe.Cr, shifted the CRCs of Ca(2+) to the right with suppression of the maximum response, similar to nifedipine or dicyclomine. The activity-directed fractions of Pe.Cr showed a combination of Ca(2+) antagonist and anticholinergic like components in all fractions but with varying potency. CONCLUSION: These results indicate that the Phyllanthus emblica fruit extract possesses antidiarrheal and spasmolytic activities, mediated possibly through dual blockade of muscarinic receptors and Ca(2+) channels, thus explaining its medicinal use in diarrhea.

3D-QSAR studies of 2,2-diphenylpropionates to aid discovery of novel potent muscarinic antagonists.

Muscarinic acetylcholine receptors (mAChRs) consisting of five known subtypes, are widely distributed in both central and peripheral nervous systems for regulation of a variety of critical functions. The present theoretical study describes correlations between experimental and calculated molecular properties of 15 alpha-substituted 2,2-diphenylpropionate antimuscarinics using quantum chemical and pharmacophore generation methods to characterize the drug mAChR properties and design new therapeutics. The calculated stereoelectronic properties, such as total energies, bond distances, valence angles, torsion angles, HOMO-LUMO energies, reactivity indices, vibrational frequencies of ether and carbonyl moieties, and nitrogen atom proton affinity were found to be well correlated when compared with experimentally determined inhibition constants from the literature using three muscarinic receptor assays: [(3)H]NMS receptor binding, alpha-amylase release from rat pancreas, and guinea pig ileum contraction. In silico predicted toxicity on rat oral LD(50) values correlated well with the [(3)H]NMS binding in N4TG1 cells and alpha-amylase release assays, but not the ileum contraction assay. Next, to explore the functional requirements for potent activity of the compounds, we developed a preliminary 3D pharmacophore model using the in silico techniques. The resulting model contained a hydrogen bond acceptor site on the carbonyl oxygen atom and a ring aromatic feature on one of the two aromatic rings in these compounds. This model was used as a template to search an in-house database for novel analogs. We found compounds equal in inhibition potency to atropine and, importantly, six not reported before as antimuscarinics. These results demonstrate that this QSAR approach not only provides a basis for understanding the molecular mechanism of action but a pharmacophore to aid in the discovery and design of novel potent muscarinic antagonists.

Bronchodilatory effects of S-isopetasin, an antimuscarinic sesquiterpene of Petasites formosanus, on obstructive airway hyperresponsiveness.

In the presence of neostigmine (0.1 microM), S-isopetasin competitively antagonized cumulative acetylcholine-induced contractions in guinea pig trachealis, because the slope [1.18+/-0.15 (n=6)] of Schild's plot did not significantly differ from unity. The pA2 value of S-isopetasin was calculated to be 4.62+/-0.05 (n=18). The receptor binding assay for muscarinic receptors of cultured human tracheal smooth muscle cells (HTSMCs) was performed using [3H]-N-methylscopolamine ([3H]-NMS). Saturation binding assays were carried out with [3H]-NMS in the presence (non-specific binding) and absence (total binding) of atropine (1 microM). Analysis of the Scatchard plot (y=0.247-1.306x, r2=0.95) revealed that the muscarinic receptor binding sites in cultured HTSMCs constituted a single population (n(H)=1.00). The equilibrium dissociation constant (Kd) and the maximal receptor density (B(max)) for [3H]-NMS binding were 766 pM and 0.189 pmol/mg of protein, respectively. The -logIC50 values of S-isopetasin, methoctramine, and 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) for displacing 0.4 nM [3H]-NMS-specific binding were 5.05, 6.25, and 8.56, respectively, which suggests that [3H]-NMS binding is predominantly on muscarinic M3 receptors of cultured HTSMCs. The inhibitory effects of S-isopetasin on enhanced pause (P(enh)) value were similar to that of ipratropium bromide, a reference drug. The duration of action of S-isopetasin (20 microM), also similar to that of ipratropium bromide (20 microM), was 3 h. In contrast to ipratropium bromide, which non-selectively acts on muscarinic receptors, S-isopetasin preferentially acts on muscarinic M3 receptors. In conclusion, S-isopetasin may be beneficial as a bronchodilator in the treatment of chronic obstructive pulmonary disease and asthma exacerbations.

Determination of (R,R)-glycopyrronium bromide and its related impurities by ion-pair HPLC.

A simple, rapid and specific ion-pair HPLC method for the determination of (R,R)-glycopyrronium bromide and its related impurities is presented, and parameters affecting the chromatographic properties of these compounds are discussed. Optimal analyte separation was achieved on base deactivated Nucleosil at 40 degrees C, using phosphate buffer pH 2.30 with sodium-1-decanesulfonate (0.01 M)/methanol (35/65; v/v) as eluent for isocratic elution at a flow rate 1 ml x min(-1). The analytical assay was validated according to international guidelines. The methodis suitable for in-process control and as stability indicating assay.

Selective antagonism activity of alkaloids from bulbs Fritillariae at muscarinic receptors: functional studies.

15 alkaloids were isolated from five Fritillariae species and 6 derivatives were synthesized. Alkaloids having anticholinergic effect on guinea-pig tracheal smooth muscle were screened out and their mechanism was further studied on the cAMP formation in Chinese hamster ovary cells stably expressing human muscarinic M2 receptor (CHO-hM2 cells) and intracellular calcium ([Ca(2+)](i)) transient in Chinese hamster ovary cells stably expressing human muscarinic M3 receptor (CHO-hM3 cells). In normal Krebs-Henseleit (KH) solution, imperialine (15), 3beta-acetylimperialine (16) and sinpeinine A (17) concentration-dependently relaxed 1 microM carbachol-induced contraction of guinea-pig tracheal rings with EC(50) of 4.19, 1.71 and 4.67 microM, respectively. In Ca(2+)-free KH solution, 10 microM 3beta-acetylimperialine (16), imperialine (15) and sinpeinnine A (17) caused 97.42%, 5.45% and 6.55% inhibition, respectively, which indicated that the three components might inhibit muscarinic receptor in different mechanism. Results of muscarinic M2 receptor-inhibited cAMP formation in CHO-hM2 cells showed that imperialine (15) and sinpeinine A (17) could potently elevate the cAMP formation whereas 3beta-acetylimperialine (16) only had weak effect on antagonism of cAMP inhibition. Furthermore, the investigations of muscarinic M3 receptor-stimulated [Ca(2+)](i) transient in CHO-hM3 cells revealed that imperialine (15) and sinpeinine A (17) could not antagonize [Ca(2+)](i) transient, but 3beta-acetylimperialine (16) significantly inhibited [Ca(2+)](i) peak elevation with an IC(50) of 5.26 microM. The functional studies suggest that the mechanism of relaxant action of imperialine (15) and sinpeinine A (17) is due to their selective inhibitory effects on muscarinic M2 receptors and the mechanism of 3beta-acetylimperialine (16) originates from its selective muscarinic M3 receptors antagonism.

Nocardimicins G, H and I, siderophores with muscarinic M3 receptor binding inhibitory activity from Nocardia nova JCM 6044.

In the screening for muscarinic M3 receptor binding inhibitors from microbial secondary metabolites, the extract of Nocardia nova JCM 6044 was found to be highly active. Bioassay-guided isolation led to the identification of three siderophores, nocardimicins G (1), H (2) and I (3). Their chemical structures were determined by spectroscopic analysis using NMR and MS. 1 and 2 inhibited the binding of tritium-labeled N-methylscopolamine to the muscarinic M3 receptor with Ki values of 0.44 microM and 0.37 microM, respectively, whereas 3 showed no inhibition at 10 microM. 1 and 2 also showed weak binding inhibitory activity to the M5 receptor but not to the M1, M2 and M4 receptors at 10 microM.

Nocardimicins A, B, C, D, E, and F, siderophores with muscarinic M3 receptor inhibiting activity from Nocardia sp. TP-A0674.

In the screening for muscarinic M3 receptor binding inhibitors from microbial secondary metabolites, the extract of Nocardia sp. TP-A0674 was found to be highly active. Bioassay-guided fractionation of it led to the isolation of six new siderophores, nocardimicins A (1), B (2), C (3), D (4), E (5), and F (6), as active principles. Their chemical structures were determined by spectroscopic and degradation analysis. Of these congeners, nocardimicin B (2) inhibited the binding of tritium-labeled N-methylscopolamine to the muscarinic M3 receptor most potently with a Ki value of 0.13 microM. Compound 2 showed more selective activity to M3 and M4 receptors than other subtypes.

Comparison of capillary electrophoresis and reversed-phase liquid chromatography for determination of the enantiomeric purity of an M3 antagonist.

The chiral separation of an M3 antagonist was investigated using capillary electrophoresis (CE) with various sulfated cyclodextrins and by reversed-phase liquid chromatography with derivatized cellulose, derivatized amylose, and two protein stationary phases. Operational parameters for each technique, such as the concentration of the chiral selectors, background electrolyte (or mobile phase) pH and type, organic modifiers, injection mode and temperature were varied in order to achieve a desired elution order and to meet a 0.1% limit of quantitation (LOQ) criteria. Based on the advantages and disadvantages of each technique, a practical CE method using sulfated gamma-cyclodextrin was selected. The method was validated in terms of linearity, LOQ, accuracy, ruggedness and precision.

Main constituents of a commercial Drosera fluid extract and their antagonist activity at muscarinic M3 receptors in guinea-pig ileum.

The range of known constituents of Drosera species is extended by identification of myricetin 3-O-galactoside, from D. madagascariensis, and (+)-cis-isoshinanolone, obtained from a commercial fluid extract. They are accompanied by the naphthoquinones droserone and plumbagin, typical of this taxon, and a series of ubiquitous flavonols, including the rarely found gossypitrin present in the latter source. Conspicuously, the commercial form of D. peltata, non-accepted by the commission E, was found to be devoid of flavonoids. In addition, the fortuitous availability of the authentic enigmatic sample 'CON', previously isolated from D. rotundifolia, led to its characterization as common quercetin. Experiments performed on isolated guinea-pig ileum demonstrated that quercetin respectively 'CON' moderately inhibited carbachol-induced contractions at 10 microM (pD'2 5.09 +/- 0.02), while (+)-cis-isoshinanolone (100 microM) was inactive. This result indicates that quercetin derivatives may well contribute to the therapeutic use of Drosera preparations.

A snake venom inhibitor to muscarinic acetylcholine receptor (mAChR): isolation and interaction with cloned human mAChR.

An inhibitor to the muscarinic acetylcholine receptor (mAChR) was purified from the venom of Crotalus atrox (western diamondback rattlesnake). The inhibitor was found to be a 30-kDa homodimer protein with phospholipase A2 activity. In order to determine the subtype selectivity of the purified inhibitor, the inhibitory effect on the binding of two orthosteric antagonists, [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine methyl chloride ([3H]NMS), to five subtypes of cloned human mAChR was tested. The purified inhibitor reduced the binding of [3H]QNB and/or [3H]NMS to all subtypes of the mAChR while showing the highest inhibitory effect on the M5 subtype. The Kd values of the receptors for the antagonists were increased in the presence of the inhibitor; however, the Bmax values were not changed. The effects of the purified inhibitor on the dissociation of [3H]NMS from the receptors were also investigated. Dissociation of the antagonist was remarkably slowed down by addition of the inhibitor. These findings may suggest an allosteric action of the purified inhibitor. In addition, the present study indicates that the presence of mAChR inhibitors is quite common in snake venoms.

Isolation of a muscarinic alkaloid with ocular hypotensive action from Trophis racemosa.

A muscarinic alkaloid with a quaternary nitrogen was isolated from Trophis racemosa. Aqueous solutions (0.5%-2%) of the chloride salt of the alkaloid produced dose-dependent reductions of intra-ocular pressure ranging from 6.6 +/- 0.7 mmHg to 15.7 +/- 0.3 mmHg, (p < 0. 001, n = 5) in dogs. Atropine (0.1 mL of a 1% solution) and pirenzepine at a non selective antagonist dose (0.1 mL of 0.5% solution) for M(1) and M(3) receptors blocked the reduction of intra-ocular pressure, but alpha-adrenoceptor blockade with phenoxybenzamine (0.1 mL of a 1% solution) did not block the reduction of intra-ocular pressure. On the isolated guinea-pig ileum and trachea, the alkaloid produced contractions which were inhibited by atropine (6 x 10(-7) M or 0.4 microg/mL) and by pirenzepine at a non-selective antagonist dose (3.1 x 10(-6) M or 1.3 microg/mL) for M(1) and M(3) receptors. But neither selective blockade of M(2) receptors with gallamine (1.7 x 10(-6) M or 1.5 microg/mL) nor selective blockade of M(1) receptors with pirenzepine (7 x 10(-9) M or 3 ng/mL) inhibited the alkaloid-induced contractions. There was also no inhibition of the alkaloid-induced contractions in the presence of ganglionic nicotinic receptor blockade with pentolinium (5.6 x 10(-7) M or 0.3 microg/mL) and hexamethonium (1.7 x 10(-6) M or 0.6 microg/mL), but nicotine-induced contractions were inhibited by these ganglionic blockers. These results suggest that a muscarinic alkaloid from Trophis racemosa produced ocular hypotension via M(3) receptor stimulation in dogs.