Hydroxypropyl ß-cyclodextrin nanohybrid monoliths for use in capillary electrochromatography with UV detection: application to the enantiomeric separation of adrenergic drugs, anticholinergic drugs, antidepressants, azoles, and antihistamine.

Two kinds of hydroxypropyl ß-cyclodextrin nanohybrid monoliths were synthesized and applied in capillary electrochromatography with UV detection. One column was fabricated by concurrently using glycidyl methacrylate-bonded hydroxypropyl ß-cyclodextrin (GMA-HP-ß-CD), sodium 3-mercaptopropanesulphonate, and alkoxysilanes in the "one-pot" process. The other was prepared by free radical polymerization of GMA-HP-ß-CD, vinylmethylcyclosiloxane, ethylene dimethacrylate, and 2-acrylamido-2-methyl propane sulfonic acid. Compared to the former hybrid monolith, the latter one displayed improved enantiomeric separation. For ten adrenergic drugs, six anticholinergic drugs, two antidepressants, six azoles, and one antihistamine enantiomeric separation was obtained on the monolith synthesized by free radical polymerization. Twelve out of twenty-five drugs were baseline-separated. Especially, anisodamine with two chiral centers was successfully separated with resolution values of 3.06, 2.11, and 2.17. The nanohybrid monoliths were characterized by optical microscopy, scanning electron microscopy, FT-IR, nitrogen adsorption analysis, and thermogravimetric analysis. Relative standard deviation values less than 5% were obtained through run-to-run, day-to-day, and column-to-column investigations (n = 3). Graphical abstract Schematic representation of two kinds of hydroxypropyl ß-cyclodextrin nanohybrid monoliths based on "one-pot" approach (route I) and free radical polymerization approach (route II), respectively.

Phytochemical Content, Antidiabetic, Anticholinergic, and Antioxidant Activities of Endemic Lecokia cretica Extracts.

The aim of this work was to investigate the enzyme inhibition, antioxidant activity, and phenolic compounds of Lecokia cretica (Lam.) DC. Acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glycosidase enzymes were strongly inhibited by the L. cretica extracts. IC50 values for the three enzymes were found as 3.21 mg/mL, 2.1 mg/mL, and 2.07 mg/mL, respectively. Antioxidant activities were examined in both aqueous and ethanol (EtOH) extracts using CUPRAC, FRAP, and DPPH method. Also, the phenolic compounds of the endemic plant were identified and quantified by using HPLC/MS/MS. According to the results, the extracts have remarkable antioxidant activities. The most abundant phenolic acids of L. cretica in EtOH extract were determined as quinic acid (12.76 mg/kg of crude extract), chlorogenic acid (3.39 mg/kg), and malic acid (2.38 mg/kg).

Study of the enantiomeric separation of the anticholinergic drugs on two immobilized polysaccharide-based chiral stationary phases by HPLC and the possible chiral recognition mechanisms.

In this work, the enantiomeric separation of ten anticholinergic drugs was first examined on two derivative polysaccharide chiral stationary phases (CSPs), i.e., Chiralpak ID and Chiralpak IA in the normal phase mode. Except for scopolamine hydrobromide, the remaining nine analytes could be completely separated with good resolutions using both columns under the optimized mobile phase conditions. And the enantiomeric discrimination ability of the studied CSPs towards nine analytes was in the order of Chiralpak ID > Chiralpak IA. The influences of organic modifier types, alcohol content, and base/acid additives on the enantiomeric separation were evaluated and optimized. According to the experimental results, the effect of the structures of analytes on enantiomeric separation was discussed. Additionally, the chiral recognition mechanisms were proposed based on the thermodynamic analysis of the experimental data.

Comparison of three S-ß-CDs with different degrees of substitution for the chiral separation of 12 drugs in capillary electrophoresis.

Three kinds of sulfated ß-cyclodextrin (S-ß-CD), including a single isomer, heptakis-6-sulfato-ß-cyclodextrin (HS-ß-CD), degree of substitution (DS) of 7, which was synthesized in our laboratory and another two commercialized randomly substituted mixtures, a sulfated ß-cyclodextrin with DS of 7 to 11, as well as a highly sulfated-ß-cyclodextrin with DS of 12 to 15, were used for the enantioresolution of 12 drugs (the ß-blockers, phenethylamines, and anticholinergic agents) in capillary electrophoresis. The enantioseparation under varying concentrations of S-ß-CD and background electrolyte pH were systematically investigated and compared. Based on the experimental results, the effect of the nature of S-ß-CD and analyte structure on the enantioseparation is discussed.

Anticholinergic, antihistaminic, and antiserotonergic activity of n-hexane extract of Zanthoxylum alatum seeds on isolated tissue preparations: An ex vivo study.

OBJECTIVES: The aim of this study was to evaluate anticholinergic, antihistaminic, and antiserotonergic activity of the n-hexane extract of the seeds of Zanthoxylum alatum (ZAHE) on isolated ileum of rat and guinea pig and fundus of rat. MATERIALS AND METHODS: ZAHE was prepared using soxhlet extraction and cumulative concentration response curves were constructed using various doses on the tissues for acetylcholine (ACh), 5-hydroxytryptamine (5-HT), and histamine with or without n-hexane extract. Atropine, ketanserin, and pheniramine maleate were used as antagonists for ACh, serotonin, and histamine, respectively. RESULTS: ZAHE-induced concentration-dependent inhibition of isolated ileum and fundus in rat and ileum of guinea pig. The half maximal effective concentration (EC50) of ACh in the presence of atropine (10-6 M; P < 0.05) and ZAHE (1000 µg/ml; P < 0.01) was significantly higher than EC50of ACh alone. The EC50of 5-HT in the presence of ketanserin (10-5 M; P < 0.01) and ZAHE (1000 µg/ml; P < 0.05) was higher than EC50of 5-HT alone. Similarly, the EC50of histamine in the presence of pheniramine maleate (10-6 M; P < 0.01) and ZAHE (300 µg/ml; P < 0.01 and 1000 µg/ml; P < 0.05) was also significantly higher than EC50of histamine alone. CONCLUSION: From the study, it was observed that ZAHE shows significant anticholinergic, antiserotonergic, and antihistaminic activity. The study provides sufficient evidence that the seeds can be used in gastric disorders, cough, chest infection, etc., as per folklore claims.

A pharmacological evidence for the presence of antihistaminic and anticholinergic activities in Equisetum debile Roxb.

OBJECTIVE: The study was designed to evaluate possible antihistaminic and anticholinergic activities of Equisetum debile. MATERIALS AND METHODS: Effects of crude ethanolic (Ed.Eth) and effects of crude aqueous (Ed.Aq) extracts of E. debile were studied using isolated guinea pig ileum, rabbit jejunum, and rabbit trachea. Tissue responses were recorded using isotonic and isometric transducers, connected with PowerLab data acquisition system. RESULTS: A dose-dependent (0.1-0.3 mg/ml) rightward shift was demonstrated in histamine concentration-response curves. Whereas a complete relaxation of carbachol (1 µM)-induced contractions in isolated rabbit jejunum (3 mg/ml) and tracheal (10 mg/ml) preparations was observed, similar to dicyclomine at 1 and 3 µM, respectively. However, no significant difference between the effects of Ed.Eth and Ed.Aq was observed. CONCLUSION: Study provides pharmacological evidence for the presence of antihistaminic and anticholinergic activities in crude extracts of E. debile and also highlight its medicinal significance in the management of airway and gastrointestinal disorders.

Anti-cholinergic and Ca2+-antagonist mechanisms explain the pharmacological basis for folkloric use of Sisymbrium irio Linn. in gastrointestinal, airways and vascular system ailments.

ETHNOPHARMACOLOGICAL RELEVANCE: Seeds of Sisymbrium irio Linn has been used traditionally in different regions of Pakistan for the treatment of gastrointestinal, airways and vascular system ailments. To insight the pharmacological basis, in vitro study was conducted in order to validate its folkloric uses. MATERIAL AND METHODS: 70% aqueous-methanolic extract of seeds from S. irio (Si.MEs) was tested on isolated rabbit aorta, jejunum and trachea strip hanged in tissue bath having physiological solutions aerated with carbogen and their responses were measured and recorded via Power Lab. RESULTS: The Si.MEs exhibited the transient spasmogenic effect (0.01-1.0mg/mL) on spontaneous jejunum contractions, followed by the spasmolytic effect. The addition of atropine resulted in blocking in spasmogenic effect while the spasmolytic effect was originated, suggesting the presence of an antimuscarinic effect. Likewise verapamil, Si.MEs (0.03-5mg/mL) repressed the high concentration K+(80mM)-induced contraction and also drifted the Ca2+ concentration-response curves toward right (0.3-3.0mg/mL), possibly signifying the Ca2+ channel blockade. Furthermore, Si.MEs exhibited nonspecific relaxant effect on carbachol (1µM)- and high concentration K+(80mM)-induced tracheal contractions in a way comparable to dicyclomine, suggesting the coexistence of Ca2+-antagonistic and/or antimuscarinic properties. Additionally, Si.MEs also relaxed the phenylephrine(1µM)- and high concentration K+(80mM)-induced aortic contraction (0.01-3mg/mL), suggesting blockade of Ca2+ channel. Moreover, oral administration of Si.MEs, as high as 6g per kg, did not produce lethality among the treated groups of mice. CONCLUSIONS: Aqueous-methanolic extract of seeds from S. irio (Si.MEs) exhibited the bronchodilator and gut modulator (spasmogenic and spasmolytic) activities, probably through dual blockade of muscarinic receptors and Ca2+ channels, whereas, vasodilator effect may be due to Ca2+ channels blockade.

Antioxidant and antiatherogenic properties of phenolic acid and flavonol fractions of fruits of 'Amari' and 'Hallawi' date (Phoenix dactylifera L.) varieties.

Date (Phoenix dactylifera L.) fruit phenolic-acid or flavonol fractions were examined in vitro for antioxidant and antiatherogenic properties. Two fractions of each subgroup were prepared from two date varieties, 'Amari' and 'Hallawi', by solid phase extraction on C18. The fractions were analyzed for phenolics composition by RP-HPLC and tested for ferric-reducing antioxidant power, free radical scavenging capacity, inhibition of Cu(2+)-induced LDL oxidation, and enhancement of HDL-mediated cholesterol efflux from macrophages. All four fractions exhibited variable capacities to reduce ferric ions, scavenge radicals, and inhibit LDL oxidation. Flavonol fractions were considerably better inhibitors of LDL oxidation compared to phenolic acid fractions, with IC50's of 9-31 nmol GAE mL(-1) compared to 85-116 nmol GAE mL(-1), respectively. Only the flavonol fractions stimulated cholesterol removal from macrophages. Within each subgroup, the levels of all the activities varied with fraction composition. The results demonstrated strong structure-activity relationships for date phenolics and identified date flavonols as potential antiatherogenic bioactives.

Three new lanostanoid triterpenes from the fruiting bodies of Ganoderma tropicum.

Three new lanostanoid triterpenes, 3ß,7ß,15ß-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid (1), 3ß,7ß,15ß-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid methyl ester (2), and 3ß,15ß-dihydroxy-7,11,23-trioxo-lanost-8,16-dien-26-oic acid methyl ester (3) were isolated from the EtOAc extract of the fruiting bodies of Ganoderma tropicum. Their structures were elucidated on the basis of 1D and 2D NMR spectroscopy as well as MS. The bioassay of inhibitory activity against acetylcholinesterase (AChE) of these isolates was evaluated and compound 2 exhibited definite inhibitory activity against AChE.

Detection of singly- and doubly-charged quaternary ammonium drugs in equine urine by liquid chromatography/tandem mass spectrometry.

Quaternary ammonium drugs (QADs) are anticholinergic agents some of which are known to have been abused or misused in equine sports. A recent review of literature shows that the screening methods reported thus far for QADs mainly cover singly-charged QADs. Doubly-charged QADs are extremely polar substances which are difficult to be extracted and poorly retained on reversed-phase columns. It would be ideal if a comprehensive method can be developed which can detect both singly- and doubly-charged QADs. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous detection and confirmation of 38 singly- and doubly-charged QADs at sub-parts-per-billion (ppb) to low-ppb levels in equine urine after solid-phase extraction. Quaternary ammonium drugs were extracted from equine urine by solid-phase extraction (SPE) using an ISOLUTE(®) CBA SPE column and analysed by LC/MS/MS in the positive electrospray ionisation mode. Separation of the 38 QADs was achieved on a polar group embedded C18 LC column with a mixture of aqueous ammonium formate (pH 3.0, 10 mM) and acetonitrile as the mobile phase. Detection and confirmation of the 38 QADs at sub-ppb to low-ppb levels in equine urine could be achieved within 16 min using selected reaction monitoring (SRM). Matrix interference of the target transitions at the expected retention times was not observed. Other method validation data, including precision and recovery, were acceptable. The method was successfully applied to the analyses of drug-administration samples.

Liquid chromatography-tandem electrospray mass spectrometry method for determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma.

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma. After a simple protein-precipitation using methanol, the post-treatment samples were separated on a CAPCELL UG120 column with a mobile phase of a mixture of methanol and water (35:65) containing 0.1% formic acid. The serial chiral analytes and internal standard (IS) were all detected by the use of selected reaction monitoring mode (SRM). The method of all serial chiral analytes developed was validated in rat plasma with a daily working range of 0.5-100 ng/ml with correlation coefficient, R(2) > or = 0.99 and a sensitivity of 0.5 ng/ml as lower limit of quantification, respectively. This method was fully validated for the accuracy, precision and stability studies for all serial chiral analytes. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of serial chiral novel anticholinergic compounds of phencynonate in rat plasma.

Chiral separation of anticholinergic drug enantiomers in nonaqueous capillary electrophoresis.

Nonaqueous capillary electrophoretic (NACE) method for the separation of nine structurally similar chiral anticholinergic drugs was developed. The eight drug enantiomers were separated on baseline within 18 min using 20mM phosphoric acid and 10 mM NaOH, containing 10 mM heptakis(2,3-dimethyl-6-sulfato)-4beta-cyclodextrin (HDMS-beta-CD) in methanol. The results were compared with those obtained in the high performance liquid chromatography system.

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone.

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the alpha-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr16]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

Structure-activity relationships of imperialine derivatives and their anticholinergic activity.

In order to check the structure-activity relationship and prepare more potent derivatives of imperialine with anticholinergic activity, imperialinol (2), 3 beta-acetoxyimperialine (3), 3 beta-propionoxyimperialine (4), and 3 beta-butyroxyimperialine (5) were prepared. Compounds 4 and 5 displayed better anticholinergic activity against muscarinic receptors of the heart and brain than imperialine (1). The decrease in activity in 2 showed the importance of the 6-keto functionality in imparting the anticholinergic activity.

New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum.

Three new steroids, zhankuic acids A [1], B [2], and C [3], were isolated from the fruiting bodies of Antrodia cinnamomea by bioassay-guided fractionation. The structures of these compounds were elucidated by chemical reactions and detailed analysis of their 1H- and 13C-nmr spectra. Biological studies revealed that 1 exhibited cytotoxic activity against P-388 murine leukemia cells and 2 showed weak anticholinergic and antiserotonergic activities.

Microbore liquid chromatography of tertiary amine anticholinergic pharmaceuticals with tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection.

The post-column chemiluminescent reaction of six anticholinergic alkaloid compounds with tris(2,2'-bipyridine)ruthenium(III) (Ru(bpy)3(3+)) is applied to microbore high-performance liquid chromatography (HPLC). At flow rates less than 200 microL/min, the capillary mixing cell in which Ru(bpy)3(3+) and the analyte are mixed directly allows for good light detection. In contrast, a diminished signal occurs at these low flow rates with conventional post-column mixing in a tee. Optimal chemiluminescent pH conditions for atropine, scopolamine, dicyclomine, cyclopentolate, cyclobenzaprine, and procyclidine are determined at moderately basic conditions (pH 7 to 9). 2-Butanone is found to be compatible with the chemiluminescent reaction, whereas tetrahydrofuran and propionitrile cause an increase in background noise and a chemiluminescent signal loss. As 2-butanone is more nonpolar than acetonitrile, it assists in the elution of these hydrophobic anticholinergic compounds. Five anticholinergic compounds are resolved successfully with a PRP-1 polymeric column and a slightly basic mobile phase, but a C8 silica column is better suited for the more hydrophobic compounds (cyclobenzaprine, procyclidine, and dicyclomine).

Synthesis and separation of optically active compounds. Part I.

Biological processes involve a high degree of stereoselectivity. Considering that pharmacological activity is very often associated with only one enantiomer, regulation authorities have defined new guidelines in requiring chiral drugs to be marketed as pure individually safe and effective enantiomers. If large scale production of pure stereoisomers has met until recently a great difficulty to become economically acceptable, the current state of the art in asymmetric synthesis and chiral separation, provides however many industrial applications. Among the methods currently used, asymmetric synthesis, using chiral auxiliaries or chiral catalysts such as enzymes or metal complexes, allows to create new asymmetric centers, starting from prochiral molecules. Regarding racemate resolution, asymmetric synthesis offers the real economical advantage to produce exclusively the targeted enantiomer. However, resolution methods allowing access to both enantiomers, are very useful at preparative scale for new chiral drugs when preclinical studies are requested for each enantiomer. Large scale racemate resolution can be achieved by crystallization, enzymatic resolution and even more recently by liquid chromatography. Although the "unwanted" enantiomer is also produced, the feasibility of its racemization for recycling has made resolution methods widely used in industry. It is clear today that in most cases, pharmacologically active molecules having an asymmetric center will have to be marketed as optically pure drugs.