Discovery of ß2- adrenoceptor agonists in Curcuma zedoaria Rosc using label-free cell phenotypic assay combined with two-dimensional liquid chromatography.

Traditional Chinese Medicines (TCMs) have been widely used in clinical practice, and provided a rich source for discovering new drug leads. However, efficient identification of active molecules responsible for the therapeutic effects of complex TCMs is still highly challenging. Here, we combined label-free cell phenotypic assay with two dimensional liquid chromatography (2DLC) to identify potential ß2-adrenoceptor (ß2-AR) agonists related to anti-asthmatic effect of Curcuma zedoaria Rosc (C.zedoaria), a commonly used TCM. The ethyl acetate extract of C.zedoaria was first fractionated into 26 fractions. Label-free cell phenotypic profiling was then used to locate the active sites. Orthogonal second-dimensional (D2) separation was performed on two fractions displaying agonistic effect at the ß2-AR, combined with screening of the D2 fractions to track the activity. Finally, this approach led to the isolation of three known diarylheptanoids, among which diarylheptanoid b exhibited the most potent agonistic activity with an EC50 value of 5.93 µM. This result was further demonstrated through the chemical synthesis of diarylheptanoid b. It is the first time to discover that diarylheptanoids could activate the ß2-AR, which may be responsible for the anti-asthmatic effect of C.zedoaria observed traditionally and in clinical application. This study also demonstrates the potential of this integrated strategy for identifying active ingredients and determining the basis of therapeutic materials in complex TCMs.

Development of retention prediction models for adrenoreceptor agonists and antagonists on a polyvinyl alcohol-bonded stationary phase in hydrophilic interaction chromatography.

Retention prediction models based on multiple linear regression (MLR) and artificial neural network (ANN) for adrenoreceptor agonists and antagonists chromatographed on a polyvinyl alcohol-bonded stationary phase under hydrophilic interaction chromatography were described. The models showed the combined effects of solute structure and mobile phase composition on the retention behavior of the analytes. Using stepwise MLR, the retentions of the studied compounds were satisfactorily described by a five-predictor model; the predictors being the %ACN, the logarithm of the partition coefficient (log D), the number of hydrogen bond donors (HBD), the desolvation energy for octanol (FOct), and the total absolute atomic charge (TAAC). The inclusion of the solute-related descriptors suggested that hydrophilic interactions such as hydrogen bonding and also ionic interactions are possible mechanisms by which analytes are retained on the studied system. ANN prediction models were also derived using the predictors derived from MLR as inputs and log k as outputs. The best network architectures were found to be 5-3-1 for the datasets at pH 3.0 and 4.0, and 5-4-1 for the dataset at pH 5.0. The optimized ANNs showed better predictive properties than the MLR models for both training and test sets under all pH conditions.

Retention prediction of adrenoreceptor agonists and antagonists on unmodified silica phase in hydrophilic interaction chromatography.

The development of retention prediction models for adrenoreceptor agonists and antagonists chromatographed on an unmodified silica stationary phase under the hydrophilic interaction chromatographic (HILIC) mode at three pH conditions (3.0, 4.0 and 5.0) is described. The models were derived using multiple linear regression (MLR) and an artificial neural network (ANN) using the logarithm of the retention factor (log k) as the dependent variable. In addition to the effects of the solute-related variables (molecular descriptors), the percentage of acetonitrile (%ACN) was also used as a predictor to gauge the influence of the mobile phase on the retention behavior of the analytes. Using stepwise MLR, the retention behavior of the studied compounds at pH 3.0 were satisfactorily described by a four-predictor model; the predictors being the %ACN, the logarithm of the partition coefficient (log D), the number of hydrogen bond acceptors (HBA), and the magnitude of the dipole moment (DipolMag). In addition to these four predictors, the total absolute atomic charge (TAAC) was found to be a significant predictor of retention at pH 4.0 and 5.0. Among the five descriptors, %ACN had the strongest effect on the retention, as indicated by its higher standardized coefficient than those obtained for the other four predictors. The inclusion of these four predictors which are related to the molecular properties of the compounds (log D, HBA, DipolMag, and TAAC) suggested that hydrophilic interactions, hydrogen bonding and ionic interactions are possible mechanisms by which analytes are retained on the studied system. The reliability and predictive ability of the derived MLR equations were tested using cross-validation and a test set which was not used when fitting the model. The models derived from MLR produced adequate fits, as proven by the high R2 values obtained for all calibration and training sets (0.9497 and above), and their good predictive power, as indicated by the high cross-validated q2 (0.9465 and above) and high R2 (0.9305 and above) values obtained for the test sets. ANN prediction models were also derived using the predictors derived from MLR as inputs and log k as output. A comparison of the models derived from both ANN and MLR revealed that the trained ANNs showed better predictive abilities than the MLR models, as indicated by their higher R2 values and their lower root mean square error of predictions (RMSEP) for both training and test sets under all pH conditions. The derived models can be used as references and they provide a useful tool for method development and the optimization of chromatographic conditions for the separation of adrenoreceptor agonists and antagonists.

Study of retention parameters obtained in RP-TLC system and their application on QSAR/QSPR of some alpha adrenergic and imidazoline receptor ligands.

The retention constant (R(0)(m)) is determined for 11 selected adrenergic and imidazoline receptor ligands by reverse-phase-thin layer chromatography. It is established that the retention behavior of investigated compounds mostly depends on geometrical, electrostatic, and hydrogen bonding properties. Good correlations among hydrophobic parameters R(0)(m) versus log P for all eleven tested compounds are obtained. The satisfactory correlations are found between R(0)(m) versus apparent partition coefficient octanol-buffer pH 7.4 (log P') or apparent partition coefficient in four liposome systems (log K'(M)) and hypotensive activity (pC(25)) for five imidazolines. The results confirm the suitability of this parameter in quantitative structure-property and structure-activity relationships studies of these drugs.

The effects of epiphyseal peptides on the release of immunoglobulins in Peyer's patches in rats in vitro.

The effects of epiphyseal peptides (1 microg/ml) on the release of immunoglobulins into the incubation medium by isolated Peyer's patches from non-immunized mice and mice immunized orally against ovalbumin were studied during 40-min incubations. The possibility that epiphyseal peptides act on adrenoreceptors of cells in secondary lymphoid organs in the small intestine was assessed using alpha- and beta-adrenoreceptor blockers, i.e., phentolamine HCl (0.02 mg/ml) and anaprilin (0.06 mg/ml) respectively. Basal levels of secretory activity in control Peyer's patches from immunized rats were 2.4 times (p < 0.01) greater than for the lymphoid organs of non-immunized animals. The effects of epiphyseal peptides on the secretory activity of antibody-forming cells depended on the functional state of Peyer's patches. Application of epiphyseal peptides led to a 35% increase (p < 0.05) in the release of immunoglobulins from Peyer's patches in non-immunized rats and a 25% decrease (p < 0.05) in the release of antibody into the incubation medium from the lymphoid organs of immunized animals. These data lead to the suggestion that the activatory effect of epiphyseal peptides on antibody-forming cells in Peyer's patches from non-immunized animals is associated with alpha-adrenoceptors, while their inhibitory action on immunoglobulin release by the small intestine lymphoid organs from immunized animals is not mediated via adrenoceptors.

The use of nonendcapped C18 columns in the cleanup of clenbuterol and a new adrenergic agonist from bovine liver by gas chromatography-tandem mass spectrometry analysis.

More specific official methodology is needed to survey the illegal use of clenbuterol in animal production plus the synthesis of new compounds that currently elude routine analytical methods. The identification of a new adrenergic agonist, N1-(2-(4-amino-3,5-dichlorophenyl)-2-hydroxyethyl)-N1-isopropyl-propanamide (known as compound A) in animal feed has prompted studies to verify if the existing cleanup procedures developed for clenbuterol are really effective. This study considers the ion-exchange mechanism on cyanopropyl (CN), sulfonic cation exchange (SCX), mixed phase (MPH) (C8 + SCX), and nonendcapped C18 (C18NE) solid-phase extraction (SPE) columns. Results indicate that compound A (by contrast with clenbuterol) is not efficiently retained on the CN, SCX, and MPH SPE columns (recovery <10%). This finding thus leads to the development of a gas chromatography-tandem mass spectrometry procedure based on C18NE SPE that is able to purify both agonists from bovine livers spiked at 0.5, 1.0, and 2.0 ppb with a mean recovery of 93% for clenbuterol and 92% for compound A.

Separation of basic drug enantiomers by capillary zone electrophoresis using glucuronyl glucosyl beta-cyclodextrin as a chiral selector.

Separations of basic drug enantiomers have been investigated using glucuronyl glucosyl beta-cyclodextrin (GUG beta-CD) as a chiral selector in the background electrolyte by capillary zone electrophoresis. The effects of GUG beta-CD concentration and running buffer pH on the migration times and resolution of 16 basic drug enantiomers were precisely examined using a linear polyacrylamide-coated capillary. High resolution of 16 basic drug enantiomers was generally attained with a running buffer pH 2.5 or 3.5 containing 10 mM GUG beta-CD. Next, we compared the chiral resolution abilities of GUG beta-CD with those of beta-CD and maltosyl beta-CD (G2 beta-CD). GUG beta-CD showed higher resolution for basic drug enantiomers tested than beta-CD and G2 beta-CD. This could be due to that hydrogen bonding or ionic interactions of uncharged and charged glucuronyl glucosyl groups of GUG beta-CD with an analyte could stabilize the inclusion complex.

[Enantiomeric high performance liquid chromatographic separation of epinephrine and analogous using chiral mobile phase additives].

The enantiomeric resolution of epinephrine, isoproterenol, ephedrine was studied by reversed phase HPLC. beta-CD, DM-beta-CD, TM-beta-CD were used as chiral mobile phase additives. The effects of the concentration of DM-beta-CD and the concentration of methanol on resolution of D,L-epinephrine were investigated. The results showed: D,L-epinephrine and D,L-isoproterenol can be separated by beta-CD, DM-beta-CD, TM-beta-CD, but D,L-ephedrine can't. Hydrogen bonding plays an important role in the chiral recognition mechanism. In the separation of isoproterenol, the enantioselectivity of TM-beta-CD was better than those of beta-CD and DM-beta-CD. As the concentration of DM-beta-CD changed from 0.04 mmol/L to 1.00 mmol/L, the resolution varied slightly. The experiment of the effect of methanol concentration on Rs showed: when the ratio of methanol/water was 40/60, the Rs reached the maximum. The coefficients of variation of retention time of D-epinephrine and L-epinephrine were 1.3% and 1.4% respectively. It is demonstrated that the chromatographic systems with a dynamically-generated stationary phase with methylated-beta-cyclodextrin are versatile tools for enantiomer separation. This technique leads to column with excellent time stability and good reproducibility of the enantio-selectivity.

Liquid chromatographic separation of enantiomers of adrenergic agonists.

Many established and experimental adrenergic agonists are derivatives of 2-aminoethanol with a phenol or catechol moiety in the 1-position. There is considerable interest in the stereochemical aspects of the actions of such chiral drugs. Surprisingly, however, little has been published on the chromatographic separation of the enantiomers of these compounds, and what has been published involved nearly exclusively capillary gas-liquid chromatography. In this report, the resolution of the racemates of ten adrenergic agents using reversed-phase liquid chromatography is described. The procedure is based on derivatizing the racemic mixture of each agent with the chiral reagent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate, followed by separation of the resulting diastereomers on octadecylsilane columns using methanol-aqueous ammonium acetate mixtures as mobile phase. Detection was at 254 nm. The separation of the enantiomers of norphenylephrine and of octopamine was less than complete; the resolution of the other agents, i.e., synephrine, N-ethylnorphenylephrine, p-hydroxyephedrine, p-hydroxynorephedrine, metanephrine, normetanephrine, isoproterenol, and nordefrin, was complete. The derivative of (-)-isoproterenol eluted before that of its antipode. The procedure may be applicable to other similar agents.