Separation and identification of cannabis components by different planar chromatography techniques (TLC, AMD, OPLC).

The use of cannabis is illicit in numerous countries, and the increasing consumption has led to a multiplication of scientific studies. New methods of planar chromatography such as automated multiple development (AMD) and optimum performance laminar chromatography (OPLC) techniques can be used as a substitute for the traditional thin-layer chromatography for the identification and quantitation of the Indian hemp components. Each method offers its own advantage: high resolution with neither diffusion nor spot stretching for AMD and speed, efficiency, and the possibility of working in the semipreparative mode for OPLC.

Chemometrical analysis of 18 metallic and nonmetallic elements found in honeys sold in france.

The elemental analysis of 86 honeys sold in France was performed with an inductively coupled plasma atomic emission spectrometer in order to measure significant concentrations of Ag, Ca, Cr, Co, Cu, Fe, Li, Mg, Mn, Mo, P, S, Zn, Al, Cd, Hg, Ni, and Pb. Principal component analysis, correspondence factor analysis, and hierarchical cluster analysis were used to rationalize and interpret the analytical data. Crude relationships were found between the elemental profiles of the honeys and their botanical origin. Some honeys were highly polluted by heavy metals and/or other xenobiotics. Explanations for these contaminations are proposed.

OPLC and AMD, recent techniques of planar chromatography: their interest for separation and characterization of extractive and synthetic compounds.

It is always important for the chemist to have good methods of separation, characterization and quantitative evaluation for one or several compounds resulting from a chemical reaction or an extraction procedure. In this domain the chromatographic techniques are choice methods, in particular Over Pressured Layer Chromatography (OPLC) and Automated Multiple Development (AMD). They are relatively recent methods whose use is unfortunately not yet generalized although they give very clean separation. In this paper we present numerous examples of the use of these two new types of planar chromatographies and especially the results we have obtained in the field of natural products on a wide variety of different structures: coumarins, flavonoids, anthocyanins, alkaloids and essential oils.

Comparison of planar chromatographic methods (TLC, OPLC, AMD) applied to essential oils of wild thyme and seven chemotypes of thyme.

Essential oils analysis is more often realized by gas chromatography. However, thin-layer chromatography (TLC) remains the reference for Pharmacopoeia. Nevertheless classical TLC has its own limitations but it is always a good technique because it is simple, rapid and less expensive. Actually the reproducibility, the separation quality and the possibility to obtain good and reproducible quantitative determinations have been improved significantly with automated sample applicator, scanner densitometers and two new chromatographic planar methods: the optimum performance laminar chromatography (OPLC) and automated multiple development (AMD). In this work, we show and compare the performance of these methods and TLC through a study of seven thyme chemotypes and wild thyme essential oil.

A comparative study of the effects of sparteine, lupanine and lupin extract on the central nervous system of the mouse.

Lupin is toxic because of its alkaloid content, sparteine and lupanine in particular. Although the pharmacological properties of sparteine are well known those of lupanine have not been much studied. This paper reports procedures for extraction, purification and crystallization of lupanine, and methods for the preparation of an extract for injection of Lupinus mutabilis Sweet, and for the determination of the acute toxicity and maximum non-lethal dose (DL0) of lupanine, sparteine and lupin extract in the mouse. The three substances were tested on the central nervous system (CNS) for locomotor activity, for interaction with specific drugs used for treatment of the CNS (the stimulant drugs amphetamine and pentetrazol and the depressant drugs pentobarbital and chlorpromazine) and for analgesic activity. The results indicate that lupanine and lupin extract are less toxic than sparteine and that at the doses studied the three products have a weak sedative effect on the CNS.

Enhancement of the endothelial production of prostacyclin by inhibitors of protein synthesis.

1. Pretreatment of bovine aortic endothelial cells with cycloheximide enhanced their capacity to release prostacyclin in response to adenosine 5'-triphosphate (ATP) and bradykinin. 2. The action of cycloheximide was time-dependent; it became detectable after a 1 h exposure to the cells and was maximal after 3 h. 3. Puromycin mimicked the effect of cycloheximide. For these two agents, the enhancement of prostacyclin release was obtained at concentrations producing a partial inhibition of protein synthesis. 4. Cycloheximide increased the mobilization of free arachidonic acid induced by ATP in bovine aortic endothelial cells. 5. In conclusion, the synthesis of new proteins is not involved in the stimulatory action of ATP and bradykinin on prostacyclin production by bovine aortic endothelial cells. Despite the short half-life of prostaglandin H synthase in endothelial cells, cycloheximide and puromycin enhanced the release of prostacyclin induced by agonists. Our data suggest that this release might be under the control of rapidly turning-over phospholipase inhibitory proteins.

Enhanced release of prostacyclin from quin 2-loaded endothelial cells.

The acetoxymethyl ester (AM) of quin-2 (quin-2/AM) enhanced the release of prostacyclin (PGI2) from bovine aortic endothelial cells stimulated by ATP, bradykinin or ionophore A23187. It also increased the mobilization of free arachidonic acid in response to ATP. Ca2+-clamping with a combination of EGTA and quin-2/AM abolished the response to ATP. The effect of quin-2/AM was mimicked by a structural analog, the acetoxymethyl ester of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), but not by the heavy metal chelator, tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and only slightly by fura-2/AM. The mechanism of this pharmacological action of quin-2/AM and its potential for the design of PGI2-stimulating drugs remain to be explored.

Adrenergic stimulation of vascular prostacyclin: role of alpha 1-receptors in smooth muscle cells.

Epinephrine and norepinephrine (1-10 microM) stimulated the release of prostacyclin (PGI2) from the rabbit aorta in vitro. The stimulation was maintained for at least 2 h in the continuous presence of epinephrine. Phenylephrine mimicked this effect, whereas the selective alpha 2-agonist UK-14,304 was completely ineffective. The action of epinephrine was abolished by prazosin (1 microM) and was maintained in the presence of yohimbine. Epinephrine or phenylephrine neither increased the basal release of PGI2 from bovine aortic endothelial cells nor potentiated the stimulatory action of adenine nucleotides, which is mediated by P2-purine receptors. The response to epinephrine was lost in freshly deendothelialized strips of rabbit aorta, possibly because of cyclooxygenase self-inactivation. The response recovered however following overnight incubation of these strips in a cell culture medium. The response to epinephrine was mimicked by neither phorbol 12-myristate,13-acetate nor ionophore A23187. It was not inhibited by pretreatment with pertussis toxin. It is concluded that adrenergic agents stimulate the vascular production of PGI2, by activating alpha 1-receptors located on smooth muscle cells.

Prostacyclin production by the deendothelialized rabbit aorta.

The acute effect of in vitro deendothelialization on the production of prostacyclin (PGI2) by the rabbit aorta has been investigated. The effectiveness of removing endothelium by rubbing it against filter paper or scraping it with a scalpel was demonstrated by scanning electron microscopy and en face examination after silver staining. Endothelium removal produced an immediate stimulation of PGI2 release, resulting in 408% of the control after rubbing and 367% of the control after scraping, during the first 30-min period of incubation. This increased production of PGI2 gradually declined over time to reach values similar to the control after 2h. At that time, the deendothelialized aorta was totally unresponsive to the stimuli that increase PGI2 release in the intact aorta (acetylcholine, ADP, ionophore A23187, and arachidonic acid). The enhanced production of PGI2 in the deendothelialized aorta was associated with an increased release of free arachidonic acid (353% of the control): in contrast with PGI2, this stimulation was maintained for at least 150 min. A transient exposure of the deendothelialized aorta to ibuprofen (250 microM) was followed by a rebound of PGI2 production, which was also prolonged by BW-755C (3-10 microM). In conclusion, removal of the endothelium triggered an immediate and sustained mobilization of free arachidonic acid in the rabbit aorta: the resulting increase of PGI2 production was short-lived, probably as a consequence of cyclooxygenase self-inactivation. Our results indicate that the subendothelium has a significant capacity to produce PGI2, but that this capacity is expressed only briefly.

Prostaglandin-9-ketoreductase activity in erythrocytes of normal and hypertensive subjects.

The PGE2/PGF2 alpha balance, controlled in part by prostaglandin-9-ketoreductase, seems to be involved in the regulation of sodium excretion by the kidney. A decreased PGE2/PGF2 alpha ratio has been observed in the urine of hypertensive subjects. This suggests that an alteration of prostaglandin metabolism might be involved in the pathogenesis of essential hypertension. In order to test this hypothesis, prostaglandin-9-ketoreductase (PG-9-KR) and prostaglandin-15-dehydrogenase (PG-15-DH) activities were measured in erythrocytes of normotensive controls and patients with essential hypertension. The two enzyme activities were highly correlated in the two groups, supporting the hypothesis that they are alternate expressions of a single enzyme. These two enzyme activities were not significantly different in hypertensive subjects as compared to controls. Human essential hypertension does not appear to be linked to a generalized defect of prostaglandin catabolic enzymes.

Cholinergic stimulation of vascular prostacyclin synthesis.

The production of prostacyclin by rings of rabbit aorta was assessed by the radioimmunoassay of 6-keto-PGF1 alpha. In steady-state conditions, the rings released 11 ng 6-K-PGF1 alpha per 100 mg tissue in 30 min. Acetylcholine increased this output: a significant effect was detected at 1 microM and at 10 microM the amplitude of stimulation was 10-fold. The production of PGE2 and PGF2 alpha was also increased, but to a lesser extent. The stimulatory action of acetylcholine was mimicked by carbamylcholine and inhibited by atropine; it was abolished in a calcium-free medium. Dog and rat aorta also produced more 6-K-PGF1 alpha in response to cholinergic agonists. A short rubbing of the intimal surface of the aorta removed the layer of endothelial cells and completely abolished the cholinergic effect. It is concluded that in the aorta, cholinergic agonists, acting on a muscarinic receptor, stimulate the production of prostacyclin by endothelial cells.

Stimulation of vascular prostacyclin synthesis by extracellular ADP and ATP.

Extracellular ADP and ATP stimulated the synthesis of prostacyclin - as reflected by the release of 6-keto-PGF1 alpha - in the rabbit aorta, the rabbit pulmonary artery and the rat aorta. A doubling of 6-keto-PGF1 alpha output was produced by 3 microM ADP. Adenosine had no effect and the stimulation by ADP was blocked by quinidine, but not by theophylline. This stimulation was abolished by indomethacin and lost after mechanical removal of the endothelium. Stimulation of vascular prostacyclin synthesis by ADP released from aggregating platelets could help localize thrombus formation to areas of vascular damage.

In vitro stimulation of prostaglandin synthesis in the rat pancreas by carbamylcholine, caerulein and secretin.

Rat pancreas pieces spontaneously released PGE2 (2.3 ng/100 mg x 45 min) and PGF2 alpha (7.6 ng/100 mg x 45 min). This release corresponds probably to a neo-synthesis since it was abolished by indomethacin. Carbamylcholine (greater than or equal to 10 microM), caerulein (greater than or equal to 10 nM) and secretin (greater than or equal to 10 nM) stimulated the release of PGE2 and PGF2 alpha: the concentrations of stimulators required to increase PGs release were thus much higher than those which trigger enzyme secretion. Atropine specifically inhibited the cholinergic stimulation, whereas indomethacin blocked the stimulatory effects of all secretagogues. Stimulation of PGE2 and PGF2 alpha release was reduced in a Ca++-free medium, abolished by EGTA and mimicked by the ionophore A23187, underscoring the crucial role of Ca++ in the regulation of PGs synthesis by the pancreas. Neither PGE2 nor PGF2 alpha stimulated enzyme secretion in this system and indomethacin did not inhibit the secretory effect of carbamylcholine. Increased synthesis of prostaglandins in response to pancreatic secretagogues does not appear to be involved in the process of enzyme secretion.

Prostaglandin formation in bacteria : a reevaluation.

We report here that production and release of PGE2 do not occur in common bacteria. The apparent production in the presence of arachidonic acid, previously reported (1) may be explained by PGE2 contamination and autooxidation of the AA used. The presence of PGE2 like material in some but not all isolates of Propionibacterium acnes is confirmed.

Inhibition by iodide of the cholinergic stimulation of prostaglandin synthesis in dog thyroid.

Iodide is shown to inhibit the cholinergic stimulation of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) synthesis in dog thyroid slices. The inibitory effect of iodide was already detectable at 1 micron and reached its maximal level at 10 microns. At this concentration, iodide inhibited the carbamylcholine-stimulated release of PGE2 and PGF2 alpha by 59% and 73%, respectively (eight experiments). The effect of iodide was neither immediate nor rapidly reversed after a change of incubation medium. The unstimulated release of PGE2 and PGF2 alpha and the stimulation by epinephrine and ionophore A23187 were not modified by iodide in concentrations up to 0.1 mM. The effect of iodide was suppressed in the presence of methimazole (0.1 mM) but not NaClO4 (2 mM). Iodid (0.1 mM) did not inhibit the stimulation by carbamylcholine of PGF2 alpha and PGE2 release by rat pancreas in vitro. These data demonstrate a new action of iodide on the thyroid and establish a link among iodide metabolism, PG synthesis, and cholinergic action in the dog thyroid.