Psychoactive Plant Database - Neuroactive Phytochemical Collection

1. Anal Chim Acta. 2010 Nov 8;680(1-2):32-40. doi: 10.1016/j.aca.2010.09.018.
Epub  2010 Sep 21.

High-performance liquid-chromatographic tandem-mass spectrometric methods for 
atropinesterase-mediated enantioselective and chiral determination of R- and 
S-hyoscyamine in plasma.

John H(1), Eyer F, Zilker T, Thiermann H.

Author information:
(1)Bundeswehr Institute of Pharmacology and Toxicology, Department of Analytical 
Chemistry, Neuherbergstrasse 11, 80937 Munich, Bavaria, Germany. 
HaraldJohn@bundeswehr.org

S-hyoscyamine (S-hyo) is a toxic tropane alkaloid from plants of the solanacea 
family, which is extracted for pharmaceutical purposes thereby undergoing 
racemization (atropine). Merely the S-hyo enantiomer acts as an antagonist of 
muscarinic receptors (MR). Nevertheless, racemic atropine is clinically 
administered in e.g. ophthalmology and for symptomatic therapy of acute 
poisoning with organophosphorus compounds (OPCs, e.g. pesticides, nerve agents). 
However, very limited data are available of comparative pharmacokinetics of S- 
and R-enantiomers in humans or other species. Therefore, we developed an 
enantioselective LC-ESI-MS/MS assay making use of rabbit serum containing 
atropinesterase (AtrE, EC 3.1.1.10) which is suitable for stereospecific 
hydrolysis of S-hyo into tropine and tropic acid while R-hyo is unaffected. For 
sample preparation plasma was incubated with human serum (not containing AtrE, 
procedure A) and with rabbit serum (procedure B). Afterwards, hyoscyamines were 
quantified by a validated previously published non-chiral LC-ESI-MS/MS method. 
Following procedure A the concentration of total hyo and following procedure B 
remaining R-hyo were determined. S-hyo was calculated by the difference between 
these concentrations. This assay design allowed reproducible, precise (RSD 
2-9%), accurate (93-101%) and selective determination of total and individual 
hyoscyamines. Potential therapeutics for OPC poisoning (carbamates, oximes) and 
thiono-pesticides did not interfere with the assay whereas some oxon-pesticides 
inhibited S-hyo hydrolysis. A control experiment was designed allowing to be 
aware of such interferences thus avoiding the use of false results. To validate 
this assay, results were compared to those from a novel isocratic chiral 
LC-ESI-MS/MS method. Separation of S-hyo (t(R) 31.1 ± 0.2 min) and R-hyo (t(R) 
33.4 ± 0.2 min) was achieved on α-glycoprotein (AGP) chiral stationary phase at 
40°C (selectivity factor α 1.07). Ammoniumformate (0.01 M, pH 8.0) with 3.75% 
(v/v) acetonitrile served as mobile phase (300 μL min(-1)). Hyoscyamines were 
detected in the positive multiple reaction monitor mode. The enantioselective 
assay was applied to the analysis of atropine degradation in diluted rabbit 
serum in vitro as well as to human in vivo plasma samples from a 
pesticide-poisoned patient treated with atropine.

Copyright © 2010 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.aca.2010.09.018
PMID: 20969988 [Indexed for MEDLINE]


2. Naunyn Schmiedebergs Arch Pharmacol. 2006 Jun;373(3):230-6. doi: 
10.1007/s00210-006-0054-5. Epub 2006 May 3.

The presence of atropinesterase activity in animal plasma.

Harrison PK(1), Tattersall JE, Gosden E.

Author information:
(1)Department of Biomedical Sciences, Dstl, Porton Down, Salisbury, Wiltshire, 
SP4 0JQ, UK. pkharrison@dstl.gov.uk

The enzyme atropinesterase (EC 3.1.1.10) causes the rapid hydrolysis of tropane 
alkaloids such as atropine and scopolamine. This enzyme is known to occur in a 
certain proportion of rabbits and some plants, although its presence in other 
animal species remains controversial. The potential presence in some animals but 
not others of an enzyme which can rapidly hydrolyse compounds such as atropine 
is a potential unwanted experimental variable in many experiments. Because of 
the uncertainty surrounding the enzyme and the paucity of data, it was decided 
to examine whether we could detect and characterise atropinesterase activity in 
the plasma of dogs, goats, guinea-pigs, humans, pigs, rabbits and rhesus by 
separating and quantitating the substrate (atropine) and one of the products 
(tropic acid) by high performance liquid chromatography (HPLC). It was found 
that plasma from some but not all rabbits possessed a capacity to breakdown 
large quantities of atropine; an effect that was apparently enantiomer-specific. 
Plasma from other rabbits, and plasma from all other species investigated, 
proved capable of hydrolysing atropine at a rate exceeding that of non-specific 
breakdown. It remains to be determined whether this effect is due to a low 
expression of atropinesterase or an alternative hydrolysing enzyme.

DOI: 10.1007/s00210-006-0054-5
PMID: 16736160 [Indexed for MEDLINE]


3. Drug Chem Toxicol. 1992;15(2):127-43. doi: 10.3109/01480549209032295.

Acetylcholinesterase inhibition and anti-soman efficacy of homologs of 
physostigmine.

Harris LW(1), Anderson DR, Pastelak AM, Bowersox SL, Vanderpool BA, Lennox WJ.

Author information:
(1)US Army Medical Research Institute of Chemical Defense, Aberdeen Proving 
Ground, MD 21010-5425.

Inhibition of acetylcholinesterase (AChE) activity by physostigmine (PHY) is 
reversible due to spontaneous decarbamylation. Physostigmine has been shown to 
be effective as a pretreatment against potent anticholinesterase poisons (e.g., 
soman) in experimental animals, yet it is short acting and causes undesirable 
side effects in mammals. The two-fold purpose of this study was 1) to determine 
whether extension of the N-substituted alkyl chain (N-SAC) of PHY from N-methyl 
to N-ethyl (I), N-propyl (II), N-isopropyl (III), N-butyl (IV) or N-heptyl (V) 
affects anti-AChE potency and spontaneous decarbamylation of inhibited AChE of 
guinea pig blood in vitro and in vivo, and 2) to see whether chain extension 
affects efficacy as pretreatment in poisoning by soman. The in vitro AChE 
inhibition studies were done using whole blood incubated at 37 degrees C for 30 
min. All 5 homologs possessed anti-AChE activity with I50s ranging from 1.1 to 
27.6 x 10(-7)M; compound III was the least potent in vitro and in vivo. 
Lengthening of the N-SAC of PHY markedly extended the duration of anti-AChE 
activity when compared to PHY, but rendered the modified compounds ineffective 
as pretreatments against soman. These data support the premise that the decrease 
in decarbamylation rates observed upon extending the N-SAC of PHY is responsible 
for the loss of effectiveness of pretreatment regimens against soman. Perhaps, 
these homologs of PHY may have potential use in instances where sustained action 
of acetylcholine is required at cholinergic junctions because of disease 
conditions or drug overdosage.

DOI: 10.3109/01480549209032295
PMID: 1597127 [Indexed for MEDLINE]


4. Biochim Biophys Acta. 1987 Apr 8;912(2):167-77. doi: 
10.1016/0167-4838(87)90085-9.

Hydrodynamic characterization of the size and shape of atropinesterase from 
Pseudomonas putida.

van der Drift AC, Sluiter W, Berends F.

Atropinesterase from Pseudomonas putida has been investigated by means of 
different ultracentrifugation methods under native and denaturing conditions. 
The following quantities were determined: sedimentation coefficient, 
translational diffusion and friction coefficient, partial specific volume and 
molecular weight. From these data the size, shape and hydration of the enzyme 
molecule in solution were estimated. The results suggest that atropinesterase is 
a globular protein which consists of a single polypeptide chain with a molecular 
weight of about 30,000. In solution under non-denaturing conditions, it occurs 
mainly as a dimer which hydrodynamically behaves as a rigid impenetrable 
particle. Calculations based on the spheroid model indicate that this particle 
resembles a hydrated sphere with a diameter of 6.1 +/- 0.2 nm and a hydration of 
0.4 +/- 0.1 g of H2O/g of protein rather than a significantly less hydrated 
ellipsoid of revolution. Under denaturing conditions dissociation into monomers 
takes place. The effects of sodium dodecyl sulphate (SDS) on size and shape 
suggest that dimerization results from side-by-side association of two elongated 
monomers rather than from end-to-end association. Approximately 57 molecules of 
SDS are bound per dimer before dissociation occurs concomitant with the 
additional binding of about 19 molecules of detergent.

DOI: 10.1016/0167-4838(87)90085-9
PMID: 3828356 [Indexed for MEDLINE]


5. Naunyn Schmiedebergs Arch Pharmacol. 1987 Feb;335(2):103-8. doi: 
10.1007/BF00177709.

The effects on Schild regressions of antagonist removal from the receptor 
compartment by a saturable process.

Kenakin TP, Beek D.

A theoretical model of the effects of a saturable removal mechanism for an 
antagonist diffusing into the receptor compartment of a tissue is used to 
calculate expected deviations in Schild regressions. At concentrations of 
antagonist which do not saturate the removal mechanism, there can be a deficit 
of antagonist in the receptor compartment as compared to the concentration of 
antagonist bathing the tissue. This results in a shift to the right of the 
Schild regression and a corresponding underestimation of antagonist potency. The 
model predicts that as the concentration of antagonist exceeds the Km for 
removal (saturation of the removal process), this concentration deficit is 
eliminated, resulting in a proportionate increase in antagonist concentration at 
the receptor and a concomitant increase in receptor antagonism. This results in 
a steepening of the Schild regression; the slope in the region of saturation is 
greater than one. Experimental evidence in support of this model was found in 
studies of the antagonism of responses to bethanechol by atropine in rabbit 
ileum; this species is known to have an atropinesterase capable of hydrolyzing 
atropine. The Schild regression for atropine was curvilinear with an overall 
slope of 1.42 (1.34-1.5) and pKB = 8.5 (8.36-8.8); in the ileum from guinea 
pigs, a species which does not possess this enzyme, the Schild regression for 
atropine was linear, had a slope not significantly different from unity (1.1; 
0.95-1.2) and a pKb of 9.0 (8.9-9.2). The slope of the regression in rabbit 
ileum was corrected to unity by the addition of an excess concentration of 
methylbutyrate, an alternate substrate for atropinesterase.(ABSTRACT TRUNCATED 
AT 250 WORDS)

DOI: 10.1007/BF00177709
PMID: 3561525 [Indexed for MEDLINE]