Physostigmine extraction

Materials required The cholinesterase preparation (biotests or simple extracts from biological tissues), the reagents for a reaction medium choosen (see section 3.2.), alkaloid physostigmine salycilate and its derivative neostigmine ("Sigma"). 

Frolich and colleagues (1998) analyzed ACh in human CSF by different methods, which included thermospray/mass spectroscopy, HPLC/mass spectroscopy, HPLC-EC Pt electrode and gas chromatogra-phy/mass spectroscopy (GC/MS). An SPE extraction was used for cleanup and concentration. Samples were run with and without the IMER to rule out any interference by physostigmine, a cholinesterase inhibitor, in the HPLC-EC assay. HPLC-EC and GC-MS gave data correlations with similar sensitivities, but the HPLC-EC values were 39% lower. Analysis using thermospray/mass spectroscopy and HPLC/ mass spectroscopy did not provide adequate sensitivity and the data obtained were inconsistent. 

The classic cholinesterase inhibitor is the alkaloid physostigmine (6)/ eserine (7). It was first isolated from the calabar bean, the seeds of Physostigma venenosum. Many novel natural products with AchE-inhibifing properties have shown promise as therapeutics for AD. Some examples of such products (pure compounds/plant extracts or formulations) are briefly reviewed here. 

The classic cholinesterase inhibitor is the alkaloid physostigmine (33), also called eserine. Investigations carried out in the nineteenth century on the ordeal poison esere, which consisted of an extract of the Calabar Bean, the seeds of Physostigma venenosum Balf, resulted in the isolation of (33)... 

Extracts of plants have been used as insecticides by humans since before the time of the Romans. Some of these extracts have yielded compounds useful as sources (e.g., pyrethrins, rotenoids, alkaloids), others as models (e.g., pyrethrins, physostigmine) of commercial insecticides. Recent technological advances which facilitate the isolation and identification of the bioactive constituents of plants should ensure the continued usefulness of plant compounds in commercial insect control, both as sources and models of new insect control agents and also as components in host plant resistance mechanisms. The focus in this paper will be on several classes of compounds, including limonoids, chromenes, ellagitannins, and methyl ketones, which were found to be components of the natural defenses of both wild and cultivated plants and which may be useful in commercial insect control. 

A series of tricyclic indole alkaloids (physostigmine alkaloids) has been obtained from the cheilostome bryozoan Flustrafoliacea (90). In addition, flustramide B (83) and flustrarine B (84) were isolated from this bryozoan (91). Flustrarine B (84) was prepared from previously known fiustramine B (85) (92) via oxidation with hydrogen peroxide. Five fiustramine derivatives, dihydroflustramine C (86) and its N-oxide (87), fiustramine D (88) and its N-oxide (89), and isoflustramine D (90), were isolated from the methylene chloride fraction of the aqueous methanol extract of a Canadian F. foliacea, and these alkaloids were found to be responsible for the antimicrobial activity of the extract (93). Oxidation of dihydroflustramine C (86) and fiustramine D (88) with m-chloroperbenzoic acid afforded the corresponding A-oxides (87 and 88, respectively). 

In the mid-19th century the Calabar bean was sent to Scotland and the two principal alkaloids were extracted in a number of European laboratories. There the formulae were determined and toxic and medicinal properties intensively studied. By the early 20th century the formula for physostigmine and the presence of two... 

Upon Julian s return to the United States, DePauw University offered him a faculty position and research support. His first project was the total synthesis of physostigmine, an indole alkaloid extracted from the Calabar bean, used in the treatment of glaucoma. Sir Robert Robinson at Oxford University in England was also working on this synthesis. Julian achieved the synthesis of physostigmine, and showed that Robinson s hypotheses about its structure were wrong. 

Lawrence, G.D. Yatim, N. Extraction of physostigmine from biologic fluids and analysis by liquid chromatography with electrochemical detection. J. Pharmacol. Meth. 1990, 24, 137-143. 

F. foliacea also occurs in the Bay of Fundy, Canada. Bioassay-guided fractionation based on antibacterial activity of the extract of this bryozoan has lead to the isolation of a different series of bromoal-kaloids of the physostigmine class, two of which were N-oxides (39]. The major alkaloids were dihydroflustramine C, 90, ( 60%), which had been reported previously by these authors [40) and flustramine D, 91, (-40%). Minor alkaloids found were dihydroflustramine C N-oxide, 92. flustramine D Af-oxide, 93, and isoflutramine D, 94. The JV-oxides were also synthesised by oxidation of the corresponding free bases with m-chloroperbenzoic acid. These alkaloids were shown to be responsible for the wide spectrum of antibacterial activity exhibited by this bryozoan extract. 

Polak Well, this is where our results obtained with cortical slices from rat brain seem to be different from your results. We always used a cholinesterase inhibitor but our results depended on the choice of the cholinesterase inhibitor. When the organo-phosphorous compound Soman was used, stimulation of the release and synthesis of ACh by atropine was followed by an increase in the amounts of ACh which could be extracted from the tissue after incubation. This increase appeared to be caused by partial re-uptake of the relatively large amounts of ACh released under the influence of atropine. We found that physostigmine largely prevented this re-uptake With physostigmine in the medium, atropine also stimulated the release and synthesis of ACh but now these effects were accompanied by a decrease in the total extractable ACh content of the tissue. 

Possibly the earliest recorded use of a substance that works, like nerve agents, by inhibiting cholinesterase (ChE) is by native tribesmen of western Africa who used the Calabar bean as an ordeal poison in witchcraft trials.1,2 An extract, the elixir of the Calabar bean, was later used medicinally,3 and in 1864, the active principle was isolated by Jobst and Hesse and called physostigmine.1 Vee and Leven independently isolated this same substance in 1865 and named it eserine,1 hence its dual nomenclature. 

Pilocarpine is used principally as a diaphoretic, that is, to induce sweating, especially in nephritis, to relieve the kidneys and to remove toxic metabolites. The secretion of 3 liters of sweat can easily be achieved, and as much as 8 g. of nitrogen can be eliminated. The alkaloid is also employed as a milder substitute for physostigmine in the treatment of ocular diseases. Pilocarpine is reputed to stimulate the growth of hair, and jaborandi extracts are therefore employed to some extent as constituents of hair lotions. 

Arundinine (127), was obtained from the epigeal parts, and the strueture was established by spectroscopic methods as well as by X-ray analysis. The moleeule is constituted from the union of two tryptamine moieties, a physostigmine-type unit and another tryptamine unit. The two units are linked by an ether bridge from C(3a) of the former to the aromatic C(5 ) of the other unit (79). A number of related bisindoles have been subsequently isolated from the root extract of the same plant, viz., arundamine (128) (80,81), arundacine (129) (82), arundanine (130) (83), arundavine (131) (84), arundaphine (132) (85), and arundarine (133) (86). The structures of 128,131, and 132 have also been confirmed by X-ray analysis, while the NMR spectrum of arundacine (129) showed the existence of two equilibrating con-formers due to restricted rotation about the amide C-N bond. 

Table 2 Physostigmine-internal standard ratio in 0.1 ml fractions collected from five solid-phase extraction columns...

P.R. Hurst and R. Whelpton, Solid phase extraction for an improved assay of physostigmine in biological fluids, Biomed. Chromatogr., 1989, 3, 226-232. 

Plant-originated anti-ChEs, such as huperzine A, have been used for thousands of years in the treatment of aging-induced memory impairment (Haviv et al., 2007). In 1877, prior to the discovery of ACh as a neurotransmitter in the brain, physostigmine (eserine)— a carbamate extracted from the seeds of Physostigma venenosum—became the first cholinesterase inhibitor used therapeutically to control increased ocular pressure caused by wide-angle glaucoma (Taylor, 1996). 

Physostigmine (a naturally occurring carbamate anticholinesterase compound extracted from the Calabar bean) may be used as an antidote to the effects of BZ. It should be administered intravenously with care in severely affected patients in doses of 2-4 mg given at hourly intervals. 

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