Dioscorine

Origin and uses

Dioscorine was first isolated from the tubers of Dioscorea hirsute by Boorsma in 1894, and the tubers of Dioscorea hispida by Levya and Gutierrez in 1937. It was obtained in a crystalline condition by Schutte. In tropical lands, tubers from varieties of these species are eaten, but the alkaloid-bearing species are of toxicological interest because of their poisoning abilities. Dioscorine produces insecticidal and antifeedant responses in various species of insects, but has more interesting historical applications. These are dependent on the geographical location of the specific tuber (Table 1). Poisoning from dioscorine first appeared from accidental food poisoning from the yam, especially during periods of severe drought in many parts of Africa. People then began making the distinction between edible and toxic plants, and put the toxins to use in hunting. Cases of poisoning have officially been reported since the 1930s but had been happening earlier.

Chemical Properties

Dioscorine is an alkaloid with a 6-membered nitrogen-containing heterocycle. Pinder extensively discussed the method of extraction of and the chemical substitution of dioscorine (Figure 1). From his studies, Pinder also concluded that 2-oxotropane is a degradation product of dioscorine and described the formula of the alkaloid.

Dioscorine derives its basic nature and nucleophilicity from the tertiary amine and carbonyl functional groups.

Dioscorine is completely soluble in a number of hydrophilic solvents (water, ethanol, acetone) but only slightly soluble in hydrophobic and mostly polar solvents (chloroform, ether, benzene, petroleum ether).

Alkaloids are generally pale yellow liquids with an aromatic smell. Dioscorine is opalescent, that is, it appears yellowish-red in transmitted light and blue in scattered light perpendicular to the transmitted light.

Biosynthesis

Dioscorine is one of few alkaloids to possess an isolated isoquinuclide nucleus that is not part of a condensed ring system, unlike catharanthine or other indole alkaloids. Attempts towards elucidating the structure of the molecule used the lactone ring as a starting point. Chemists typically injected into leaves of tubers radioactively labeled precursor molecules and tracked how these got incorporated into the molecule after dioscorine was synthesized in-situ. Degradation experiments involving the analysis of radioactive fragments thus generated were important in putting chemical blocks together in a temporal manner, and in obtaining the exact sequence of the mechanism.

The initial hypothesis was that dioscorine was formed by condensation between piperidine (formed from the amino acid lysine in higher plants) and a branched eight-carbon unit derived from four acetate units. This was however disproved by partial degradation results leading researchers to speculate that nicotinic acid served as precursor to dioscorine in Dioscorea Hispida.

Leete at al further refined this model in 1989, and showed that it was trigonelline (nicotinic acid methylated at the nitrogen) that served as an intermediate in the biosynthesis of dioscorine. Trigonelline enhances the nucleophilic nature of nicotinic acid. Nucleophilic attack of trigonelline was a known reaction at that time. A series of reactions involves reduction, production of a beta imino acid, and decarboxylation. The last synthetic step leading to the generation of dioscorine is the reaction of an iminium ion with lactone.

As with any biosynthesis reproduced in lab, not benefiting of enzymatic action, side products are bound to form. These yield from the reduction of the ketone and the tetrahydropyridine ring. Further lactone formation gives another side product, dumetorine, which is an alkaloid that can be isolated from Dioscorea dumetorum.

The key steps of the biosynthesis have been highlighted in figure 2. A complete step-wise reaction including an arrow-pushing mechanism can be found in Leete’s paper.

Biological Effects

Dioscorine is a neurotoxin. It acts as an antagonist of the nicotinic acetylcholine receptor (nAChR) by physically blocking an open ion channel, leading to hyperpolarization of the neuron. Nagata et al. studied the effects of dioscorine on the nicotinic acetylcholine receptor in rat clonal phaeochlomocytoma cells (mixture of neuroblasts and eosinophils). They found that dioscorine at concentrations of 0.45-450 μM accelerated the desensitization of current induced by 100 uM acetylcholine, suppressing the current in a dose-dependent manner. Dioscorine itself did not induce any current at concentrations between 0.45 and 450 μM, suggesting that it might act as an antagonist for the nAChR (as opposed to agonist or inverse agonist). Co-application of dioscorine and acetylcholine at the surface of the ion channel decreased the mean open time and mean closed time, as well as the duration of the current burst. These changes in single-channel kinetics by dioscorine significantly reduce the total charge carried through the open channels, explaining the suppressive effect of dioscorine on the nAChR, and its toxicity.

At the molecular level, dioscorine enters and physically blocks the ion channels when they are open, causing a conformational change in the channel proteins. This increases the affinity of dioscorine for its binding site. The ion channels involved are normally those associated with the N-methyl-D-aspartate (NMDA) and GABA receptors that are modulated by Ca²⁺ ions. The Ca²⁺ ions enter through the nAChR in the presypnatic membranes. Therefore, apart from physical blocking of the ion channel, dioscorine could also be indirectly inhibiting the activity of ion channels through the secondary messenger system mediated by Ca²⁺ ions and a cascade of various synaptic events.

Symptoms

In humans, physiological responses range from dizziness, nausea, vomiting and sleepiness. At large doses, convulsions result, and death usually occurs in extensor spasms. The interaction of dioscorine with the nAChR also results in local anesthetic effects: dioscorine in 0.5% solution has approximately the same activity as 0.05% cocaine. Dioscorine also shows antidiuretic activity and depressant actions.

Toxicity

Dioscorine is reported to be one of the most potent alkaloid toxins isolated from yam. It has an LD₅₀ of 60 mg/kg in mice through an intraperitoneal route of administration. When injected into monkeys, it has a mydriatic action (that is, it causes the pupils to dilate), and resembles the pharmacological action of picrotoxin and cardiac glycosides.

Diagnostic tests

Van Itallie and Bylsma, in 1930, described the following chemical tests for dioscorine:

1) A solution of this alkaloid in sulfuric acid turns yellow when a small amount of iodic acid is added to it. From the edge, the yellow color changes slowly to reddish-violet. Which in turn changes to bluish-violet.

2) When a drop of diluted solution of sodium nitroprusside and a few drops of sodium hydroxide are mixed with dioscorine, a reddish-violet color appears after a short while.

3) If dioscorine is heated with sulfuric acid on a water bath, a reddish-violet color appears slowly.

Treatment (Antidote)

Since dioscorine is as a cholinergic receptor ligand, any stronger agonist of the nAChR can serve as valid antidote of dioscorine. If added in a concentration higher than dioscorine, it can competitively displace the latter from the receptor. Several developed antidotes are aza-bridged bicyclic amine derivatives.

An anesthetic, pentobarbital sodium, was often administered to mice during toxicity experiments involving dioscorine. Convulsions in humans can be readily antagonized with this compound.