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ligands

The wide spectrum of structures that interact with GABA receptors encompasses both naturally-occurring and synthetic compounds. Many well-known GABA receptor ligands are native to plant and/or animal tissue and GABA itself is a significant component of the free amino acid pool in most pro- and eukaryotes. Although it is not known whether GABA performs a physiological role in plants, interestingly its synthesis is induced during stress conditions [228], and therefore could have a protective or reparatory role, or may be a precursor to compounds with such a function.

Benzodiazepines o f the type used as drugs in the treatment o f anxiety, sleep disturbances and seizures are found in mammalian tissue in trace amounts, although the possibilities for biological effect at the concentrations found are largely speculative [215]. The origin of these benzodiazepines is also in dispute: they may, in fact, originate from ingested microorganisms or plant material, both of which are known to synthesize them [100]; recendy the production o f benzodiazepines by plant tissue rather than by their associated flora was confirmed by observing the synthesis o f delorazepam and temazepam from sterile plant regenerates in tissue culture [133]. The positive aUosteric modulator ethanol is, of course, best known as a product o f yeast fermentation. Furthermore, many endogenous steroids and steroid metaboUtes modulate GABA^, such as the two progesterone

metaboUtes aUopregnanolone and 5a-pregnanediol, although so far no activity o f insect steroids, such as ecdysone, on insect GABARs has been noted [206].

Some of the most widely used tools for studying the GABAR are naturaUy-occurring: muscimol, a fuU agonist which binds with high affinity to GABA^ receptors, is found in the fungus Amanita muscaria (Fly Agaric); bicucuUine, an antagonist used to distinguish between the two major types o f ionotropic GABAR in both insects and mammals, is an isoquinoUne alkaloid found in species of Corydalis and Dicentra (Fumariaceae) [70]. Picrotoxin has been isolated from plants o f the moonseed famUy, Menispermaceae; it is a mixture of picrotin and picrotoxinin, the latter o f which is GABAR-active. The picrotoxinin pharmacophore, as predicted by molecular modeUing, was the basis for the successful insecticidal compound BIDN [209].

In the last few years, there have been increasing numbers o f investigations o f plant extracts and their constituents on GABARs, especially those derived from herbs having

physiological effects reminiscent o f other GABAergic compounds, such as pro-convulsant or hypnotic activities. Earlier studies commonly only explored binding activity, such as the numerous reports o f flavonoids binding to the GABA^-Hnked benzodiazepine receptor

[152,214,257]. Some studies focussed on the reversal of natural product-induced

physiological effects by application of known GABAergic ligands; Rubus brasiliensis Martis (Rosaceae) hexanic fraction has anxiolytic, hypnotic, anti-convulsant and muscle relaxant effects in mice, aU o f which are reversed by pre-treatment with flumazenil [186]. Only in recent years have studies on ethnopharmacologically interesting plants become more comprehensive and included binding, electrophysiological and behavioural experiments. The majority o f these studies have been carried out on small mammals an d /o r mammalian tissue; there are, at present, far fewer investigations o f natural products, insecticidal or otherwise, at insect GABA receptors. A selection o f the work on GABAergic natural products is given below and comprises plants and compounds with both convulsant and benzodiazepine-like properties.

Water hemlock, Cicuta virosa (UmbeUiferae) is toxic to mammals, causing convulsions and respiratory paralysis. It contains several chemicals which inhibit the binding o f fH]EBOB (ethynylbicycloorthobenzoate), a GABAR antagonist, to rat brain cortex, and the efficacy o f inhibition was positively correlated with toxicity [252]. One o f these toxins, virol A, inhibited GABA induced Cl flux in rat hippocampal CAl neurones, apparently acting both at the GABA binding domain and the PTX site in the channel [253]. Anisatin, a toxic and insecticidally active component of the Sikimi plant was studied electrophysiologically by whole and single cell patch-clamp on rat dorsal root ganglion; suppression o f Cl flux was only attained during or after application o f GABA, not before, and PTX attenuated anisatin-suppression o f GABA-induced currents; in binding studies anisatin inhibited [^H]EBOB binding. Both lines o f evidence suggest that anisatin acts at the PTX site in the channel [126]. The alkaloid ricinine, from BJanus communis^ was identified as having a novel mode o f inducing seizures, via the benzodiazepine site rather than the via convulsant site; induction o f seizures in mice was blocked by diazepam (but not phenobarbitone), and ricinin blocked fH] fiunitrazepam binding to cerebral cortex membranes [85].

St John’s Wort (SJW), Hypericum perforatum L. (Hypericaceae), is used for many purposes in herbal medicine, such as for insomnia, and its anti-depressant activity has been proven in clinical trials. Hypericin used to be considered the only active principle. However, recent

studies o f total SJW extract showed an anxiolytic activity in mammals which was blocked by pre-treatment with flumazenil; in electrophysiological studies on GABA^^ receptors, hypericin reduced GABA activated Cl currents, whereas pseudohypericin, another SJW constituent, enhanced them. Pseudohypericin is therefore more likely to be responsible for the anxiolytic effect o f SJW extract than hypericin [255].

Kava is an intoxicating beverage prepared from the pepper Piper methjsticum^ it is widely consumed by people native to the South Pacific island. It’s most popular use in traditional medicine is as an anxiolytic, and in this respect it is reported to perform as effectively as benzodiazepines. The kavalactones (also known as kavapyrones) were reported to be the active ingredients, as four o f these showed significant anaesthetic and analgesic effects. However, an apparently non-kavalactone-related GABA^ binding activity from Hawaiian kava has been described in methanolic leaf extracts [77,80].

The bark from the roots and stems of various Magnolia species are used in Traditional Chinese Medicine to treat a variety of disorders including anxiety and nervous disturbances. Honokiol and magnolol are major constituents o f this material; both o f these biphenolic compounds have anxiolytic and CNS depressant effects in mammals and both were found to potently enhance [^H]muscimol binding to various mammalian CNS tissues [4,239]. As propofol, a monophenolic, also increases muscimol binding at GABARs, honokiol and magnolol may also potentiate the GABA-induced Cl' current at GABA^Rs, but this effect has yet to be studied.

Extracts o f Valerian, the common name given to the crude drug made from underground organs o f plants from the species Valeriana (Valerianaceae), are used in folklore medicine for their sedative, hypnotic, tranquiiising, and anti-convulsive effects. There appears to be a dual activity with relation to their GABAR activity, as low concentrations o f extracts enhance [^H] fiunitrazepam binding to GABA^Rs but higher concentrations inhibit it [191].

Ginkgo biloba and ginseng are herbal drugs reputed to have remedial effects on various neurological disorders. Bilobalide is a sesquiterpene isolated from the leaves o f Ginkgo biloba L.; it has anti-depressant qualities, shortens pentobarbital-induced sleeping-time in mice [34], enhances the excitability of rat CAl pyramidal neurones and reduces the neuronal inhibitory actions o f muscimol [218]. The major component o f Vietnamese ginseng saponin, majonoside-RZ has a preventative effect against psychological stress induced lipid peroxidation in mouse brain, an effect that was blocked by flumazenil and

pregnenolone sulfate (a negative aUosteric GABA^^ modulator) [278] suggesting an action via the benzodiazepine site.

The dried flowers o f Matricaria chamomilla L, (a species o f chamomile) are used in herbal preparations to provide sedative and spasmolytic effects. A study o f one o f its biological constituents revealed confUcting bioactivities: the flavonoid apigenin was isolated and found to act as an anxiolytic and a sUght sedative in mice, but did not have anticonvulsant or myo-relaxant activities. It also displaced fH] fiunitrazepam from central benzodiazepine sites [257]. However, electrophysiological analysis revealed that apigenin reduces GABA activated Cl currents in cultured cerebeUar granule ceUs, and this was reversed by flumazenil, suggesting a negative modulatory activity via the GABA^i^R-Unked benzodiazepine binding site [13].

O f course, not aU GABAergic activity is via GABA receptors and studies into natural products have also addressed this. For example Caesalpinia sappan wood extracts have anti­ convulsant properties; further investigation revealed two compounds, sappanchalcone and braziUn, both o f which were found to inhibit GABA degradative enzymes [15].