In this thesis, mechanistic studies of pediculicidal monoterpenoids focus on the phenolics, thymol, and carvacrol. Eugenol, a structurally similar insecticidaUy-active phenylpropanoid derived from the shikimic acid pathway, was also investigated. To date, mechanistic studies on the interaction o f these compounds with insect neuronal receptors have not been carried out. However, there are various studies that examine the effects of thymol and eugenol on vertebrate neurotransmission and/or cellular ionic composition; some o f the physiological effects of these compounds may be related to events at the molecular level.
Eugenol and oil of cloves are renowned for their anaesthetic qualities, especially when applied locally as analgesics. Eugenol is still widely used for this purpose in dentistry, being present in periodontal dressings and eugenates (cements containing eugenol), which are employed in potentially painful restorative dentistry. Eugenol anaesthetises mice when administered intraperitoneally [226], and suppresses the electrical activity o f some nerve or neuromuscular preparations, as expected from an anaesthetic agent: eugenol elicited
reversible inhibition o f the compound action potential o f rat phrenic nerve [35], and also extinguished impulse transmission in the bullfrog sciatic nerve [139].
In rat dorsal root ganglion (DRG) neurons, 1 mM eugenol activated both Ca^^ and Cl conductances [188] and, although at lower concentrations (0,1-2.5 mM) eugenol blocked K^-induced contracture o f toad skeletal muscle, higher concentrations (3-12 mM) induced contractures, probably via release of Ca^"^ from sarcoplasmic reticulum [146]. In guinea pig heart muscle, eugenol had a negative inotropic effect and reduced resting state contractions due to inhibition o f the Ca^^ current, probably via Ca^^ channel block; a conflicting effect o f current inhibition was also detected [227]. However, in one study involving in vitro and in vivo tests for anaesthetic action, the rat phrenic nerve hemidiaphragm preparation and rabbit conjunctival reflex, respectively, eugenol was inactive on both accounts [95]. The effects o f eugenol on electricaUy-active cells are clearly diverse. Despite having an inhibitory action on many preparations, eugenol elicits responses in olfactory receptor neurones distributed in the nasal cavity [31]. Olfactory neurons respond to volatile compounds as part o f their function in recognising potential food sources [165].
Furthermore, the enhancing action of eugenol at heterologously expressed vertebrate ion channels was investigated by injection o f rat whole brain mRNA into Xenopus oocytes. Ionotropic receptor-mediated GABA responses achieved after injection were potentiated by eugenol and also by the monoterpenoids pinene, citronellol and citronellal [285].
Thymol is used as a stabilising agent in halothane anaesthetic preparations, but is not considered anaesthetic itself; thymol is best known as an antimicrobial component of preparations such as mouthwashes and toothpastes. The local anaesthetic effects of thymol, and other monoterpenoids, have been studied in the rat phrenic nerve hemidiaphragm preparation, where (+)/(-)-menthol, but not thymol or (-)-menthone, increased the number o f stimuli needed to elicit a response, and in the rabbit conjunctival reflex test where both menthol isomers, but neither thymol nor (-)- menthone, suppressed responses [93]. However, these experiments were the same as those described in the previous paragraph in which eugenol was also found to be inactive, and therefore other tissues or experimental models may yield positive results. Other monoterpenoids that were active in these tests included terpineol and trans-anethole, inactive compounds included + /- citronellal, a-terpinene and (+)-carvone [95].
The activity o f thymol in releasing Ca^"^ from intracellular stores has also been well- documented. In neurones o f the snail Helixpomatia, thymol induces release o f stored Ca^"^
[138], and thymol is also known to release Ca^^ from sarcoplasmic reticulum vesicles, a property shared by menthol and also chloroform and halothane [195]. Furthermore, the dye thymol blue was shown to be a potent inhibitor o f InsPj (inositol 1,4,5 trisphosphate) binding activity [211].
Recently, the moUuscicidal activity of thymol was attributed to its effects on the activity of various cellular enzymes of the snail hymnaea acuminata^ thymol was found to have in vitro and in vivo acetylcholinesterase-inhibiting actions in this organism [232], and, after 96 h of exposure to sub-lethal thymol concentrations, 5-hydroxytryptamine (5-HT) and dopamine (DA) levels in vivo were also reduced. More new research has demonstrated the direct agonist activity o f thymol and six related phenolic compounds at a neuronal receptor, the rat a l (32y2 GABA^R, expressed in H EK (human embryonic kidney) cells. At these receptors, only compounds with a phenolic -OH attached directly to the benzene ring and aliphatic substituents in ortho position with respect to the phenolic -OH were able to elicit currents [176].
Carvacrol, like thymol, has been assayed on numerous occasions for its anti-bacterial activity, but evidence for biological properties that may influence neurotransmission, or indicate the existence o f a neuronal target, is rare. One study showed that both carvacrol and thymol were the main components contributing to the anti-spasmodic activity of Origanum compactum [254]. Again, the stimulatory actions o f carvacrol on sensory nerves have been documented: carvacrol and several other monoterpenoids were shown to induce fast wave bursts o f activity in the rat rhinencephalic cortex, an activity which appears to be a common feature o f many compounds that are anti-feedant towards herbivores, including another monoterpenoid, eucalyptol [283].
So far, numerous studies have indicated similarities in mechanisms o f action between monoterpenoids and anaesthetic compounds; many anaesthetics are also physicochemically similar to the mono terpenoids, being volatile and lipophilic. However, just as there are non-volatile anaesthetics there are also non-volatile monterpenoids with certain anaesthetic properties: the valepotriates, isolated from Valerian, possess sedative [261] and spasmolytic activity [260], the mechanism o f action is currently unknown although valepotriates bind to, and therefore may block, CNS dopamine receptors [121]. Furthermore, not aU monoterpenoids have anaesthetic-like biological activities; the essential oil o f wormwood has convulsant activity and also kills worms and insects, the most likely constituent
GABAR modulator: it gives poisoning signs in mice similar to those induced by
picrotoxinin, and the symptoms due to both PTX and a-thujone are alleviated by diazepam and phénobarbital; Drosophila melanogaster flies o f the 'Rdl strain (with the A302S mutation) are resistant to a-thujone as well as PTX; a-thujone is a competitive blocker o f ^H[EBOB] binding to mouse brain membranes; most conclusively, GABA-induced, Cl -mediated, peak currents in rat dorsal root ganglion neurones are suppressed (reversibly) by a-thujone [110]. These results suggest that vertebrate GABA^ and insect bicucuUine-insensitive GABARs may be simUarly susceptible to a-thujone inhibition. The effects o f a-thujone on heterologously expressed GABARs have not yet been investigated.
Summary and direction
Natural products have a firm place in the history o f human louse control. Among the chemicals o f this type that provide avenues for future exploitation as pedicuUcides, the monoterpenoids and related essential oil constituents appear promising. So far, the activity o f these compounds on human Uce and eggs has not been investigated extensively.
Particular monoterpenoids are already known to be especiaUy influential towards the viability o f certain other insects, but the mechanism by which monoterpenoid lethaUty is caused is not known. Many other insecticidaUy active compounds have a neuronal mode of action and there is already evidence that certain monoterpenoids are active at mammaUan GABA receptors.
In Chapter 2, the activity o f monoterpenoids against human Uce and their eggs wiU be explored. Later chapters wiU discuss a possible mode o f action at insect GABA receptors.