Toxicity and behavioural effects of diet-borne alkaloids on larvae of the black blowfly,Phormia regina

Larvae of the black blowfly, Phormia regina (Meigen) (Diptera: Calliphoridae) were exposed for 24 h to artificial diets that contained one of the following alkaloids: arecoline, caffeine, nicotine, quinine, sparteine or strychnine at either 1000 or 100 p.p.m. Each of the alkaloids caused reduced weight gain, relative to a control population in a no-choice bioassay and, with the exception of quinine, all alkaloids caused reduced larval weights in a choice bioassay. Larvae were unable to move away from diets containing arecoline (1000 and 100 p.p.m) and congregated away from diets containing 1000 p.p.m. quinine. Arecoline (1000 p.p.m) and both concentrations of nicotine caused significant mortality of larvae. Over a longer period (120 h), 10 and 1 p.p.m. nicotine resulted in significant numbers of larvae congregating away from a treated diet. Ten p.p.m. nicotine caused reduced weight gain over 120 h, although larvae provided with a choice were less affected. Exposure of larvae to dried residues of nicotine for 2 h did not affect subsequent development.     

Toxicity of quinoline alkaloids to cultured Cinchona ledgeriana cells

The toxicity of Cinchona alkaloids to cell cultures of C. ledgeriana has been studied in relation to alkaloid uptake and possibilities for selecting high-yielding cell lines. The most toxic, quinine, was completely toxic at 5.5 mM. Both quinine and quinidine were more toxic than their unmethoxylated precursors, cinchonidine and cinchonine. The permanently-charged metho-chlorides of quinine and cinchonidine were less toxic than the parent alkaloids, despite showing similar accumulation ratios in 5-day uptake experiments at sub-toxic concentrations (ca 1.7mM). The toxicity of the natural quinoline alkaloids appears to be a non-specific effect which may be caused by intracellular alkalinisation following uptake of the uncharged bases. The use of precursors of quinine and quinidine as toxic agents for the selection of cell lines with enhanced quinine and quinidine production is ruled out by the lower toxicity of these precursors and by the correlation of an apparently non-specific toxicity with uptake.     

Organ-specific distribution of dietary alkaloids in the marine opisthobranch Tylodina perversa

Specimens of the opisthobranch Tylodina perversa that were observed while feeding on the sponge Aplysina aerophoba were transferred to seawater tanks along with their prey and kept under controlled conditions. After one week the opisthobranchs were anaesthetized, dissected and studied for sequestered sponge-derived brominated alkaloids. All parts of T. perversa analyzed including feces, mucus and egg masses that had been produced during captivity contained alkaloids derived from A. aerophoba. The highest total alkaloid concentration (24.6 mg g⁻¹ dry wt) was found in mantles of T. perversa (compared to 51.2 mg g⁻¹ dry wt of total alkaloids in A. aerophoba). Hepatopancreas, egg masses and mucus (respective total alkaloid concentrations ranging from 20.4 to 12.5 mg g⁻¹ dry wt) were also rich in alkaloids. Whereas in A. aerophoba the isoxazoline alkaloids aerophobin-2 and isofistularin-3 were present in almost equal concentrations, aerophobin-2 constituted by far the major alkaloid (amounting to approximately 70% of all identified alkaloids) in mantles, mucus and egg masses of T. perversa, indicating selective sequestration by the opisthobranchs. Mantles as well as mucus also contained appreciable concentrations (approximately 20% of all identified compounds) of the brominated alkaloid aerothionin; this is not detected in A. aerophoba. It is possible that aerothionin originates from a previous encounter of T. perversa with the sponge A. cavernicola, the latter being closely related to A. aerophoba. The enrichment of aerophobin-2 (and of aerothionin) in mantles, mucus and egg masses that are vulnerable and exposed (mantles and egg masses) to predators and/or pathogens argues for defensive functions of the respective alkaloids even though this hypothesis still needs to be experimentally corroborated.     

An improved synthesis of 10,11-didehydro Cinchona alkaloids

A revised procedure for the conversion of the four major Cinchona alkaloids (quinine, quinidine, cinchonidine, and cinchonine) into their respective 10,11-didehydro derivatives is described. The reported protocol offers several advantages over a recently published synthetic route. These include (i) enhanced robustness (ii) ready scalability (iii) reduced operational complexity and number of steps (iv) chromatography-free work-up. In addition, toxic solvents were replaced by environmentally less problematic alternatives.     

Conformational spaces of Cinchona alkaloids

A systematic and comprehensive study of the conformational spaces of the Cinchona alkaloids quinine, quinidine, cinchonine, cinchonidine, epiquinine, epiquinidine, epicinchonine, and epicinchonidine using the semiempirical PM3 method is described. The results were analyzed in terms of syn/anti and open/closed/hindered and alpha/beta/gamma conformations. Special emphasis was given to the torsion angles T(1) (C(4a')-C(4')-C(9)-C(8)), T(2) (C(4')-C(9)-C(8)-N(1)) and T(3) (H-O(9)-C(9)-C(8)) that define the backbone and the hydroxy conformation, respectively. The results reveal the quasi-enantiomeric relationships between quinine and quinidine and between epiquinine and epiquinidine, and the main structural differences that exist between the therapeutically active Cinchona alkaloids, quinine and quinidine, and their inactive epimers, epiquinine and epiquinidine. The lowest energy conformation of quinine and quinidine is anti-closed-alpha. The lowest energy conformations of epiquinine and epiquinidine are anti-open-beta and anti-open-alpha, respectively. Low energy conformations with an intramolecular hydrogen bond (N(1.)H(.)O(9)) were found in epiquinine (the global minimum) and epiquinidine, but not in quinine and quinidine.     

A novel action of quinine and quinidine on the membrane conductance of neurons from the vertebrate retina

The cinchona alkaloids quinine and quinidine have been shown to block a broad range of voltage-gated membrane conductances in a variety of excitable tissues. Using the whole-cell version of the patch clamp technique, we examined the effects of these compounds on voltage- dependent currents from horizontal cells dissociated enzymatically from the all-rod retina of the skate. We report here a novel and unexpected action of quinine and quinidine on isolated horizontal cells. In addition to blocking several of the voltage-activated currents of these cells, the introduction of the alkaloids evoked a large outward current when the cells were held at depolarized potentials. Using tail current analysis, the reversal potential of the outward current was close to O mV, and the current was markedly suppressed by extracellularly applied cobalt, acetate, and halothane. Depolarization in the presence of quinine also permitted entry into the cells of extracellularly applied Lucifer yellow (MW = 443 D), whereas a 3-kD fluorescein-dextran complex was excluded. These findings suggest that the large, apparently nonselective conductance induced by quinine and quinidine results from the opening of hemi-gap junctional channels.     

Berberine contents and alkaloid profile of Berberis species from different altitudes

Roots, stem and leaves of eight species of Berberis collected from different altitudes in Garhwal, Himilaya, were analysed for berberine. Lowland species and types were found to have considerably higher berberine contents in all plant parts than sub-alpine and alpine species. In all, 12 or more alkaloids were separable in these species. Based on the similarity coefficient of leaf alkaloids, B. nepalensis was found to be identical to B. asiatica and B. vulgaris was identical to B. lycium. It is suggested that the concentration of berberine is probably a result of adaptive response to altitudinal gradient in addition to it being a genetic character of different species of Berberis.     

Simultaneous stressors: interactive effects of an immune challenge and dietary toxin can be detrimental to honeybees

Recent large-scale mortality of honeybee colonies is believed to be caused by multiple interactions between diseases, parasites, pesticide exposure, and other stress factors. To test whether a dual challenge has an additive effect in reducing survival, we experimentally stimulated the immune system of caged Apis mellifera scutellata workers from six colonies by injecting saline or Escherichia coli lipopolysaccharides (LPS), and additionally fed them the alkaloid nicotine (0 μM, 3 μM and 300 μM in 0.63 M sucrose). Workers did not increase their sucrose intake to compensate for the immune system activation, and those injected with E. coli LPS decreased their intake on the highest nicotine concentration. In the single challenges, injection and high nicotine doses negatively affected survival. All injected worker groups showed reduced survival. Without nicotine, survival of the saline and E. coli LPS worker groups was similar, but survival of E. coli LPS-challenged workers dropped below that of the saline groups when additionally challenged by nicotine, with bees dying earlier at higher nicotine concentrations. In the dual challenge of saline injection and dietary nicotine, a reduced effect on survival was observed, with lower mortality than expected from the summed mortalities due to the single challenges. However, additive and synergistic effects on survival were observed in workers simultaneously challenged by E. coli LPS and nicotine, indicating that interactive effects of simultaneous pathogen exposure and dietary toxin are detrimental to honeybee fitness.     

Crystal and molecular structures of new enantiopure quinuclidines

X-ray crystal structure analysis was performed on single crystals of two diastereomeric enantiopure quinuclidines, (3R,8R)-3-vinyl-8-hydroxymethyl-quinuclidine (quincoridine, QCD) and (3R,8S)-3-vinyl-8-hydroxymethyl-quinuclidine (quincorine, QCI) as their salts with tartaric and p-toluenesulphonate anions, respectively. The molecules of these quinuclidine derivatives are considered here as fragments of the Cinchona alkaloids, quinidine and quinine. A comparison of the conformational features of QCD, QCI, and Cinchona alkaloids in the crystalline state shows that the molecular geometry of the title compounds is similar to that of threo-alkaloids (e.g., R,R isomer of epicinchonine) rather than to quinidine and quinine. The packing of the molecules in both structures is dominated by intermolecular hydrogen bonds.     

Iron(III) protoporphyrin IX complexes of the antimalarial Cinchona alkaloids quinine and quinidine

The antimalarial properties of the Cinchona alkaloids quinine and quinidine have been known for decades. Surprisingly, 9-epiquinine and 9-epiquinidine are almost inactive. A lack of definitive structural information has precluded a clear understanding of the relationship between molecular structure and biological activity. In the current study, we have determined by single crystal X-ray diffraction the structures of the complexes formed between quinine and quinidine and iron(III) protoporphyrin IX (Fe(III)PPIX). Coordination of the alkaloid to the Fe(III) center is a key feature of both complexes, and further stability is provided by an intramolecular hydrogen bond formed between a propionate side chain of Fe(III)PPIX and the protonated quinuclidine nitrogen atom of either alkaloid. These interactions are believed to be responsible for inhibiting the incorporation of Fe(III)PPIX into crystalline hemozoin during its in vivo detoxification. It is also possible to rationalize the greater activity of quinidine compared to that of quinine.     

Lethal and sublethal effects of cypermethrin and methoxyfenozide on the larvae of Rachiplusia nu (Guenee) (Lepidoptera: Noctuidae)

Rachiplusia nu is a defoliator lepidopteran species considered a potential pest in soybean and other crops. Cypermethrin is the main insecticide used in Argentina. Worldwide trends in pest control promote the use of insecticides with high specificity like methoxyfenozide; however, their usage in South America is still incipient. The effectiveness of cypermethrin and methoxyfenozide was studied on fifth larval instar of R. nu under chronic exposure in laboratory conditions. Four dilutions (between 10% and 80%) of the maximum field recommended concentration for each insecticide were assessed. Methoxyfenozide caused 100% larval mortality in all tested concentrations, while at tested cypermethrin concentrations, larval mortalities were between 75% and 98%. Cypermethrin treatments showed higher mean survival time values than methoxyfenozide. Both insecticides inhibited larval growth according to weight loss assessment. Cypermethrin also inhibited diet consumption during the first 24-h exposure.     

Sensory inhibition as a mechanism of feeding deterrence: effects of three alkaloids on leaf beetle feeding

ABSTRACT. Effects of three alkaloids, sparteine, nicotine and quinine on galeal sensilla of larvae and adults of Entomoscelis americana Brown (Coleoptera: Chrysomelidae) are described. Each of these deterrent compounds stimulates a cell which is stimulated also by glucosinolates (Mitchell & Gregory, 1979; Sutcliffe & Mitchell, 1980) which are feeding stimulants. They inhibit also the response of the sugar-sensitive cell. It is suggested that the latter effect may be mainly responsible for the observed feeding deterrence caused by sparteine and implicated in the other two compounds. Possible mechanisms for the actions of these compounds are discussed with reference to work on other invertebrates. The results also lead us to suggest modification of recent interpretations of the role of secondary plant compounds in the evolution of insect chemosensitive systems.     

Susceptibility of Eldana saccharina (Lepidoptera: Pyralidae), Busseola fusca and Sesamia calamistis (Lepidoptera: Noctuidae) to Bacillus thuringiensis Cry toxins and potential side effects on the larval parasitoid Cotesia sesamiae (Hymenoptera: Braconidae)

Laboratory trials were conducted to determine the efficacy of four Bacillus thuringiensis (Bt) Cry toxins at five different concentrations (0.016, 0.08, 0.4, 2, 10 μg/mL) for controlling three lepidopteran stem-borer species (i.e., the pyralid Eldana saccharina and the noctuids Busseola fusca and Sesamia calamistis) as well as to evaluate their indirect effect on the braconid larval parasitoid Cotesia sesamiae. In addition, larvae from the treatments above, after having been parasitized, were either fed a contaminated (group 1) or a toxin-free (group 2) diet and compared with a control (i.e., parasitized larvae which have never fed on Bt-toxin). All Bt Cry toxins induced larval feeding inhibition. Compared with the control, significant mortality resulted at all concentrations and for all species of Lepidoptera. Cry1Ab was the most toxic with 10 days post treatment mortalities ranging from 81% in B. fusca and S. calamistis to 100% in E. saccharina. In contrast, Cry1Ac had comparatively low toxicity particularly for B. fusca and S. calamistis (e.g., respectively, 54 and 74% mortality at 10 days at the highest concentration). In the toxin-treated group 1, percentages of C. sesamiae cocoon-producing moth larvae were higher compared to group 2 and the control, whereas clutch size was higher than in group 2 but similar to the control. In both groups, 0.016, 0.08 μg/mL CrylAc yielded lower female-biased sex ratios than the control. It was suggested that paralyses of the moth larvae caused by the toxin may have facilitated parasitization leading to higher parasitism and clutch size.     

Survival of larvae of the ragwort flea beetle, Longitarsus flavicornis (Coleoptera: Chrysomelidae), in water-logged soil

Water-logged soil could be a major factor affecting populations of the ragwort biological control agent, Longitarsus flavicornis, in Australia. Survival of L. flavicornis larvae in potted ragwort plants after inundation with water for up to 264h (11days) was compared to larval survival in control plants not exposed to inundation. Probit analysis indicated that 35.6% mortality of the larval population occurred after 24h inundation and 50.4% after 72h. As high mortality was recorded at periods of inundation known to occur in the field, it is concluded that winter flooding is likely to be detrimental to populations of L. flavicornis.     

The effect of varying alkaloid concentrations on the feeding behavior of gypsy moth larvae, Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Nine alkaloids (acridine, aristolochic acid, atropine, berberine, caffeine, nicotine, scopolamine, sparteine, and strychnine) were evaluated as feeding deterrents for gypsy moth larvae (Lymantria dispar (L.); Lepidoptera: Lymantriidae). Our aim was to determine and compare the taste threshold concentrations, as well as the ED₅₀ values, of the nine alkaloids to determine their potency as feeding deterrents. The alkaloids were applied to disks cut from red oak leaves (Quercus rubra) (L.), a plant species highly favored by larvae of this polyphagous insect species. We used two-choice feeding bioassays to test a broad range of biologically relevant alkaloid concentrations spanning five logarithmic steps. We observed increasing feeding deterrent responses for all the alkaloids tested and found that the alkaloids tested exhibited different deterrency threshold concentrations ranging from 0.1 to 10 mM. In conclusion, it appears that this generalist insect species bears a relatively high sensitivity to these alkaloids, which confirms behavioral observations that it avoids foliage containing alkaloids. Berberine and aristolochic acid were found to have the lowest ED₅₀ values and were the most potent antifeedants. Handling Editor: Joseph Dickens.     

Combined use of [TBA][L-ASP] and hydroxypropyl-β-cyclodextrin as selectors for separation of Cinchona alkaloids by capillary electrophoresis

In this paper, a new capillary electrophoresis (CE) separation and detection method was developed for the chiral separation of the four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) using hydroxypropyl-β-cyclodextrin (HP-β-CD) and chiral ionic liquid ([TBA][L-ASP]) as selectors. Separation parameters such as buffer concentrations, pH, HP-β-CD and chiral ionic liquid concentrations, capillary temperature, and separation voltage were investigated. After optimization of separation conditions, baseline separation of the three analytes (cinchonidine, quinine, cinchonine) was achieved in fewer than 7 min in ammonium acetate background electrolyte (pH 5.0) with the addition of HP-β-CD in a concentration of 40 mM and [TBA][L-ASP] of 14 mM, while the baseline separation of cinchonine and quinidine was not obtained. Therefore, the first-order derivative electropherogram was applied for resolving overlapping peaks. Regression equations revealed a good linear relationship between peak areas in first-order derivative electropherograms and concentrations of the two diastereomer pairs. The results not only indicated that the first-order derivative electropherogram was effective in determination of a low content component and of those not fully separated from adjacent ones, but also showed that the ionic liquid appeared to be a very promising chiral selector in CE.     

The effect of alkaloids on sugar receptors and the feeding behaviour of the blowfly

ABSTRACT. The deterrent effect of alkaloids on feeding by the black blowfly Phormia regina Meigen (Diptera: Calliphoridae) was tested by determining tarsal thresholds for mixtures of sucrose and alkaloids. The following alkaloids were used: atropine sulphate, berberine sulphate, quinine mono-hydrochloride, caffeine, yohimbine sulphonic ester, pilocarpine hydrochloride, coniine hydrochloride and codeine. The same alkaloids were tested electrophysiologically on tarsal chemoreceptors (D hairs). Both behaviourally and electrophysiologically alkaloids reduced response to sucrose. Deterrence and peripheral inhibition could be blocked by increasing the concentration of sucrose. Application of kinetic analyses to the electrophysiological data ruled out competitive, non-competitive, and uncompetitive inhibition at receptor sites. There is no correlation of thresholds with available data on lipid solubility or octanol/water partition coefficients. The diverse pharmacological properties of alkaloids suggest that there is no uniform limiting mechanism for this multiform array of compounds.     

Alkaloidal variation in the genus Pearsonia

Several quinolizidine alkaloids, including various angelate esters, are known from the genus Pearsonia. In a detailed variation study which included 98 samples from nine of the 11 species, large qualitative and quantitative differences were recorded. The observed variation is ascribed to the following: 1, species (the alkaloids of some species and subspecies are diagnostically different); 2, provenance (various populations of the same species may have unique combinations of alkaloids); 3, developmental stage (in P. cajanifolia there is a marked decreased in esterification towards the end of the growing season); 4, plant parts extracted (seeds, for example, have high concentrations of hydroxylated lupanine-type alkaloids and only small amounts of esters). These results highlight some of the problems associated with the use of alkaloids as taxonomic characters.     

Comparative effects of the diastereoisomers, quinine and quinidine in producing phenocopy debrisoquine poor metabolisers (PMs) in healthy volunteers

1. A single oral dose (50 mg) of quinidine significantly increased the debrisoquine metabolic ratio in six healthy volunteers. For four of the volunteers the metabolic ratio changed to that typical of the poor metaboliser (PM) phenotype. 2. The effect of quinidine in producing debrisoquine oxidation "poor metaboliser" phenocopies persisted for at least 3 days but had disappeared by 1 week. 3. The debrisoquine metabolic ratios for the same six subjects were not significantly altered by the oral administration of quinine (200 or 400 mg), the diastereoisomer of quinidine. 4. The plasma pharmacokinetic parameters of both nortriptyline and desipramine in healthy volunteers were all changed to those more typical of the debrisoquine PM phenotype following the concomitant administration of quinidine (50 mg). 5. It is concluded that quinidine, but not its diastereoisomer quinine, is a potent selective inhibitor of the in vivo oxidation of debrisoquine and can produce an artifactual PM phenocopy in persons who are phenotypically extensive metaboliser (EM) phenotype status. The clinical implications of this observation are discussed.     

Effect of temperature and treatment conditions on the mortality of Epiphyas postvittana (Lepidoptera: Tortricidae) exposed to ethanol

Morality responses were determined for 5th-instar lightbrown apple moth, Epiphyas postvittana (Walker), in the presence and absence of apples to immersion in ethanol solutions and exposure to ethanol vapor at a range of concentrations, treatment times, and temperatures. Ethanol may have caused an initial knock-down effect in E. postvittana larvae because there was a trend for larval mortality to reduce with increased periods between treatment and assessment time when immersed at 20 degrees C in 30 or 50% ethanol solutions. Mortality for larvae immersed on apples in a range of ethanol concentrations was higher than for larvae in the absence of apples. Increasing treatment temperature from 20 to 45 degrees C during ethanol immersion significantly increased larval mortality. During ethanol vapor exposure, longer treatment times were required to achieve 99% E. postvittana mortality for larvae on apples compared with those in the absence of apples.