Analysis of limonoid glucosides from citrus by electrospray ionization liquid chromatography-mass spectrometry.

An electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) method for the detection and quantitation of limonoid glucosides has been developed. Negative ions [M - H(+)](-) characteristic of six limonoid glucosides can be detected and quantified from selected ion monitoring chromatograms using carminic acid as an internal standard. The described method has been applied to the analysis of limonoid glucoside content in various liquid and solid Citrus spp. samples as well as complex mixtures of partially purified limonoid glucosides. Rapid and sensitive qualitative screening of samples for limonoid glucosides can also be accomplished with slight modifications of the method.

Three new toxic norditerpenoid alkaloids from the low larkspur Delphinium nuttallianum.

Three new N-(methylsuccinimido)anthranoyllycoctonine norditerpenoids, given the names bearline (1), 14-acetylbearline (2), and 16-deacetylgeyerline (3), were isolated from the extract of the low larkspur Delphinium nuttallianum. The structures of the individual alkaloids were determined by (1)H and (13)C NMR spectroscopy and HRMS. These alkaloids are structurally related to the neurotoxic alkaloid methyllycaconitine (4) and may be important in establishing the toxicity of low larkspurs to cattle. A mouse bioassay was used to measure the LD(50) values for two of the new alkaloids (1 and 2), as sufficient quantities of 3 were not available for toxicity testing. A structurally related alkaloid, geyerline (7), was isolated from D. geyeri in sufficient quantities for toxicity testing. The toxicities of 1, 2, and 7 were found to be comparable to that of 4, with calculated LD(50) values in mice of 5.7, 3.3, and 6.2 mg/kg, respectively.

Effects of Delphinium alkaloids on neuromuscular transmission.

The Delphinium alkaloids methyllycaconitine (MLA), nudicauline, 14-deacetylnudicauline (14-DN), barbinine, and deltaline were investigated for their effects on neuromuscular transmission in lizards. The substituent at C14 provides the only structural difference among the alkaloids MLA, nudicauline, 14-DN, and barbinine. Deltaline lacks the N-(methylsuccinyl)anthranilic acid at C18 common to the other four alkaloids. Each alkaloid reversibly reduced extracellularly recorded compound muscle action potential (CMAP) amplitudes in a concentration-dependent manner. The IC(50) values for CMAP blockade were between 0.32 and 13.2 microM for the N-(methylsuccinimido)anthranoyllycacotonine-type alkaloids and varied with the C14 moiety; the IC(50) value for deltaline was 156 microM. The slopes of the concentration-response curves for CMAP blockade were similar for each alkaloid except barbinine, whose shallower curve suggested alternative or additional mechanisms of action. Each alkaloid reversibly reduced intracellularly recorded spontaneous, miniature end-plate potential (MEPP) amplitudes. Alkaloid concentrations producing similar reductions in MEPP amplitude were 0.05 microM for 14-DN, 0.10 microM for MLA, 0.50 microM for barbinine, and 20 microM for deltaline. Only barbinine altered the time constant for MEPP decay, further suggesting additional or alternative effects for this alkaloid. MLA and 14-DN blocked muscle contractions induced by exogenously added acetylcholine. All five alkaloids are likely nicotinic receptor antagonists that reduce synaptic efficacy and block neuromuscular transmission. The substituent at C14 determines the potency and possibly the mechanism of nicotinic acetylcholine receptor blockade for MLA, nudicauline, 14-DN, and barbinine at neuromuscular synapses. The lower potency of deltaline indicates that the N-(methylsuccinyl)anthranilic acid at C18 affects alkaloid interactions with nicotinic acetylcholine receptors at neuromuscular junctions.

Larkspur (Delphinium spp.) poisoning in livestock.

Larkspurs (Delphinium spp.) are toxic plants that contain numerous diterpenoid alkaloids which occur as one of two structural types: (1) lycotonine, and (2) 7,8-methylenedioxylycoctonine (MDL-type). Among the lycoctonine type alkaloids are three N-(methylsuccinimido) anthranoyllycoctonine (MSAL-type) alkaloids which appear to be most toxic: methyllycaconitine (MLA), 14-deacetylnudicauline (DAN), and nudicauline. An ester function at C-18 is an important structural requirement for toxicity. Intoxication results from neuromuscular paralysis, as nicotinic acetylcholine receptors in the muscle and brain are blocked by toxic alkaloids. Clinical signs include labored breathing, rapid and irregular heartbeat, muscular weakness, and collapse. Toxic alkaloid concentration generally declines in tall larkspurs with maturation, but alkaloid concentration varies over years and from plant to plant, and is of little use for predicting consumption by cattle. Knowledge of toxic alkaloid concentration is valuable for management purposes when cattle begin to eat larkspur. Cattle generally begin consuming tall larkspur after flowering racemes are elongated, and consumption increases as larkspur matures. Weather is also a major factor in cattle consumption, as cattle tend to eat more larkspur during or just after summer storms. Management options that may be useful for livestock producers include conditioning cattle to avoid larkspur (food aversion learning), grazing tall larkspur ranges before flowering (early grazing) and after seed shatter (late grazing), grazing sheep before cattle, herbicidal control of larkspur plants, and drug therapy for intoxicated animals. Some potentially fruitful research avenues include examining alkaloid chemistry in low and plains larkspurs, developing immunologic methods for analyzing larkspur alkaloids, developing drug therapy, and devising grazing regimes specifically for low and plains larkspur.

The characterization and structure-activity evaluation of toxic norditerpenoid alkaloids from two Delphinium species.

A new N-(methylsuccinimido)anthranoyllycoctonine norditerpenoid alkaloid, geyerline, has been isolated and characterized from extracts of the poisonous larkspur Delphinium glaucum. A previously described norditerpenoid alkaloid, grandiflorine, has also been isolated from Delphinium geyeri. Both alkaloids are closely related structurally to the potent neurotoxin methyllycaconitine, established as the primary toxin in many larkspurs poisonous to cattle. Mouse bioassay tests showed grandiflorine to possess toxicity comparable to methyllycaconitine, while its synthetically derived monoacetate, grandiflorine acetate, and geyerline are significantly less toxic.

Influence of alkaloid concentration on acceptability of tall larkspur (Delphinium spp.) to cattle and sheep.

Tall larkspur (Delphinium spp.) is a serious toxic plant problem on western U.S. ranges. The major toxins in tall larkspur are methyllycaconitine (MLA) and 14-deacetylnudicauline (14-DAN); the sum of both is termed the toxic alkaloid concentration. Toxic alkaloids comprise about 20-50% of the total alkaloid concentration in tall larkspur. Toxic and total alkaloid concentration generally declines with maturity, whereas cattle and sheep consumption of larkspur typically increases with plant maturity. We hypothesized that cattle and sheep consumption of tall larkspur was negatively related to higher concentrations of total or toxic alkaloid. We compared consumption of several collections of dried, ground larkspur and fresh larkspur in a series of trials. In another trial, a crude alkaloid fraction was extracted with ethanol, added to alfalfa hay, and consumption compared to untreated alfalfa hay, alcohol-treated hay, and the essentially alkaloid-free plant residue. In all cases we correlated amounts eaten with total and toxic alkaloid concentration. A grazing trial was also conducted to relate larkspur consumption over time to alkaloid concentrations. Total alkaloid concentrations in dried, whole-plant collections ranged from 9.3 to 38.8 mg/g of dry weight, whereas toxic alkaloid concentrations varied from 0.0 to 7.1 mg/g. In one pen trial, cattle preferred a larkspur collection (P<0.01) that contained no toxic alkaloids but had a high total alkaloid concentration (39 mg/g). There was no correlation (P>0.05), however, between concentrations of total or toxic alkaloids and amount of dry plant consumed in this or any other trial. Conversely, sheep consumption tended to be negatively influenced by total and toxic alkaloid concentration (P≤0.08). In the trials with extract, cattle preferred the alcohol-treated hay and rejected the alkaloid-free residue (P<0.01), whereas the alkaloid-treated hay was of intermediate acceptability. Cattle preferred the alkaloid-treated hay over the alkaloid-free residue, indicating that alkaloids did not deter consumption. Conversely, the alkaloid-treated hay was less preferred than either untreated or alcohol-treated hay, suggesting a negative effect on acceptability. There was no correlation between alkaloid concentration and amount of treated feed eaten. In field trials, the amount of composited, fresh leaves or flowers eaten by cattle was influenced by plant part (P=0.04), but was not related (P>0.05) to alkaloid concentration. Cattle preferred leaves over flowers when offered individual plants differing in phenological stage and/or amount of shade, but alkaloid concentration was not related to consumption. We conclude that knowledge of the concentration of total or toxic alkaloid in tall larkspur will give little or no indication of plant acceptability to cattle. Even though accurate predictions can be made about the potential toxicity of larkspur based on the concentration of toxic alkaloids, predictions about consumption must be based primarily on plant phenology.

Structure-activity relationships of norditerpenoid alkaloids occurring in toxic larkspur (Delphinium) species.

Fourteen norditerpenoid alkaloids present in larkspur (Delphinium) species associated with cattle poisoning on grazing land in the western United States have been toxicologically assessed in a mouse bioassay. Toxicity data for these alkaloids have established the tertiary nitrogen atom and anthranilic acid esterification as important structural features necessary to impart toxicity to lycoctonine-type norditerpenoid alkaloids. Variation in C-14 functionality of the toxic alkaloids is also a factor that influences toxicity in these compounds. The relationship of the structure-activity information of this study to previous in vitro neuromuscular studies is discussed.

Reversal of tall larkspur (Delphinium barbeyi) poisoning in cattle with physostigmine.

Tall larkspur poisoning of cattle is a serious problem on western US rangelands. Single oral doses of tall larkspur ranging from 1.5 to 3 g/kg body weight were administered to steers. These doses caused clinical signs of muscular tremors and collapse. Physostigmine was administered iv, ip or sc at 0.04 to 0.08 mg/kg body weight when animals were sternally or laterally recumbent. Physostigmine given iv rapidly reversed the larkspur toxicity. Serial injections of physostigmine were generally necessary to reverse acute toxicity. Administration of physostigmine to grazing animals poisoned on larkspur was also effective. Physostigmine can be effective treatment for intoxicated cattle consuming tall larkspurs.

Effective dose in cattle of toxic alkaloids from tall larkspur (Delphinium barbeyi).

We determined the po toxicity of tall larkspur (Delphinium barbeyi) of known alkaloid composition to cattle. We calculated the effective dose at which the alkaloids methyllycaconitine and 14-deacetylnudicauline, collectively termed total toxic alkaloid, caused collapse and sternal recumbency. The effective dose of total toxic alkaloid that produced sternal recumbency in steers was 11.2 mg/kg bw. Based on this dose, we hypothesize that the LD50 for a po dose of total toxic alkaloids in cattle will be 25 to 40 mg/kg bw.

Effect of temperature and sucrose concentration on hydroquinone toxicity in leafy spurge suspension culture cells.

Euphorbia esula (leafy spurge) suspension culture cell bioassays were used to determine whether sucrose accumulation enhanced the glucosylation (detoxification) of hydroquinone in this noxious weed. The bioassay results indicate that cold temperatures and exogenous hydroquinone represent a dual stress to spurge cell growth that can be partially ameliorated by hydrolysis of sucrose. The persistent susceptibility of leafy spurge suggests that hydroquinone-producing forage plants (which are not toxic to animals) might be used as natural competitors.

Differential allelochemical detoxification mechanism in tissue cultures ofAntennaria microphylla andEuphorbia esula.

Callus and suspension cultures ofAntennaria microphylla (small everlasting) and the noxious weedEuphorbia esula (leafy spurge) can glucosylate benzene-1,4-diol (hydroquinone) to the corresponding monoglucoside, arbutin. HPLC analysis of extracts from callus tissue corroborates the presence of hydroquinone in the cells of small everlasting. Constitutive levels of a UDPG-dependent glucosyltransferase were detected in cell-free extracts of this tissue. Although this detoxification enzyme was induced in leafy spurge suspension culture cells grown in the presence of hydroquinone, the activity was six-fold lower than that measured in small everlasting. Differential ability to detoxify hydroquinone provides a basis for the observed allelopathic interaction between small everlasting and leafy spurge.

Allelopathy of small everlasting (Antennaria microphylla) : Phytotoxicity to leafy spurge (Euphorbia esula) in tissue culture.

Media and media extracts from callus cultures of small everlasting (Antennaria microphylla) inhibited leafy spurge (Euphorbia esula L.) callus tissue and suspension culture growth (50 and 70% of control, respectively) and were phytotoxic in lettuce and leafy spurge root elongation bioassays (64 and 77% of control, respectively). Hydroquinone, a phytotoxic compound previously isolated from small everlasting, was also biosynthesized by callus and suspension cultures of this species. Exogenously supplied hydroquinone (0.5 mM) was toxic to leafy spurge suspension culture cells and was only partially biotransformed to its nontoxic water-soluble monoglucoside, arbutin, by these cells. This report confirms the chronic involvement of hydroquinone in the allelopathic interaction between small everlasting and leafy spurge.

Ingestion of tall larkspur by cattle : Separating effects of flavor from postingestive consequences.

Tall larkspur (Delphinium spp.) is a palatable but toxic poisonous plant in the western United States. The toxins in tall larkspur are diterpenoid alkaloids. We examined the influences of food flavor and postingestive consequences on consumption of a 33% larkspur pellet during 30-min feeding periods for five days using esophageally fistulated cattle that were sham-fed larkspur pellets. Consumption by the sham-fed group was compared to a control group fed alfalfa pellets, and a larkspur group fed only larkspur pellets. Sham-fed cattle did not decrease (P > 0.1) feed consumption compared to controls, indicating no significant difference in food flavor. The larkspur group decreased (P < 0.05) feed consumption by 41% relative to controls and by 31% relative to sham-fed animals (P = 0.08). This reduction in feed consumption indicates the adverse postingestive consequences of tall larkspur ingestion, as the larkspur group apparently developed a conditioned taste aversion to the larkspur pellet. Even though these animals were averted to the pellets, they showed none of the classical signs of intoxication from ingestion of tall larkspur.

Plant-animal interactions in larkspur poisoning in cattle.

Larkspur (Delphinium spp.) toxicity in cattle seriously impedes the efficient use of productive mountain rangelands. Larkspurs contain complex diterpenoid alkaloids that cause acute intoxication and death from respiratory paralysis. Alkaloids and their concentrations vary among larkspur species, plant parts and phenological growth stages, thus causing great variability in toxicity. Ingestion rate of larkspur by the cow, alkaloid toxicity and concentration in the plant and the kinetics of absorption and excretion interact to determine whether a cow is poisoned. Plant and animal factors influencing consumption and subsequent intoxication must be further elucidated to devise management strategies to reduce liverstock losses.

In vivo exposure to plant flavonols. Influence on frequencies of micronuclei in mouse erythrocytes and sister-chromatid exchange in rabbit lymphocytes.

No consistent increases in the micronucleus frequency were observed in bone marrow or peripheral blood erythrocytes from mice treated with quercetin, rhamnetin, neohesperidin dihydrochalcone, or hesperetin dihydrochalcone under various exposure and sampling conditions. Over the dose range of 100-1000 mg/kg, quercetin failed to increase significantly erythrocyte micronucleus frequencies either (1) in bone marrow of male mice at 6 h after the second of 2 i.p. or oral doses given 24 h apart, or at 48, 96 or 192 h after a single i.p. or oral dose, or (2) in peripheral blood of male or female mice sampled for 7 consecutive days following a single i.p. dose. Feeding 5% or 10% quercetin for 8 days also failed to increase the micronucleus frequency in bone marrow erythrocytes of female or male mice. Hesperetin dihydrochalcone and neohesperidin dihydrochalcone, at p.o. doses of 100-1000 mg/kg, did not increase the micronucleus frequency in bone marrow erythrocytes 6 h after the second of 2 doses 24 h apart, nor did rhamnetin at 48 or 96 h after a single i.p. dose of 1000 mg/kg. Galangin, in contrast, did significantly increase the micronucleus frequency in bone marrow and blood erythrocytes under certain conditions, but the largest increases were only between 2 and 3 times control values and these were observed at highly toxic doses. Rabbits given up to 250 mg/kg quercetin i.p. showed no treatment-related increase in the sister-chromatid-exchange frequency in peripheral blood lymphocytes sampled at 1 and 7 days after treatment. These results fail to confirm published data which report a markedly increased frequency of micronuclei in bone marrow erythrocytes from quercetin-treated mice, show no quercetin-related alterations in the sister-chromatid-exchange frequency in rabbit lymphocytes, and indicate that clastogenesis in bone marrow erythroblasts due to oral or i.p. administration of the flavonols studied is at most very weak.