Cardenolide Increase in Foxglove after 2,1,3-Benzothiadiazole Treatment Reveals a Potential Link between Cardenolide and Phytosterol Biosynthesis.

Cardenolides are steroidal metabolites in Digitalis lanata with potent cardioactive effects on animals. In plants, cardenolides are likely involved in various stress responses. However, the molecular mechanism of cardenolide increase during stresses is mostly unknown. Additionally, cardenolides are proposed to arise from cholesterol, but indirect results show that phytosterols may also be substrates for cardenolide biosynthesis. Here, we show that cardenolides increased after methyl jasmonate (MJ), sorbitol, potassium chloride (KCl) and salicylic acid analog [2,1,3-benzothiadiazole (BTH)] treatments. However, the expression of three known genes for cardenolide biosynthesis did not correlate well with these increases. Specifically, the expression of progesterone-5ß-reductases (P5ßR and P5ßR2) did not correlate with the cardenolide increase. The expression of 3ß-hydroxysteroid dehydrogenase (3ßHSD) correlated with changes in cardenolide levels only during the BTH treatment. Mining the D. lanata transcriptome identified genes involved in cholesterol and phytosterol biosynthesis: C24 sterol sidechain reductase 1 (SSR1), C4 sterol methyl oxidase 1, and 3 (SMO1 and SMO3). Surprisingly, the expression of all three genes correlated well with the cardenolide increase after the BTH treatment. Phylogenetic analysis showed that SSR1 is likely involved in both cholesterol and phytosterol biosynthesis. In addition, SMO1 is likely specific to phytosterol biosynthesis, and SMO3 is specific to cholesterol biosynthesis. These results suggest that stress-induced increase of cardenolides in foxglove may correlate with cholesterol and phytosterol biosynthesis. In summary, this work shows that cardenolides are important for stress responses in D. lanata and reveals a potential link between phytosterol and cardenolide biosynthesis.

Authentication of tejocote (Crataegus mexicana) dietary supplements based on DNA barcoding and chemical profiling.

Tejocote (Crataegus mexicana, Mexican hawthorn), known as a weight-loss supplement, has been marketed online and is easily available for overseas direct purchase. Alipotec (brand name) is known as one of the most popular products containing tejocote in Mexico and other countries. However, adverse effects have been reported by users of these supplements. Therefore it is necessary to find the reason for the side effect. Dietary supplement samples labelled as containing tejocote were analysed using mass spectrometry and DNA barcoding analysis. Our results demonstrate that Alipotec samples contained ingredients from different species, yellow oleander instead of tejocote. The rpoB barcode region was able to differentiate between tejocote and yellow oleander species. Moreover, it was also observed that three compounds, including thevetin B, neriifolin, and digitoxigenin, clearly distinguish between tejocote and yellow oleander samples. This is the first and preliminary investigation to use an integrated approach of both chemical and genomic profiling for the authentication of dietary supplement containing tejocote.

Structural Analysis of Diastereomeric Cardiac Glycosides and Their Genins Using Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

Ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) is an economical and indispensable tool in natural product research to investigate novel metabolites, biomarker discovery, chemical diversity exploration, and structure elucidation. In this study, the structural analysis of 38 naturally occurring cardiac glycosides (CGs) in various tissues of Nerium oleander was achieved by the extensive use of mass spectrometry. The chemical diversity of CGs was described on the basis of characteristic MS/MS fragmentation patterns, accurate mass measurement, and published scientific information on CGs from Nerium oleander. It was observed that only six genins, viz., Δ16anhydrogitoxigenin, Δ16adynerigenin, gitoxigenin, oleandrigenin, digitoxigenin, and adynerigenine, produce 38 diverse chemical structures of CGs. Among them, 20 were identified as diastereomers having a difference in a sugar (l-oleandrose, ß-d-diginose, and ß-d-sarmentose) unit. However, the differentiation of diastereomeric CGs was not possible by only MS/MS fragments. Thus, the diastereomer's chromatographic elution order was assigned on the basis of the relative retention time (RRt) of two reference standards (odoroside A and oleandrin) among their diastereomers. Besides this, the in-source fragmentation of CGs and the MS/MS of m/z 325 and 323 disaccharide daughter ions also exposed the intrinsic structure information on the sugar units. The daughter ions m/z 162, 145, 113, 95, and 85 in MS/MS spectra indicated the abundance of l-oleandrose, ß-d-diginose, and ß-d-sarmentose sugars. At the same time, m/z 161, 143, 129, and 87 product ions confirmed the presence of a ß-d-digitalose unit. As a result, the UPLC-ESI/TQD system was successfully utilized for the structure characterization of CGs in Nerium oleander tissues.

Fatal poisoning by ingestion of a self-prepared oleander leaf infusion.

An unusual case of poisoning by the ingestion of oleander leaves is reported. A 71 year old male laboratory technician committed suicide at home in this unusual manner. At the death scene a steel pan and other paraphernalia, used for the extraction of oleandrin and other cardiac glycosides from the leaves of the Nerium oleander plant were found.Toxicological investigations for oleandrin, oleandrigenin, neritaloside, and odoroside were performed by LC-MS/MS on all biological samples (peripheral blood, vitreous humor, urine, liver, gastric contents) and on the yellow infusion found at the death scene.In all samples, toxic levels of oleandrin were detected (blood 37.5 ng/mL, vitreous humor 12.6 ng/mL, urine 83.8 ng/mL, liver 205 ng/mg, gastric content 31.2 µg/mL, infusion 38.5 µg/mL). Qualitative results for oleandrigenin, neritaloside, and odoroside were obtained. Oleandrigenin was present in all tissue samples whereas neritaloside and odoroside were absent in the blood and vitreous humor but present in urine, liver, gastric content, and in the leaf brew.The purpose of this study was the identification of oleandrin and its congener oleandrigenin, detected in the vitreous humor. The blood/vitreous humor ratio was also calculated in order to assess of the likely time interval from ingestion to death. According to the toxicological results death was attributed to fatal arrhythmia due to oleander intoxication. The manner of death was classified as suicide through the ingestion of the infusion.

Negligible Oleandrin Content of Hot Dogs Cooked on Nerium oleander Skewers.

INTRODUCTION: The Nerium oleander plant contains cardenolides that may cause human poisoning when ingested. A long-standing belief holds that it is possible to be poisoned by eating hot dogs or other foods cooked on Nerium oleander branch skewers. Oleandrin levels in frankfurters cooked on fresh and dry Nerium oleander skewers were measured. METHODS: Hot dogs were cooked separately on either dried or fresh oleander branch skewers using a disposable charcoal grill. The hot dogs were then frozen and transported to an analytical laboratory where oleandrin content was measured via liquid chromatography/mass spectroscopy (LC/MS). RESULTS: The oleandrin content of hot dogs cooked on dried and fresh skewers did not exceed 343 ng and 701 ng, respectively. CONCLUSION: Hot dogs cooked on Nerium oleander skewers contain a negligible amount of oleandrin with respect to that sufficient to cause human poisoning. Reports of poisonings occurring in this manner are most likely the result of an urban myth.

Outbreak of Oleander (Nerium oleander) Poisoning in Dairy Cattle: Clinical and Food Safety Implications.

Oleander is a spontaneous shrub widely occurring in Mediterranean regions. Poisoning is sporadically reported in livestock, mainly due to the ingestion of leaves containing toxic cardiac glycosides (primarily oleandrin). In this study, 50 lactating Fleckvieh cows were affected after being offered a diet containing dry oleander pruning wastes accidentally mixed with fodder. Clinical examination, electrocardiogram, and blood sampling were conducted. Dead animals were necropsied, and heart, liver, kidney, spleen, and intestine were submitted to histological investigation. Oleandrin detection was performed through ultra-high performance liquid chromatography-tandem mass spectrometry in blood, serum, liver, heart, milk, and cheese samples. Severe depression, anorexia, ruminal atony, diarrhea, serous nasal discharge, tachycardia, and irregular heartbeat were the most common clinical signs. The first animal died within 48 h, and a total of 13 cows died in 4 days. Disseminated hyperemia and hemorrhages, multifocal coagulative necrosis of the cardiac muscle fibers, and severe and diffuse enteritis were suggestive of oleander poisoning. The diagnosis was confirmed by the presence of oleandrin in serum, liver, heart, milk, and cheese. Our results confirm the high toxicity of oleander in cattle and report for the first time the transfer into milk and dairy products, suggesting a potential risk for the consumers.

Development and Validation of a UHPLC-ESI-MS/MS Method for Quantification of Oleandrin and Other Cardiac Glycosides and Evaluation of Their Levels in Herbs and Spices from the Belgian Market.

Cardiac glycosides (CGs) are naturally occurring plant secondary metabolites that can be toxic to humans and animals. The aim of this work was to develop a targeted analytical method utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of these plant toxins in a herbal-based food and human urine. The method included oleandrin, digoxin, digitoxin, convallatoxin, and ouabain. Samples of culinary herbs were extracted with acetonitrile and cleaned using Oasis® MAX solid-phase extraction (SPE), while samples of urine were diluted with acidified water and purified on Oasis® HLB SPE cartridges. Limits of quantification were in the range of 1.5-15 ng/g for herbs and 0.025-1 ng/mL for urine. The mean recovery of the method complied with the acceptable range of 70-120% for most CGs, and relative standard deviations were at maximum 14% and 19% for repeatability and reproducibility, respectively. Method linearity was good with calculated R² values above 0.997. The expanded measurement uncertainty was estimated to be in the range of 7-37%. The LC-MS/MS method was used to examine 65 samples of culinary herbs and herb and spice mixtures collected in Belgium, from supermarkets and local stores. The samples were found to be free from the analyzed CGs.

Profiling and structural analysis of cardenolides in two species of Digitalis using liquid chromatography coupled with high-resolution mass spectrometry.

Plants of the Digitalis genus contain a cocktail of cardenolides commonly prescribed to treat heart failure. Cardenolides in Digitalis extracts have been conventionally quantified by high-performance liquid chromatography yet the lack of structural information compounded with possible co-eluents renders this method insufficient for analyzing cardenolides in plants. The goal of this work is to structurally characterize cardiac glycosides in fresh-leaf extracts using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) that provides measured accurate mass. Fragmentation of cardenolides is featured by sequential loss of sugar units while the steroid aglycone moieties undergo stepwise elimination of hydroxyl groups, which distinguishes different aglycones. Using a reverse-phase LC column, the sequence of elution follows: diginatigenin→digoxigenin→gitoxigenin→gitaloxigenin→digitoxigenin for cardenolides with the same sugar units but different aglycones. A linear range of 0.8-500 ng ml-1 has been achieved for digoxigenin, ß-acetyldigoxin, and digitoxigenin with limits of detection ranging from 0.09 to 0.45 ngml-1. A total of seventeen cardenolides have been detected with lanatoside A, C, and E as major cardenolides in Digitalis lanata while seven have been found in Digitalis purpurea including purpurea glycoside A, B, and E. Surprisingly, glucodigifucoside in D. lanata and verodoxin and digitoxigenin fucoside in D. purpurea have also been found as major cardenolides. As the first MS/MS-based method developed for analyzing cardenolides in plant extracts, this method serves as a foundation for complete identification and accurate quantification of cardiac glycosides, a necessary step towards understanding the biosynthesis of cardenolide in plants.

Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae.

A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5 -3ß-hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ5 -isomerase gene from Comamonas testosteronii, (c) a mutated steroid-5ß-reductase gene from Arabidopsis thaliana, and (d) a steroid 21-hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed "CARD II yeast", was capable of producing 5ß-pregnane-3ß,21-diol-20-one, a central intermediate in 5ß-cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast.

Mycorrhizae Alter Constitutive and Herbivore-Induced Volatile Emissions by Milkweeds.

Plants use volatile organic compounds (VOCs) to cue natural enemies to their herbivore prey on plants. Simultaneously, herbivores utilize volatile cues to identify appropriate hosts. Despite extensive efforts to understand sources of variation in plant communication by VOCs, we lack an understanding of how ubiquitous belowground mutualists, such as arbuscular mycorrhizal fungi (AMF), influence plant VOC emissions. In a full factorial experiment, we subjected plants of two milkweed (Asclepias) species under three levels of AMF availability to damage by aphids (Aphis nerii). We then measured plant headspace volatiles and chemical defenses (cardenolides) and compared these to VOCs emitted and cardenolides produced by plants without herbivores. We found that AMF have plant species-specific effects on constitutive and aphid-induced VOC emissions. High AMF availability increased emissions of total VOCs, two green leaf volatiles (3-hexenyl acetate and hexyl acetate), and methyl salicylate in A. curassavica, but did not affect emissions in A. incarnata. In contrast, aphids consistently increased emissions of 6-methyl-5-hepten-2-one and benzeneacetaldehyde in both species, independent of AMF availability. Both high AMF availability and aphids alone suppressed emissions of individual terpenes. However, aphid damage on plants under high AMF availability increased, or did not affect, emissions of those terpenes. Lastly, aphid feeding suppressed cardenolide concentrations only in A. curassavica, and AMF did not affect cardenolides in either plant species. Our findings suggest that by altering milkweed VOC profiles, AMF may affect both herbivore performance and natural enemy attraction.

A Probable Fatal Case of Oleander (Nerium oleander) Poisoning on a Cattle Farm: A New Method of Detection and Quantification of the Oleandrin Toxin in Rumen.

Oleander (Nerium oleander) is an ornamental plant common in tropical and sub-tropical regions that is becoming increasingly widespread, even in temperate regions. Oleander poisoning may occur in animals and humans. The main active components contained in the plant are cardiac glycosides belonging to the class of cardenolides that are toxic to many species, from human to insects. This work describes a case of oleander poisoning that occurred on a small cattle farm and resulted in the fatality of all six resident animals. Furthermore, the investigation of the poisonous agent is described, with particular focus on the characterization of the oleandrin toxin that was recovered from the forage and rumen contents. The innovation of this study is the first description of the detection and quantification of the oleandrin toxin by liquid chromatography-high resolution mass spectrometry (LC-HRMS) in rumen.

A fatal case of self-poisoning through the ingestion of oleander leaves.

BACKGROUND: Benefits and even dangers of plants are known since time began. The ancients used plants and herbs because of their effects on the human body. Poisoning is a logical consequence of their use: history is full of episodes of plants and herbs poisoning, whether intentional or accidental. AIM: Oleander poisoning is generally accidental; an intentional assumption of its leaves to commit suicide is uncommon because the population is not aware of the harmfulness of its cardiotoxic glycosides, therefore we report a fatal case of self-poisoning through the voluntary ingestion of oleander leaves. METHODS: A diagnosis of oleander self-poisoning was highly suspected on the basis of the circumstantial evidence and the autopsy findings. Toxicological investigations were performed on the samples collected during the autopsy and aimed at confirm the presence of oleandrin at a toxic level. RESULTS: The autopsy revealed a piece of oleander leaf on the posterior third of the tongue's body and several plant residues, similar to the one recovered on the tongue, into the gastric content; petechiae on the deep surface of the scalp, multi-organ congestion, and pulmonary edema were also observed. The histological study corroborated the pulmonary edema macroscopically observed but did not provide any other information. The detection of oleandrin in biological cadaveric samples revealed high, fatal, concentrations. CONCLUSIONS: Cases of voluntary ingestion of oleander with a suicidal intent prove to be uncommon: in the case reported the victim was aware about the possibility to commit suicide through the ingestion of oleander leaves.

The demographic effects of functional traits: an integral projection model approach reveals population-level consequences of reproduction-defence trade-offs.

Quantitatively linking individual variation in functional traits to demography is a necessary step to advance our understanding of trait-based ecological processes. We constructed a population model for Asclepias syriaca to identify how functional traits affect vital rates and population growth and whether trade-offs in chemical defence and demography alter population growth. Plants with higher foliar cardenolides had lower fibre, cellulose and lignin levels, as well as decreased sexual and clonal reproduction. Average cardenolide concentrations had the strongest effect on population growth. In both the sexual and clonal pathway, the trade-off between reproduction and defence affected population growth. We found that both increasing the mean of the distribution of individual plant values for cardenolides and herbivory decreased population growth. However, increasing the variance in both defence and herbivory increased population growth. Functional traits can impact population growth and quantifying individual-level variation in traits should be included in assessments of population-level processes.

Toxicity of Milkweed Leaves and Latex: Chromatographic Quantification Versus Biological Activity of Cardenolides in 16 Asclepias Species.

Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na+/K+ ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na+/K+ ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed's diverse community of specialist cardenolide-sequestering insect herbivores.

Improved method for diagnosis of Nerium oleander poisoning in necropsy tissues / Método aprimorado para diagnóstico da intoxicação por Nerium oleander em tecidos de necropsia

Nerium oleander is an ornamental cardiotoxic plant found in tropical and subtropical areas of the World. Its toxicity is related to the content of cardioactive glycosides, mainly oleandrin, found throughout the plant. The present study aimed to describe a new and improved method for oleandrin detection in tissue samples. The determination of oleandrin was made after extraction with a modified QuEChERS technique and measurement by UFLC-MS/MS. A total of 36 guinea pigs (Cavia porcellus) were distributed into 3 groups (n=12): control group that received only water orally (CON), and two treated groups that received hydroalcoholic oleander extract at doses of 150mg.kg-1 (OLE 150) and 300mg.kg-1 (OLE 300) in single oral dose. After three hours, fragments of heart, kidneys, liver and brain were collected for determination of oleandrin levels. The extraction and chromatographic procedures were effective for oleandrin detection and quantification in tissues, with retention time of 1.2 min and detection limit of 0.001μg g-1. The chromatographic analysis of treated guinea pigs indicated that oleandrin is distributed equally among the analyzed tissues. The developed methodology is a reliable, effective and rapid form of diagnosis of N. oleander poisoning based on necropsy tissue samples.(AU)

Nerium oleander é uma planta cardiotóxica ornamental encontrada em áreas tropicais e subtropicais do mundo. Sua toxicidade é relacionada á presença de glicosídeos cardioativos, principalmente a oleandrina, encontrada em toda a planta. O presente estudo objetiva descrever um novo e aprimorado método para detecção da oleandrina em amostras de tecido. A determinação da oleandrina foi feita após extração utilizando técnica modificada de QuEChERS e mensuração por UFLC-MS/MS. Um total de 36 cobaios (Cavia porcellus) foi distribuído em três grupos (n=12): grupo controle que recebeu apenas água por via oral (CON), e dois grupos tratados que receberam extrato hidroalcóolico de oleander nas doses de 150mg.kg-1 (OLE 150) e 300mg.kg-1 (OLE 300) em uma única dose oral. Após três horas, fragmentos do coração, rins, fígado e cérebro foram coletados para determinação dos níveis de oleandrina. A extração e procedimentos cromatográficos foram eficientes na detecção e quantificação da oleandrina nos tecidos, com tempo de retenção de 1,2min e limite de detecção de 0,001μg g-1. A análise cromatográfica dos animais tratados indicou que a oleandrina é distribuída de forma equalizada pelos tecidos analisados. A metodologia desenvolvida representa uma forma de diagnóstica segura, efetiva e rápida da intoxicação por N. oleander a partir de amostras de tecidos de necropsia.(AU)

Determination of Oleandrin in Blood and Liver Samples by LC-MS/MS.

OBJECTIVES: To develop a liquid chromatography-tandem mass spectrometry （LC-MS/MS） analytical method for the determination of oleandrin in blood and liver tissues, which could be applied to the cases of death caused by oleander poisoning. METHODS: Blood or liver tissues underwent a liquid-liquid extraction （LLE） using ethyl acetate, and the extract was separated on an Agilent ZORBAX SB-C18 column and eluted with a gradient of acetonitrile and 20 mmol/L ammonium acetate （containing 0.1% formic acid）. Oleandrin was detected using electrospray positive ionization （ESI+） with multiple-reaction monitoring （MRM） mode. RESULTS: Oleandrin showed excellent linearity in both blood and liver samples in the corresponding linear range （r>0.995 0）, with detection limits 1 ng/mL and 2 ng/g, respectively, extraction recovery rates greater than 70.50%, both intra- and inter-day precisions less than 10.71%, accuracies 98.42%-111.63%, and matrix effects 91.52%-106.39%. The method was successfully applied to a case of suspected oleander poisoning. Oleandrin was detected in the blood, urine, liver tissues, bile, stomach wall tissues and stomach contents of the cadaver, with the content ranging from 65.5 to 29 600.0 ng/mL （ng/g）. CONCLUSIONS: The method developed in this study is simple and convenient to operate with good selectivity, and is suitable for the analysis of oleandrin in biological samples such as blood and liver tissues, which can provide technical support for forensic identification and clinical diagnosis and treatment of oleander poisoning.

Evading plant defence: Infestation of poisonous milkweed fruits (Asclepiadaceae) by the fruit fly Dacus siliqualactis (Diptera: Tephritidae).

To cope with toxic metabolites plants use for defence, herbivorous insects employ various adaptive strategies. For oviposition, the fruit fly Dacus siliqualactis (Tephritidae) uses milkweed plants of the genus Gomphocarpus (Asclepiadaceae) by circumventing the plant's physical (gluey latex) and chemical (toxic cadenolides) defence. With its long, telescope-like ovipositor, the fly penetrates the exo- and endocarp of the fruit and places the eggs on the unripe seeds located in the centre of the fruit. Whereas most plant parts contain high concentrations of cardenolides such as gomphoside, calotropin/calacatin and gomphogenin, only the seeds exhibit low cardenolide levels. By surmounting physical barriers (fruit membranes, latex), the fly secures a safe environment and a latex-free food source of low toxicity for the developing larvae. One amino acid substitution (Q111V) at the cardenolide binding site of the fly's Na+, K+-ATPase was detected, but the significance of that substitution: reducing cardenolide sensitivity or not, is unclear. However, poisoning of the larvae by low levels of cardenolides is assumed to be prevented by non-resorption and excretion of the polar cardenolides, which cannot passively permeate the midgut membrane. This example of an insect-plant interaction demonstrates that by morphological and behavioural adaptation, a fruit fly manages to overcome even highly effective defence mechanisms of its host plant.

Cytotoxicity and chromosomal aberrations induced by methanolic extract of Cuscuta reflexa and its pure compounds on meristematic cells of Allium species.

Cuscuta reflexa (Convolvulaceae), is commonly known as amarbel or akashbel. In Bangladesh and Nepal some of the tribes use C. reflexa against edema, body ache, cancer, skin infections and liver disorders. Despite its traditional uses there is no information regarding genotoxic effects of either the plant extract or its pure compounds. Methanolic extract of C. reflexa (MECR) and pure compounds derived from it namely, odoroside H, neritaloside, and strospeside, were evaluated in Allium cepa L. and A. sativum L. for their effects on root growth, root apical meristem mitotic index (MI) , and chromosomal aberrations (CAs). In this study, we adopted a new method of calculating percent change in root length. MECR caused a concentration- and time- dependent inhibition in root length at 100 - 10000µg/ml in A. cepa root. It was accompanied by a subsequent decline in MI which is an indicative of its cytotoxic effect. On the contrary, at low concentrations a significant rise in root length was noticeable. In A. sativum, MECR also reduced the root length having IC50 values ~8 x and 4.3 x lower than A. cepa. A variety of CAs were evident in both Allium systems after treatment with MECR, odoroside H and neritaloside. Thus in MECR, cardenolides glycosides, i.e. odoroside H and neritaloside could be accountable for its genotoxicity.

Simultaneous determination of eight alkaloids and oleandrin in herbal cosmetics by dispersive solid-phase extraction coupled with ultra high performance liquid chromatography and tandem mass spectrometry.

We utilized ultra-high performance liquid chromatography with tandem mass spectrometry and dispersive solid-phase extraction to develop a new method for the detection of nine analytes (scopolamine, cephaeline, strychnine, hyoscyamine, brucine, hydrastine, ajmalicine, colchicine, and oleandrin) in herbal cosmetics. Acetonitrile/water and 2-propylaminoethylamine were used to disperse and purify during the dispersive solid-phase extraction step. The analytes were separated by a Waters UPLC HSS T3 column and detected through electrospray ionization source in the positive mode with multi-reaction monitoring conditions. Under the optimal conditions, the calibration curves were linear in the range of 0.2-100.0 µg/L with the correlation coefficients higher than 0.995. The method limit of quantitation (S/N = 10) were 5.0 µg/kg for oleandrin and 1.0 µg/kg for the other eight alkaloids. The mean recoveries at three spiked concentration levels of 1.0-10.0 µg/kg were in the range of 86.9-116.5% with the intra-day relative standard deviations (n = 6) ranging from 2.4 to 8.8%, and inter-day relative standard deviations ranging from 2.7 to 5.7%. This method is accurate, simple and rapid, and has been applied to the quality supervision of herbal cosmetics in Guangzhou.

Effect of extraction, microbial fermentation and storage on the cardenolide profile of Strophanthus kombé Oliv. seed preparations.

Strophanthus extracts containing cardioactive cardenolides are still applied in European complementary medicine for the treatment of heart diseases. However, the cardenolide profile and the fate of individual compounds during extraction and storage are not well understood. Therefore, the objective of the present study was to characterize the cardenolide compound pattern in extracts of different polarity and their structural changes upon storage in aqueous fermented preparations. For this purpose, individual cardenolides were quantitated by a UHPLC-DAD validated method using an internal standard. Three different extraction protocols were compared: hydroethanolic extraction under reflux with and without previous defatting of the seed material and ultrasonic-assisted extraction at ambient temperature. Reflux extraction of non-defatted seeds showed maximum cardenolide yields. Differences in the cardenolide contents of seeds of the different origins Zimbabwe and Malawi were observed. The cardenolide profile and metabolization of individual compounds upon fermentation and storage of S. kombé seed extracts revealed that predominant cardenolides, mainly strophanthidin glycosides, changed upon storage over 12 months. Cardenolides exhibiting two or three saccharide moieties were degraded presumably by ß-glucosidase activities, originating from the plant material or lactobacilli, releasing the corresponding monoglycosides. The latter were further degraded into the corresponding aglycones probably by acid hydrolysis as a result of lactic acid accumulation.