ANT-Mediated Inhibition of the Permeability Transition Pore Alleviates Palmitate-Induced Mitochondrial Dysfunction and Lipotoxicity.

Hyperlipidemia is a major risk factor for vascular lesions in diabetes mellitus and other metabolic disorders, although its basis remains poorly understood. One of the key pathogenetic events in this condition is mitochondrial dysfunction associated with the opening of the mitochondrial permeability transition (MPT) pore, a drop in the membrane potential, and ROS overproduction. Here, we investigated the effects of bongkrekic acid and carboxyatractyloside, a potent blocker and activator of the MPT pore opening, respectively, acting through direct interaction with the adenine nucleotide translocator, on the progression of mitochondrial dysfunction in mouse primary lung endothelial cells exposed to elevated levels of palmitic acid. Palmitate treatment (0.75 mM palmitate/BSA for 6 days) resulted in an 80% decrease in the viability index of endothelial cells, which was accompanied by mitochondrial depolarization, ROS hyperproduction, and increased colocalization of mitochondria with lysosomes. Bongkrekic acid (25 µM) attenuated palmitate-induced lipotoxicity and all the signs of mitochondrial damage, including increased spontaneous formation of the MPT pore. In contrast, carboxyatractyloside (10 µM) stimulated cell death and failed to prevent the progression of mitochondrial dysfunction under hyperlipidemic stress conditions. Silencing of gene expression of the predominate isoform ANT2, similar to the action of carboxyatractyloside, led to increased ROS generation and cell death under conditions of palmitate-induced lipotoxicity in a stably transfected HEK293T cell line. Altogether, these results suggest that targeted manipulation of the permeability transition pore through inhibition of ANT may represent an alternative approach to alleviate mitochondrial dysfunction and cell death in cell culture models of fatty acid overload.

Mitochondrial uncoupling caused by a wide variety of protonophores is differently sensitive to carboxyatractyloside in rat heart and liver mitochondria.

Mitochondrial uncoupling by small-molecule protonophores is generally accepted to proceed via transmembrane proton shuttling. The idea of facilitating this process by the adenine nucleotide translocase ANT originated primarily from the partial reversal of the DNP-induced mitochondrial uncoupling by the ANT inhibitor carboxyatractyloside (CATR). Recently, the sensitivity to CATR was also observed for the action of such potent OxPhos uncouplers as BAM15, SF6847, FCCP and niclosamide. Here, we report measurements of the CATR effect on the activity of a large number of conventional and novel uncouplers in isolated mammalian mitochondria. Despite the broad variety of chemical structures, CATR attenuated the uncoupling efficacy of all the anionic protonophores in rat heart mitochondria with high abundance of ANT, whereas the effect was much less pronounced or even absent, e.g. for SF6847, in rat liver mitochondria with low ANT content. The fact that the uncoupling action is tissue specific for a broad spectrum of anionic protonophores is highlighted here for the first time. Only with the cationic uncoupler ellipticine and the channel-forming peptide gramicidin A, no sensitivity to CATR was found even in rat heart mitochondria. By contrast, with the recently described ester-stabilized ylidic protonophores [Kirsanov et al. Bioelectrochemistry 2023], the stimulating effect of CATR was discovered both in liver and heart mitochondria.

Synthetic Derivatives of Natural ent-Kaurane Atractyligenin Disclose Anticancer Properties in Colon Cancer Cells, Triggering Apoptotic Cell Demise.

The antitumor activity of different ent-kaurane diterpenes has been extensively studied. Several investigations have demonstrated the excellent antitumor activity of synthetic derivatives of the diterpene atractyligenin. In this research, a series of new synthetic amides and their 15,19-di-oxo analogues obtained from atractyligenin by modifying the C-2, C-15, and C-19 positions were designed in order to dispose of a set of derivatives with different substitutions at the amidic nitrogen. Using different concentrations of the obtained compounds (10-300 µM) a reduction in cell viability of HCT116 colon cancer cells was observed at 48 h of treatment. All the di-oxidized compounds were more effective than their alcoholic precursors. The di-oxidized compounds had already reduced the viability of two colon cancer cells (HCT116 and Caco-2) at 24 h when used at low doses (2.5-15 µM), while they turned out to be poorly effective in differentiated Caco-2 cells, a model of polarized enterocytes. The data reported here provide evidence that di-oxidized compounds induced apoptotic cell death, as demonstrated by the appearance of condensed and fragmented DNA in treated cells, as well as the activation of caspase-3 and fragmentation of its target PARP-1.

Investigating the Broad Matrix-Gate Network in the Mitochondrial ADP/ATP Carrier through Molecular Dynamics Simulations.

The mitochondrial ADP/ATP carrier (AAC) exports ATP and imports ADP through alternating between cytosol-open (c-) and matrix-open (m-) states. The salt bridge networks near the matrix side (m-gate) and cytosol side (c-gate) are thought to be crucial for state transitions, yet our knowledge on these networks is still limited. In the current work, we focus on more conserved m-gate network in the c-state AAC. All-atom molecular dynamics (MD) simulations on a variety of mutants and the CATR-AAC complex have revealed that: (1) without involvement of other positive residues, the charged residues from the three Px[DE]xx[KR] motifs only are prone to form symmetrical inter-helical network; (2) R235 plays a determinant role for the asymmetry in m-gate network of AAC; (3) R235 significantly strengthens the interactions between H3 and H5; (4) R79 exhibits more significant impact on m-gate than R279; (5) CATR promotes symmetry in m-gate mainly through separating R234 from D231 and fixing R79; (6) vulnerability of the H2-H3 interface near matrix side could be functionally important. Our results provide new insights into the highly conserved yet variable m-gate network in the big mitochondrial carrier family.

The newborn Fmr1 knockout mouse: a novel model of excess ubiquinone and closed mitochondrial permeability transition pore in the developing heart.

BACKGROUND: Mitochondrial permeability transition pore (mPTP) closure triggers cardiomyocyte differentiation during development while pathological opening causes cell death during myocardial ischemia-reperfusion and heart failure. Ubiquinone modulates the mPTP; however, little is known about its mechanistic role in health and disease. We previously found excessive proton leak in newborn Fmr1 KO mouse forebrain caused by ubiquinone deficiency and increased open mPTP probability. Because of the physiological differences between the heart and brain during maturation, we hypothesized that developing Fmr1 KO cardiomyocyte mitochondria would demonstrate dissimilar features. METHODS: Newborn male Fmr1 KO mice and controls were assessed. Respiratory chain enzyme activity, ubiquinone content, proton leak, and oxygen consumption were measured in cardiomyocyte mitochondria. Cardiac function was evaluated via echocardiography. RESULTS: In contrast to controls, Fmr1 KO cardiomyocyte mitochondria demonstrated increased ubiquinone content and decreased proton leak. Leak was cyclosporine (CsA)-sensitive in controls and CsA-insensitive in Fmr1 KOs. There was no difference in absolute mitochondrial respiration or cardiac function between strains. CONCLUSION: These findings establish the newborn Fmr1 KO mouse as a novel model of excess ubiquinone and closed mPTP in the developing heart. Such a model may help provide insight into the biology of cardiac development and pathophysiology of neonatal heart failure. IMPACT: Ubiquinone is in excess and the mPTP is closed in the developing FXS heart. Strengthens evidence of open mPTP probability in the normally developing postnatal murine heart and provides new evidence for premature closure of the mPTP in Fmr1 mutants. Establishes a novel model of excess CoQ and a closed pore in the developing heart. Such a model will be a valuable tool used to better understand the role of ubiquinone and the mPTP in the neonatal heart in health and disease.

A Single Cysteine Residue in the Translocation Pathway of the Mitosomal ADP/ATP Carrier from Cryptosporidium parvum Confers a Broad Nucleotide Specificity.

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

Simultaneous quantification of atractyloside and carboxyatractyloside in rat plasma by LC-MS/MS: Application to a pharmacokinetic study after oral administration of Xanthii Fructus extract.

Xanthii Fructus is extensively used as an herbal medicine. Ingestion of this herb is associated with severe hepatotoxicity and nephrotoxicity. Atractyloside and carboxyatractyloside are two dominative toxic constituents in Xanthii Fructus. However, their pharmacokinetic study is lacking. In this study, a novel high-performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously quantify the rat plasma concentrations of atractyloside and carboxyatractyloside. After protein precipitation, the analytes were chromatographic separated on a ZORBAX Eclipse Plus column (2.1 × 150 mm id, 5 µm) under gradient elute. In the negative electrospray ionization mode, the transitions at m/z 725.3→645.4 for atractyloside, m/z 769.3→689.4 for carboxyatractyloside, and m/z 479.2→121.1 for paeoniflorin (the internal standard) were acquired by multiple reaction monitoring. This analytical method showed good linearity over 1-500 ng/mL for atractyloside and 2-500 ng/mL for carboxyatractyloside with acceptable precision and accuracy. No matrix effect, instability and carryover occurred in the analysis procedure. The extraction recoveries were greater than 85.0%. This method was applied to a preliminary pharmacokinetic study by orally administering Xanthii Fructus extract (9 g/kg) to rats, which was useful to evaluate the role of these two compounds in Xanthii Fructus-induced toxicity.

Atractyligenin, a terpenoid isolated from coffee silverskin, inhibits cutaneous photoaging.

Ultraviolet (UV) light exposure-induced photoaging of the skin is a multifactorial process involving both extrinsic and intrinsic cellular mechanisms. Several naturally occurring products are known to confer protection against UV light-induced skin damage. Our preliminary studies confirmed that the ethyl acetate fraction of coffee silverskin exhibits inhibitory effects on matrix metalloproteases (MMPs). Furthermore, we previously isolated and identified atractyligenin, which has MMP-inhibitory activity, from the silverskin ethyl acetate fraction. The aim of this study was to elucidate the anti-photoaging effects of atractyligenin on human dermal fibroblasts and the underlying mechanism. Human dermal fibroblasts were exposed to 8 J/cm2 UVA radiation, and cell viability was analyzed by MTT assay. The fluorescent dye 2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) was used to measure the intracellular reactive oxygen species (ROS) levels. Our study showed that atractyligenin significantly suppressed the expression of UVA-induced MMPs by inhibiting intracellular ROS production. Atractyligenin treatment reduced c-Jun phosphorylation and c-Fos expression by inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway activated by UVA irradiation. Additionally, treatment with atractyligenin contributed to the homeostasis of collagen by restoring the loss of collagen absorption-related receptor Endo180 and altered fibroblast morphology induced by UVA irradiation. These results indicate that atractyligenin isolated from coffee silverskin inhibits multiple pathways in the human skin photoaging process and is thus a potential candidate for treatment or prevention of photoaging.

Bioavailability and Biological Effects of 2- O-ß-d-Glucopyranosyl-carboxyatractyligenin from Green Coffee in Caenorhabditis elegans.

Targeted analysis of Coffea arabica and Coffea canephora green coffees (total sample size n = 57) confirmed 2- O-ß-d-glucopyranosyl-carboxyatractyligenin (6) as the quantitatively dominating carboxyatractyligenin derivative. Its abundance in Arabicas (2425 ± 549 nmol/g, n = 48) exceeded that in Robustas (34 ± 12 nmol/g, n = 9) roughly by a factor of 70. Coffee processing involving heat (e.g., steam treatment and decaffeination) reduced concentrations of 6 and increased those of the decarboxylated derivative. The bioavailability of compound 6 in Caenorhabditis elegans was demonstrated by ultraperformance liquid chromatography-tandem mass spectrometry analysis of extracts prepared from nematode cultures incubated in a liquid medium containing 6. A toxicity assay performed to assess the impact of 6 in vivo showed a 20-fold higher median lethal dose (LD50 = 11.7 ± 1.2 mM) concentration compared to that of the known phytotoxic adenine-nucleotide transporters inhibitor carboxyatractyloside (2, LD50 = 0.61 ± 0.05 mM), whereas 1 mM 6 and 0.1 mM 2 were sufficient to decrease the survival of wild type C. elegans, already 10-20-fold lower doses reduced reproduction. Because the insulin/insulin-like growth factors signaling cascade (IIS) is a key regulator of life span and stress resistance, the impact of compound 6 on the survival of long-living daf-2 C. elegans was tested. As the susceptibility of these nematodes to 6 was as high as that in wild type, an impact on central metabolic processes independent of IIS was suggested. Analysis of the in vivo adenosine triphosphate (ATP) content of adult C. elegans revealed no changes after 1 and 24 h, but a 50% reduction after treatment with 1 mM 6 during the entire postembryonic development. These data speak for a developmental-stage-dependent modulation of the ATP pool by 6.

Chemometrics coupled with UPLC-MS/MS for simultaneous analysis of markers in the raw and processed Fructus Xanthii, and application to optimization of processing method by BBD design.

BACKGROUND: As a widely used toxic traditional herbal medicine, the quality of the Fructus Xanthii must be well controlled to ensure the clinical therapeutic efficacy and safety. AIMS: A rapid, and sensitive using ultra-high performance liquid chromatography to triple quadrupole tandem mass spectrometry (UPLC-MS/MS) in selected reaction monitoring (SRM) mode was developed and validated for simultaneous quantitation of determination active and toxic ingredients form processed by stir-frying and raw materials of Fructus Xanthii. METHODS: Chromatographic separation of all targeted compound was performed on Waters ACQUITY UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 µm). Moreover, the method was successfully applied in thirty-six samples of Fructus Xanthii collected from different sources in China. The processing method was optimized through Box-Behnken statistical design and response surface methodology. RESULTS: In this work, chemometrics was able to successfully discriminate and classify among samples. The optimal incubation conditions were as follows: under heating in a pot at 295 °C, medicine at 120 °C for 11.0 min with flipping frequently. CONCLUSIONS: Therefore, the established UPLC-QQQ-MS method in combination with chemometric analysis provides a rapid, flexible and reliable method for quality assessment of Fructus Xanthii. Importantly, the optimized experimental value of the processing process provides the basis for future research.

Acyl atractyligenin and carboxyatractyligenin glycosides from Antennaria rosea subsp. confinis.

Eight previously undescribed acyl atractyligenin and carboxyatractyligenin glycosides were isolated from whole Antennaria rosea subsp. confinis (Greene) R. J. Bayer (Compositae) [syn. Leontopodium leontopodioides (Willd.) Beauv. (Asteraceae)] plants and their structures were determined by spectroscopic and chemical methods. The compounds were trivially named leontopodiosides F-M. Seven of the compounds showed potent in vitro inhibitory activity toward pancreatic lipase with IC50 values ranging from 3.4 to 52.5 µM, suggesting that they participate in the previously observed effect this plant has in reducing triglyceride absorption in rats.

Experimental poisoning by Vernonia rubricaulis in sheep.

In order to evaluate the susceptibility of sheep to V. rubricaulis and to establish the clinical signs, serum biochemistry, and pathological findings, eight sheep were fed varying doses of V. rubricaulis. The onset of clinical signs occurred 6-48 h after the ingestion of V. rubricaulis. Clinical courses lasted 6-56 h after the ingestion of the plant. Serum activities of aspartate aminotransferase, gamma-glutamyl transferase, and alkaline phosphatase were highly elevated and glucose blood levels were low in affected sheep. Clinical signs consisted of apathy, anorexia, dry muzzle, respiratory distress, abdominal pain, and mushy feces with streaks of blood and mucus. Two sheep had neurological signs including muscle fasciculation, nystagmus, paddling movements, and blindness. Liver necrosis could be detected antemortem through liver biopsy. Five sheep died and three recovered. The liver was affected in all necropsied sheep; it increased in volume and had marked accentuation of the lobular pattern with red, depressed areas intercalated with a pale yellow network. Ascites and hydropericardium were consistent findings. Microscopically, centrilobular to massive coagulative necrosis was observed. Coagulative necrosis was also observed in a few proximal renal tubules. Microscopic lesions were not found in any other organs. The severity of liver lesions was proportional to the dose. Chemical analysis to detect carboxyatractyloside in V. rubricaulis plant material was negative. It is concluded that V. rubricaulis poisoning in sheep is clinically, biochemically, and pathologically characteristic of an acute hepatoxicosis.

UHPLC-PDA-ESI-TOF/MS metabolic profiling and antioxidant capacity of arabica and robusta coffee silverskin: Antioxidants vs phytotoxins.

A deeper knowledge of the chemical composition of coffee silverskin (CS) is needed due to the growing interest in its use as a food additive or an ingredient of dietary supplements. Accordingly, the aim of this paper was to investigate the metabolic profile of aqueous extracts of two varieties of CS, Coffee arabica (CS-A), Coffee canephora var. robusta (CS-R) and of a blend of the two (CS-b) and to compare it to the profile of Coffee arabica green coffee (GC). Chlorogenic acids, caffeine, furokauranes, and atractyligenins, phytotoxins not previously detected in CS, were either identified or tentatively assigned. An unknown compound, presumably a carboxyatractyligenin glycoside was detected only in GC. Caffeine and chlorogenic acids were quantified while the content of furokauranes and atractyligens was estimated. GC and CS were also characterized in terms of total polyphenols and antioxidant capacity. Differences in the metabolites distribution, polyphenols and antioxidant capacity in GC and CS were detailed.

Mitochondrial ATP transporter depletion protects mice against liver steatosis and insulin resistance.

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment.

To involvement the conformation of the adenine nucleotide translocase in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl(+)-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria. Using the Tl(+)-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.

Identification of new highly selective inhibitors of the human ADP/ATP carriers by molecular docking and in vitro transport assays.

Mitochondrial carriers are proteins that shuttle a variety of metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. The mitochondrial ADP/ATP carriers (AACs) specifically translocate the ATP synthesized within mitochondria to the cytosol in exchange for the cytosolic ADP, playing a key role in energy production, in promoting cell viability and regulating mitochondrial permeability transition pore opening. In Homo sapiens four genes code for AACs with different tissue distribution and expression patterns. Since AACs are dysregulated in several cancer types, the employment of known and new AAC inhibitors might be crucial for inducing mitochondrial-mediated apoptosis in cancer cells. Albeit carboxyatractyloside (CATR) and bongkrekic acid (BKA) are known to be powerful and highly selective AAC inhibitors, able to induce mitochondrial dysfunction at molecular level and poisoning at physiological level, we estimated here for the first time their affinity for the human recombinant AAC2 by in vitro transport assays. We found that the inhibition constants of CATR and BKA are 4 nM and 2.0 µM, respectively. For finding new AAC inhibitors we also performed a docking-based virtual screening of an in-house developed chemical library and we identified about 100 ligands showing high affinity for the AAC2 binding region. By testing 13 commercially available molecules, out of the 100 predicted candidates, we found that 2 of them, namely suramin and chebulinic acid, are competitive AAC2 inhibitors with inhibition constants 0.3 µM and 2.1 µM, respectively. We also demonstrated that chebulinic acid and suramin are "highly selective" AAC2 inhibitors, since they poorly inhibit other human mitochondrial carriers (namely ORC1, APC1 and AGC1).

Enhanced oxidative stress sensitizes the mitochondrial permeability transition pore to opening in heart from Zucker Fa/fa rats with type 2 diabetes.

AIMS: Obesity and diabetes mellitus type 2 (DM2) frequently coexist and increase the propensity of cardiovascular dysfunction by numerous mechanisms. Chief among them are oxidative stress and Ca(2+) dysregulation, and both are inducers of the mitochondrial permeability transition pore (MPTP). Nevertheless, it is unknown whether MPTP formation is triggered in DM2 animals, and thereby contributing to cardiac dysfunction. We assessed MPTP sensitivity and reactive oxygen species production in cardiac mitochondria, as well as cytosolic Ca(2+) handling in ventricular myocytes from rats with DM2. MAIN METHODS: Male Zucker Fa/fa rats (Fa/fa) 32weeks old presenting DM2, concentric hypertrophy, and diastolic dysfunction were used. MPTP formation was evaluated in isolated mitochondria and Ca(2+) handling in ventricular myocytes, by spectrophotometric and confocal microscope techniques, respectively. KEY FINDINGS: We found that the systolic Ca(2+) transient relaxation was ~40% slower, while mitochondrial H2O2 production increased by ~6-fold. MPTP opening in isolated mitochondria from Fa/fa (mFa/fa) was more sensitive to Ca(2+) than in mitochondria from lean rats (mLean), and correlated with increased thiol group exposure. The mFa/fa showed decreased oxidative phosphorylation capacity. The ATP content decreased in myocytes, while the PCr/ATP ratio remained unchanged and caspase 9 activity largely increased in myocytes from Fa/fa animals. SIGNIFICANCE: Our results showed that oxidative stress and diastolic Ca(2+) dysregulation increased MPTP sensitivity leading to mitochondrial dysfunction and apoptosis. Mitochondrial dysfunction could compromise ATP synthesis, and lower ATP could be linked to decreased SERCA2 activity, which might underlie diastolic dysfunction. Prolonged Ca(2+) transients might further exacerbate mitochondrial dysfunction.

Influence of Processing on the Content of Toxic Carboxyatractyloside and Atractyloside and the Microbiological Status of Xanthium sibiricum Fruits (Cang'erzi).

The dried ripe fruits of Xanthium sibiricum (Cang'erzi) are used in traditional Chinese medicine for the treatment of nasal congestion, nasal discharge, allergic rhinitis, sinusitis, and wind-cold headaches. Carboxyatractyloside and atractyloside are important constituents of the fruits because these diterpenoid glycosides are responsible for their toxicity. In order to evaluate procedures for reducing the amount of the more toxic carboxyatractyloside, the fruits were dried and heated with different methods. Carboxyatractyloside and atractyloside were analysed by a new reversed-phase high-performance liquid chromatographic method using liquid chromatography-diode array detector-tandem mass spectrometry analysis. The results revealed that temperature and drying methods have a strong influence on the content of carboxyatractyloside and atractyloside. Fruits which were treated at higher temperatures showed a lower content of carboxyatractyloside and an increased content of atractyloside, which is 50 times less toxic. This indicates that the roasting process can reduce toxicity effectively. The microbiological colonisation of Xanthium fruits is also reduced by roasting and by drying above 100 °C. For the safe use of Cang'erzi, the effect of processing should be monitored and analysis of carboxyatractyloside and atractyloside should be obligatory in quality control.

Analytical confirmation of Xanthium strumarium poisoning in cattle.

Xanthium strumarium, commonly referred to as "cocklebur," rarely causes poisoning in cattle. When mature, this robust, annual weed bears numerous oval, brownish, spiny burs. Only the seeds in the burs and young seedlings (cotyledonary leaves) contain the toxic principle, carboxyatractyloside. In the Frankfort district of the Free State Province of South Africa, a herd of 150 Bonsmara cows were allowed to graze on the banks of a small river, where mature cocklebur was growing. Four cows died while grazing in this relatively small area. Clinical signs ranged from recumbency, apparent blindness, and hypersensitivity to convulsive seizures. During necropsy, burs completely matted with ingesta were located in the rumen content. The most distinctive microscopic lesions were severe, bridging centrilobular to midzonal hepatocyte necrosis and hemorrhage. Ultrastructurally, periacinar hepatocytes were necrotic, and novel electron-dense cytoplasmic needle-like crystals were observed, often in close association with peroxisomes. Carboxyatractyloside concentrations were determined using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Carboxyatractyloside was present in rumen contents at 2.5 mg/kg; in burs removed from the rumen at 0.17 mg/kg; in liver at 66 ng/g, and was below the limit of quantitation in the kidney sample, estimated at approximately 0.8 ng/g. Based on the presence of the plants on the riverbank, the history of exposure, the clinical findings, the presence of burs in the rumen, and the microscopic and ultrastructural lesions, X. strumarium poisoning in the herd of cattle was confirmed and was supported by LC-HRMS.

A validated method for quantifying atractyloside and carboxyatractyloside in blood by HPLC-HRMS/MS, a non-fatal case of intoxication with Atractylis gummifera L.

Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle (Atractylis gummifera L.) and Zulu impila (Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavored to fill this analytical gap. Analysis was carried out using a solid-phase extraction and a high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry detection. The method was validated in the whole blood with quantification limits of 0.17 and 0.15 µg/L for ATR and CATR, respectively. The method was applied to a non-fatal case of intoxication with A. gummifera. To the best of the authors' knowledge, this is the first time that a concentration of ATR and CATR in blood (883.1 and 119.0 µg/L, respectively) and urine (230.4 and 140.3 µg/L, respectively) is reported. ATR and CATR were quantified in A. gummifera roots by the standard method addition (3.7 and 5.4 mg/g, respectively).