Pharmacokinetics and tissue distribution of cantharidin after oral administration of aqueous extracts from Mylabris in rats.

A sensitive gas chromatography-mass spectroscopy method was established for the determination of cantharidin (CTD) in rat plasma and liver homogenates. During the experiment, rats were randomly divided into two groups (low, high) and were administered aqueous extract of Mylabris compound for 7 days. Then, plasma and tissue samples were taken at different time points to study the pharmacokinetics and tissue distribution of CTD in rats. The selected reaction monitoring transitions for CTD and clofibrate (internal standard) were m/z 128 → 85 and m/z 169 → 141, respectively. The calibration curve ranged from 10.26 to 3,078 ng/ml for plasma and from 10.26 to 246.24 ng/ml for liver homogenates. The lower limits of quantification were 10.26 ng/ml for both plasma and liver. The intra- and inter-day precision and accuracy were <20% for both plasma and liver homogenates. Extraction recovery ranged from 89.21 to 103.61% for CTD in rat plasma and liver and from 83.79 to 102.74% for IS in rat plasma and liver. Matrix effects ranged from 93.06 to 110.44% for CTD and from 91.65 to 110.80% for IS.

Rapid profiling of cantharidin analogs in Mylabris phalerata Pallas by ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry.

We evaluated the protective effect and toxicity of extracts from Mylabris phalerata Pallas by measuring the activated partial thromboplastin time, prothrombin time, venous thrombosis and acute toxicity in rats. Results showed the petroleum ether and water fractions of M. phalerata inhibited thrombosis but hardly prolonged the activated partial thromboplastin time and prothrombin time in rats. The trichloromethane fraction had obvious toxicity with an LD50 of 0.2 g/kg in vivo, and contained many cantharidin analogs (CAs) by ultra-performance liquid chromatography-quadrupole ion trap-tandem mass spectrometry (UPLC-QTRAP-MS/MS). CAs are the major potential bioactivity constituent in M. phalerata. An effective and reliable UPLC-QTRAP-MS/MS method was successfully developed to separate and identify CAs. The fragmentation patterns of five purified compounds were applied to elucidate the structure of their analogs. Thirty-four CAs were characterized or tentatively identified, eight of which are proposed to be novel compounds (13-17, 20, 21, 23), and their fragmentation patterns were investigated for the first time. Most importantly, a rapid and reliable UPLC-MS method was developed to identify the CAs of M. phalerata. This method has contributed to the discovery of most of these unknown analogs or their metabolites in M. phalerata effectively and quickly, and does not rely on limited chemical structural diversity libraries.

Cloning and functional analysis of farnesyl pyrophosphate synthase (FPPS) gene from Mylabris cichorii.

Cantharidin, a defensive terpene compound synthesized by the meloid beetle (Coleoptera, Meloidae), is an important anticancer agent. However, there has been little study done on how this compound synthesized by the beetle. In this paper, a farnesyl pyrophosphate synthase (FPPS) gene, designated McFPPS, was isolated from Mylabris cichorii by reverse transcription PCR based on conserved domains in other organisms. Multiple alignment analysis showed that the deduced amino acids shared >70% homology with FPPSs from other species and contained typically seven conservative regions. Gene expression profile analysis revealed that McFPPS was expressed throughout the tested growth stages of M. cichorii adults, whereas the transcripts accumulated to the highest level at 20 days in male adults while the highest expression level appeared at 15 days in females. Tissue expression pattern analysis showed that McFPPS was expressed constitutively in all tested tissues and a relatively higher expression level in the alimentary canal of males, but no significant tissue difference in the females. For the first time, a RNA interference strategy was employed to induce a greater suppression of McFPPS mRNA, and thus a sharp decrease in the expression levels of downstream genes and the concentration of product. All these results indicated that McFPPS may be directly involved or play an essential role in the biosynthesis of cantharidin.

Quantitative characterization of cantharidin in the false blister beetle, Oedemera podagrariae ventralis, of the southern slopes of Mount Elborz, Iran.

Cantharidin, a potent vesicant and antifeedant agent, is produced by two families of beetles, Meloidae and Oedemeridae (Coleoptera). In this study, the cantharidin content of oedemerid beetles of central Iran was investigated using the GC-MS method. Cantharidin in both sexes of Oedemera podagrariae ventralis Meïneïtrieãs (Oedemeridae) was found in an average of 3.89 µg/beetle in males and 21.68 µg/beetle in females, which are amounts sufficient to irritate human skin. The average of cantharidin in virgin and coupled beetles was 1.35 and 1.62 (µg cantharidin/mg of beetle) respectively. Females had five to six times more cantharidin in their bodies than males, but there was no significant difference between the amount of cantharidin in virgin and coupled females. The results of this study revealed the production of cantharidin in both sexes of beetle.

[Study on the change of the content of cantharidin in Mylabris befere and after biortransfermation].

OBJECTIVE: To compare the change of the content of cantharidin in Mylabris before and after biotransformation. METHODS: The content of cantharidin was determined by gas chromatography (GC). The conditions of GC were as follows: HP-5 column, vanillin as internal standard, the column temperature maintained at 175 degrees for 5 min, rised to 240 degrees C at the speed of 4 degrees C/min and the remained for 5 min. RESULTS: The GC method was good reproducibility. The content of cantharidin in Myabris before and after botransformation was 0.7% and 1.29% respectively. After biotranformation, the content of cantharidin was increased. CONCLUSION: Mylabris fermentated with Trametes cinnabarina has great significance for increasing the content of cantharidn and provides experimentd basis for efficient use of Mylabris.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of cantharidin in biological specimens and application to postmortem interval estimation in cantharidin poisoning.

A rapid, sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination and quantification of cantharidin in rats liver and kidney. After grinding with methanol, the supernatant was determined by LC-MS/MS using an Thermo Accucore C18 column (100 mm×2.1 mm, 2.6 µm) with a gradient elution of 0.1% formic acid and 0.1% acetonitrile, and in the subsequent analysis using selected reaction monitoring mode, three ion transitions were monitored for analyte. The limit of detection (LOD) was 0.741 ng/ml and the limit of quantitation (LOQ) was 2.471 ng/ml. Good linearity (R2 = 0.9998) was observed for the analyte over the linear range (5-400 ng/ml). The LC-MS/MS method was applied to the analysis of rats liver and kidney in different postmortem intervals (6 h, 12 h, 24 h, 48 h, 72 h and 168 h after death) after a single dose (4 mg/kg) of cantharidin administration by gavage. At 72 h after death, the cantharidin concentration in livers and kidneys were significantly higher than that in other postmortem intervals. Linear regression equations between postmortem interval and lg postmortem cantharidin concentration in rats liver and kidney were Y = 0.007455*X + 1.332(R2 = 0.863) and Y = 0.002689*X + 1.433 (R2 = 0.115) respectively. The animal experiment demonstrated LC-MS/MS method can be used to determine the postmortem cantharidin concentration in rats liver and kidney and the determination of cantharidin in the rats liver after death has potential value for postmortem interval estimation in cantharidin poisoning.

Determination of trace cantharidin in plasma and pharmacokinetic study in beagle dogs using gas chromatography-mass spectrometry.

The blister beetle is traditional Chinese medicine that was first discovered and used as anticancer drug in China, and cantharidin proved to be its principal active ingredient. Cantharidin-based pharmaceutical preparations are now widely used in clinics in China with good therapeutic efficacy. As a toxic anticancer drug, the therapeutic dose of cantharidin is low, and no method to determine the blood cantharidin concentration under the therapeutic dose has so far been reported. Here, we present a simple, sensitive, and reliable gas chromatography-mass spectrometry (GC-MS) method to monitor the plasma cantharidin and perform the pharmacokinetic study of cantharidin in beagle dogs. After protein precipitation by hydrochloric acid, a liquid-liquid extraction procedure using ethyl acetate was applied to extract cantharidin from plasma. An elastic quartz capillary GC column DB-5MS was used in GC-MS, the temperature was kept at 60 degrees C for 1 min, then increased to 220 degrees C at the rate of 6 degrees C/min, held there for 1 min, and then to 280 degrees C at the rate of 20 degrees C/min, held for 3 min. The extraction recovery was over 80% for all the tested specimens. The linearity ranged from 2.14 to 314.2 ng/mL, the intra- and interday precisions were both below 20%, the limit of detection was 0.5 ng/mL, and the limit of quantification was 2.14 ng/mL. Cantharidin in plasma proved to be stable during the whole period of storage, treatment, and analysis. Cantharidin demonstrated as one-compartment model after i.v. administration with an elimination half-life of 0.69 +/- 0.03 h and area under curve of 204 +/- 24 h.ng/mL. This GC-MS assay proved to have high precision, accuracy, reliability, and sensitivity, and it was suitable for determination of trace cantharidin in plasma.

Mass spectra of N-substituted cantharidinimides.

The mass spectra of a series of N-substituted cantharidinimides were examined. The feature of this series compounds is a sequential double hydrogen transfer from the oxabicycloheptane unit to either the carbonyl group of the succinimide unit or the nitrogen atom of the pyridyl or thiazolyl substituent through space. The ability of the N-substituent to accept a hydrogen atom possibly leads to the different fragmentation pathway.

Antitumor imide derivatives of 7-oxabicyclo[2.2.1]heptane-2,3-dimethyl-2,3-dicarboxylic acid.

The imide and methylimide derivatives of 7-oxobicyclo[2.2.1]heptane-2,3-dimethyl-2,3-dicarboxylic acid were synthesized and shown to have antitumor inhibitory activity (growth inhibition) against the KB cell line. The compounds were prepared according to standard procedures. Interest in the respective imide derivatives stemmed from their structural relationship to antitumor-active derivatives of 7-oxobicyclo[2.2.1]heptane-2,3-dicarboxylic acid which lacked 2,3-dimethyl substituents or which were derivatives of isoindolines and lacked the carbonyl groups.

Post-mortem serum concentration of cantharidin in a fatal case of cantharides poisoning.

A patient admitted to hospital died shortly after admission without a proper diagnosis having been made. Symptoms as well as the presence of a brown powder found in the possession of the deceased indicated the possibility of cantharidin intoxication. Cantharidin was positively identified by means of a GC/MS analysis, utilizing the selected ion monitoring technique (SIM), for m/z = 197.0813, (M + H+) for cantharidin, under positive chemical ionization conditions at a resolution of 7000 and a mass window of 30 ppm. Quantitation was done by means of a GC/MS SIM analysis of a toluene extract of acidified post-mortem serum under El+ conditions at a resolution of 3000, using clofibrate as internal standard and monitoring m/z = 128.0473 and 128.0029 for cantharidin and clofibrate respectively. The post-mortem serum was found to contain cantharidin at a concentration of 72.3 ng/ml whilst the cantharides powder contained 0.87% cantharidin.

Etiologic agents, incidence, and improved diagnostic methods of cantharidin toxicosis in horses.

In addition to the 3-striped blister beetles (Epicauta temexa and E occidentalis), other sources of equine cantharidin toxicosis were identified at the Texas Veterinary Medical Diagnostic Laboratory and included E albida and E attrivittata and the previously incriminated E pardalis and E pennsylvanica. Improved methods for diagnosing cantharidin or blister beetle toxicosis involve partial purification of urine and gastric content extracts, using silica cartridges, followed by analysis, using capillary gas chromatography/mass spectrometry. During a 26-month period, 53 episodes of cantharidin toxicosis in horses were confirmed at our diagnostic laboratory. Concentrations of cantharidin in urine and gastric contents ranged from 0.0003 to 3.50 micrograms/g. Peak incidences were observed in late summer and early fall.

Evaluation of an analytical method for the diagnosis of cantharidin toxicosis due to ingestion of blister beetles (Epicauta lemniscata) by horses and sheep.

An analytical method for cantharidin, using high performance liquid chromatography, was applied to field specimens from horses and sheep with blister beetle (Epicauta lemniscata) poisoning. Stomach content and urine proved to be valuable aids in diagnosis. One incident of cantharidin toxicosis in ruminants (sheep) was confirmed.

Clinical features of blister beetle poisoning in equids: 70 cases (1983-1996).

OBJECTIVE: To document clinical signs and gross pathologic changes associated with naturally acquired cantharidiasis (blister beetle poisoning) in equids. DESIGN: Retrospective study. ANIMALS: 70 equids with laboratory-confirmed blister beetle poisoning. PROCEDURE: Medical records were reviewed to obtain history, physical examination findings, feeding practices, and diagnostic test and necropsy results. RESULTS: 32 horses and 2 donkeys died from exposure to cantharidin, whereas 36 horses survived. Diet content varied, but alfalfa hay was the common component. Onset of signs of disease was rapid. Most equids had signs of gastrointestinal tract distress. Six horses had nonspecific neurologic signs. All equids dying from cantharidiasis were in shock terminally, with duration of clinical signs ranging from 3 to 18 hours. Six horses that died had no gross lesions, whereas 14 had mild to moderate erythema of gastric, small intestinal, or colonic mucosa. Only 2 horses had gastric or duodenal ulceration, and 2 had hemorrhage of the urinary bladder mucosa. One horse had cardiac muscle necrosis. Clinicopathologic data available on 10 horses included hypocalcemia, hypomagnesemia, and azotemia. Cantharidin concentrations in urine or pooled gastric-cecal contents did not always correlate with severity of disease. CLINICAL IMPLICATIONS: Blister beetle poisoning is not universally fatal in equids. Clinical signs are related to the amount of cantharidin ingested. Every horse that survived was treated aggressively. In fatal poisonings, gross lesions may be minimal or inapparent, and diagnosis must be confirmed by chemical detection of cantharidin in urine, blood, or stomach or cecal contents.

A fatality due to the use of cantharides from Mylabris phalerata as an abortifacient.

A fatal case of attempting to procure abortion by the ingestion of the crude extract of cantharides from over 200 dried Mylabris phalerata is presented. The quantification of catharidin in blood, urine and liver by gas chromatography using trichloroacetic acid in the extraction process and butobarbitone as the internal standard is described. Ante- and post-mortem blood levels were found to be 0.27 and 0.11 micrograms/ml respectively. To conclude, the lack of legislative control in Hong Kong over Chinese herbal medicines is highlighted.

Mortality in chickens associated with blister beetle consumption.

Mortality in young chickens was associated with blister beetle consumption. Two species of these insects, Cyaneolytta sp. and Cylindrothorax sp., were found in the chickens' crops, and erosive lesions in the gastrointestinal tract were compatible with blister beetle poisoning (cantharidiasis).

[Cantharidin and its detection in forensic toxicology]. / Cantharidin a jeho prukaz ve forenzní toxikologii.

Experimental detection of deadly toxic cantharidin C10H12O4 by chemical analysis was performed in imagoes of two species of cryptotoxic Meloidae beetles--Lytta vesicatoria and Mylabris variabilis. Samples were processed by extraction and sublimation and analyzed by gas chromatography. Graphic documentation proved the presence of cantharidin. A part of microcristalline sublimate was studied microscopically and determinant features of crystalline cantharidin were identified. Moreover, cantharidin levels in single body parts of the beetle were investigated.

[Ultraviolet spectrophotometric determination of cantharidin in Mylabris].

In this study the content of cantharidin in Mylabris was analyzed quantitatively by UV. Analytical results thus obtained agree well with those by the neutralization method set forth in ChP. The average recovery of cantharidin is 99.98% and the coefficient of variation is 0.719%.

Analysis of cantharidin in false blister beetles (Coleoptera: Oedemeridae) by headspace solid-phase microextraction and gas chromatography-mass spectrometry.

A headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) method was developed to determine a type of terpenoid named as cantharidin in the false blister beetles, family Oedemeridae. The experimental parameters for HS-SPME method were optimized. Six commercial fibers for HS-SPME method development were tested and the divinylbenzene/carboxene/polydimethylsiloxane fiber was selected to provide the best detection of analyzed compound. The calibration curve showed linearity in the range of 0.1-50 µg mL(-1), correlation coefficient (R(2)=0.992), limit of detection (0.01 ng mL(-1)) and quantitation (0.04 ng mL(-1)) were obtained for the proposed method. The relative standard deviations of intra-day and inter-day assays were 7.8 and 3.4%, respectively. The recovery values, obtained after spiking the beetle samples by three concentration levels of standard solution, were higher than 87%. The results indicated the successful application of the proposed method on the analysis of cantharidin from the false blister beetles.