Antitumor effect of blister beetles: an ethno-medicinal practice in Karbi community and its experimental evaluation against a murine malignant tumor model.

ETHNOPHARMACOLOGICAL IMPORTANCE: The blister beetles Epicauta hirticornis and Mylabris cichorii are used as a folk medicine by the Karbi tribe in Karbi Anglong district of Assam, India for the treatment of different human ailments, including cancer cases. AIM OF THE STUDY: It includes field survey related to zoo-therapeutic aspects of two blister beetles in Karbi community, isolation of bio-active compound and evaluation of its antitumor potential with possible mode of action against murine Ehrlich ascites carcinoma (EAC). MATERIALS AND METHODS: The main bio-active compound of blister beetles was isolated from ethyl acetate extract and the structure was confirmed as cantharidin using NMR, IR, Mass and X-ray diffractometer. The effect of cantharidin on apoptosis, necrosis, autophagy and the apoptosis related signaling pathways were determined using different bioassays, including cell cycle analysis, mitochondrial membrane potential, western blot analysis of cytochrome c, caspases 9, 3/7 assays, and lactate dehydrogenase (LDH) assay. RESULTS: Cantharidin induced apoptosis, necrosis and autophagy cell death in EAC cells. The decrease in mitochondrial membrane potential was observed, which may help to release cytochrome c from mitochondria to cytosol. Cantharidin treatment caused up-regulation of caspases 9 and -3/7 and a decrease in LDH activity in EAC cells. CONCLUSION: The major bioactive compound of these blister beetles is cantharidin which induces severe apoptosis in EAC cells involving mitochondrial intrinsic pathway. Cantharidin-mediated inhibition of LDH activity may lead to short supply of NAD(+) and cut off energy and anabolic supply to cancer cells.

Changes in glutathione, oxidative stress and mitochondrial membrane potential in apoptosis involving the anticancer activity of cantharidin isolated from redheaded blister beetles, epicauta hirticornis.

The present work describes the anticancer activity of cantharidin isolated from red-headed blister beetles, Epicauta hirticornis and its possible mode of action involving induction of apoptosis, oxidative stress and decrease in glutathione against murine ascites Dalton's lymphoma. The structure of isolated compound was confirmed as cantharidin by X-ray diffraction method. Cantharidin treatment showed potent anticancer activity with an increase in life span (~ 87%) of tumor-bearing mice. Cantharidin treatment induced apoptosis in Dalton's lymphoma cells and also caused an oxidative stress due to generation of reactive oxygen species (ROS) and an increase in lipid peroxidation. The observed canthardin-mediated decrease in glutathione and glutathione related enzymes activities in the tumor cells may weaken the cellular antioxidant system. Moreover, cantharidin treatment also caused a significant decrease in mitochondrial cytochrome c and simultaneous increase in cytosolic cytochrome c which ultimately facilitates activation of caspase 9 and 3 to augment mitochondrial apoptotic pathway causing cancer cell death. Based on the present findings, it may be suggested that cantharidin-mediated anticancer activity could be due to decrease in the protective ability of cancer cells by ROS and subsequent activation of effecter caspases leading to apoptotic cell death.

Religious objection to cantharidin use in Jain patients.

We report a case of a Jain family that was upset with the use of cantharidin for treatment of molluscum contagiosum for religious reasons.

[Comparative study of antitumor activity of cantharidin and cantharis peptides].

OBJECTIVE: To prepare cantharidin and cantharidin peptides from Mylabris and compare their antitumor activity. METHODS: Cantharis peptides was prepared by bionic enzymolysis approach and cantharidin was prepared by alkaline water supersonic extraction. The inhibitory effects of both compounds on BEL-7402 cells proliferation of human liver cancer were tested by Prestoblue method. The influence of both compounds on the grown of tumor, thymus, spleen of S180 tumor-bearing mice were detected. RESULTS: Cantharis peptides and cantharidin could inhibit the proliferation of BEL-7402 cells (P < 0.05) in vivo, and the inhibitory effect of cantharis peptide was 12.54% lower than that of cantharidin. At the same time, they could inhibit the grown of S180 sarcoma (P < 0.05), and the inhibitory effect of cantharidin was higher than that of cantharis peptides (5.93%). Furthermore, cantharis peptides could't inhibit the grown of thymus and spleen. CONCLUSION: Both cantharis peptides and cantharidin have antineoplastic activity, but cantharidin peptides have no immunosuppression.

Bioactive component, cantharidin from Mylabris cichorii and its antitumor activity against Ehrlich ascites carcinoma.

The anticancer activity of the extract of blister beetle, Mylabris cichorii has been documented earlier by us. In the present study, the active principle of M. cichorii was isolated and its anticancer efficacy was evaluated against murine Ehrlich ascites carcinoma (EAC). The isolated bioactive compound was characterized to be cantharidin which showed potent antitumor activity and inhibited the proliferation of Ehrlich ascites carcinoma, both in vivo and in vitro. Cantharidin-treated EAC-bearing mice showed about 82% increase in lifespan at the dose of 0.5 mg/kg/day. In vitro cytotoxicity assay with the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed about 50% cell death at the concentration of 25.8 µg/ml. The fluorescence and transmission electron microscopy revealed that EAC cells treated with cantharidin depicted typical apoptotic morphology with chromatin condensation, nuclear fragmentation into discrete masses, and plasma membrane blebbing which deduce towards the death of these cells. Histological examination of the kidney of cantharidin-treated mice showed glomerular and tubular congestion with abnormal Bowman's capsule, thus, indicating a renal toxicity in the host. Cantharidin-induced renal damage in the host was also manifested by the decreased lactate dehydrogenase isozymes and its possible release from the cells.

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.

[Preparation and characterization of non-ionic surfactant vesicle of cantharidin].

OBJECTIVE: To study the preparation of cantharidin entrapped non-ionic surfactant vesicle (noisome)and evaluate its quality. METHOD: The niosome loaded with cantharidin was prepared using injection method by non-ionic surfactants as the carrier. An centrifugation separation method and HPLC analysis method of the cantharidin were established to detect the entrapment efficiency. The optimum preparation technology was established by a orthogonal experiment. The morphology, and particle size were studied to evaluate the preparation. RESULT: The average size of niosomes were (209. 8 +/- 0.5) nm. The entrapment efficiency of the CTD-NS was (27.5% +/- 2.0%) and Zeta potential was (41.5 +/- 0.65) mV. CONCLUSION: The preparation of cantharidin noisome by TweenA and SpanB is practicable and successful. These experiments can be the basement of developing targeting drug delivery system.

[Research progress on medicinal resources of Mylabris and close origin species].

The paper summarizes the research progress on the medicinal resources of Mylabris and close origin species in recent years. Besides the 45 species in 7 genus within Meloidae insects which contain cantharidin, there are also more 9 species in 7 close origin genus containing cantharidin which include Zanna, Fulgora and Lycorma within Fulgoridae of Homoptera, Oxocopis, Heliocis Xanthochroa and Oedemera within Oedemeridae of Coleoptera. New medicinal resources of cantharidin are redundant, there are biological relationships in the biosynthesis of cantharidin, the emerge of cantharidin is related to ecology and there is more attention on the new methods of utilizing Mylabris resources such as living body extraction.

Three novel cantharidin-related compounds from the Chinese blister beetle, Mylabris phalerata Pall.

Three novel cantharidin analogues were isolated from the Chinese blister beetle, Mylabris phalerata PALL. (Meloidae), which has been used in traditional Chinese medicine for the treatment of cancer. Their structures were determined on the basis of heteronuclear multiple-bond connectivity and nuclear Overhauser effect spectroscopy experiments, and chemical data confirmed them to be so-called cantharimides, in which the anhydride oxygen atoms are replaced by the basic amino acid L-lysine, L-ornithine, and L-arginine moieties.

(R)-(+)-palasonin, a cantharidin-related plant toxin, also occurs in insect hemolymph and tissues.

Gas chromatographic and mass spectroscopic analyses of extracts of cantharidin-containing meloid, clerid, and staphylinid beetles revealed the presence of minor to significant amounts of palasonin, previously only known from seeds and fruits of the Indian shrub Buteafrondosa (Leguminaceae). Unlike (S)-(-)-palasonin (> 99% ee) from B. frondosa, the insects produce palasonin of low ee with the (R)-(+)-enantiomer (0-50% ee) prevailing. The ee of palasonin from individual specimens of predatory insects (Trichodes apiarius), which acquire their chemical protection from cantharidin-producing insects, may vary considerably. The absolute configuration of (S)-(-)-palasonin, previously deduced from indirect chemical and spectroscopic methods, was confirmed by X-ray crystal structure analysis of a cyclic imide derived from (S)-(-)-palasonin and (S)-(-)- 1 -(4-nitrophenyl)-ethylamine.

Cytotoxic effects of cantharidin on the growth of normal and carcinoma cells.

Cantharidin is isolated from Mylabris phalerata Pallas and is a potent inhibitor of hepatocellular carcinoma cells (Hep 3B cells). In the present study, the IC(50) values of cantharidin on Hep 3B cells and normal Chang liver cells were found to be 2.2 and 30.2 microM for 36 h, respectively. Furthermore, cantharidin-treated Hep 3B cells induced cell death within 1 h (IC(50)=52.8 microM), suggesting that cantharidin is an acute cytotoxic agent. We found that although cantharidin could induce cell death, it could not directly inhibit the activity of nucleic acid biosynthesis by the cellular incorporation of 3H-thymidine, 3H-uridine or 3H-leucine. Cantharidin-treated Hep 3B cells showed no evidence of major alterations in the cell cycle distribution within 1 h. However, examination of cells after treatment for 36 h showed that cantharidin regulated the cell cycle at the G(2)/M phase. Moreover, the treated Hep 3B cells had a rounded and shrunken appearance. The microvilli of treated Hep 3B cells were reduced in number and replaced by numerous blebs. Other ultrastructural changes following cantharidin treatment included the presence of lipid droplets, swelling of the mitochondria and accumulation of glycogen particles. The findings of damaged mitochondria in the cantharidin treated Hep 3B cells in this study suggest that cantharidin can induce acute and lethal toxic effects on Hep 3B cells by inhibiting the mitochondria energy system. In conclusion, this study had demonstrated that cantharidin could inhibit progression of all phases of the Hep 3B cell cycle.

High pressure liquid chromatographic determination of cantharidin, using a derivatization method in specimens from animals acutely poisoned by ingestion of blister beetles, Epicauta lemniscata.

Experimental animals (rabbit, rat, goat, sheep, and pony) were given cantharidin or dried preparations of blister beetles (Epicauta lemniscata) to stimulate naturally occurring toxicosis in which beetles were ingested with alfalfa hay. A sensitive high-pressure liquid chromatographic method, involving derivatization of cantharidin with p-nitrobenzyloxyamine, was developed to detect the toxin extracts of ingesta, fluids, and tissues from these severely poisoned animals. Urine and ingesta from the upper portion of the gastrointestinal tract, containing from 1 to 20 ppm of cantharidin, were the most satisfactory samples for diagnosing toxicosis. Beetle preparations also were assayed and found to contain widely varying amounts of cantharidin (0.89% to 5.40% of dry weight). Blood chemical analyses on sera and urine samples from the sheep and pony indicated renal dysfunction.