| Cantharidin is the active ingredient of the cantharis which is Tranditional Chinese Medicines. Cantharidin is also one kind of natural defensive toxins of Terpenoid. There are a lot of researches achieved about the anti-cancer mechanisms, its pharmacology or the toxicology. And a large number of cantharidin derivatives were synthesized. However, thare are still many problems unknown, such as biosynthesis sites or rutes and the anticancer mechanism.At present, the cantharidin-detection methods such as weight, pH, UV spectrophotometry, HPLC, GC, are usually not too convenient. Some of them require expensive chromatography instruments, some of them require the sample be purified before being detected, and some of them even request the operater equipt the high techlonogy.Objectives: Extract the cantharidin from the cantharis, and besed on this, synthesis cantharidin carboxyl derivatives N-carboxyethylcantharidimide. And inquire the best conditions and the highest yields for the reaction. Then compare the toxicity of the new derivatives to that of canthsridin. Prepare the antigen by coupleing the hapten N-carboxyethylcantharidimide to the carrier protein. Prepare the specific antibody by imunning rabbits. Ultimately, under the antigen-antibody reaction principle, establish cantharidin indirect competitive ELISA method and its curve.Methods: Extract cantharidin by mixed extraction method, shocking method and rapid extraction method respectively. A series reactions of cantharidin withβ-alanine were carried out with single factor differed. Detect and identify the extracted and synthesized chemicals by GC, TLC, UV, MS and NMR. And compare the stomach poisoning toxicity by determining the two chemicals'LD50 in mice with modified Kaber method. Use couple agent NHS, DCC to couple the N-carboxyethylcantharidimide to the carrier protein by NHS ester methord, and test or confiem the product by molar absorption coefficient method. Then, immune rabbits to get antibody and detect the antibody titer by ELISA. When the antibody titer is above 1:3200, separate the antiserum, then, purify the antibody in the antiserum with CNBr-activated agarose gel 4B affinity chromatography. Then, detect the purified antibody by SDS-PAGE and ELISA. Use the purified antibody and the antigen to establish cantharidin indirect competitive ELISA method and its curve. Inquire the scope and limits of this method, and verify the accuracy and reliability of this method in the cantharidin detection by comparing the detection results of this method to that of GC.Experiment Results: The purity of the cantharidin extracted with the mixed extraction method arrived at 98.53%. And structure identifications show that the condensation product is N-carboxyethyl- cantharidimide. And the optimal reaction conditions suggest that the best ratio of cantharidin toβ-alanine is 1:1.2, that the optimal solvent and catalyst is Et3N, that the best dosage of catalyst is 72 ml Et3N for 1 mol cantharidin, and that the optimal reaction temperature and dehydration time are 125～150℃and 4～5 h respectively. And the yield will reach to 74.4% at the optimal reaction conditions. And stomach poisoning toxicity of N-carboxyethylcantharidimide is only 1/400 that of cantharidin for mice. The combined ratios of N-carboxyethy- lcantharidimide to the carrier protein BSA and OVA are 25:1and 23:1 respectively. The rabbit antiserum titer purified is 1:6400～1:12800 that is 2 to 4 times than the titer unpurified which is 1:3200. And the cantharidin indirect competitive ELISA curve equation is y=-0.0697x+0.1894, and the detection upper and lower limits of curve are 2.4 mg/ml and 2.4 mg/L. The statistical method suggests that there is no significant difference between ELISA and GC in actual detection. |
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