Studies On The Anti-Cancer Activity And The Oral Colon Delivery System Of Bee Venom

In the present study, bee venom was selected as a model drug and used to study the effects on the proliferation of cancer cells in vitro and in vivo and to determine its possible mechanisms of action in cancer cells. Lastly, an oral colon delivery system (OCDS) was developed to deliver bee venom. The scope of this thesis research includes: (1) the study of the anticancer activity of the bee venom in vitro and in vivo and its possible mechanisms of action; (2) the establishment of analytical methods of typical principles in bee venom in vitro or i/i vivo; (3) the study of the physicochemical properties and the stabilities of the active constituents in bee venom; (4) the design of the OCDS; (5) the evaluation of the OCDS in vivo.In this study MTT test showed that the proliferation of various tumor cells in vitro was inhibited by bee venom in a concentration- and time- dependent manner. The ICso ranged from 2.73 to 13.64 jj,g/ml. The inhibition of K1735M2 melanoma cells was due to the arrest at the GI stage of the cell cycle with a sub-Gi peak as analyzing a DNA histogram by flow cytometer. The bee venom induced apoptosis-like cell death as identified by histological observations and by DNA fragmentation (DNA laddering on the agarose gel electrophoresis), and long dendrite-like projections were observed and lipid and dark granules accumulated. All these results suggested that bee-venom-induced cytostatic effects caused the induction of apoptotic events, which might activate Caspase-3 to induce cells apoptosis. Circular dichroism study showed that an increased order of membrane conformation was induced by bee venom. In addition, the study showed that the adhesion of cancer cells on the extracellular matrix was decreased when treated with bee venom. The results suggested that it might be a connection of various bio-effectiveness by which bee venom inhibited tumor growth. The in vivo experiments indicated that bee venom successfully inhibited the growth of implanted B16 solid tumor proliferation and growth. Further research on the mechanism by which bee venom inhibited the growth and proliferation of the B16 melanoma in vivo is on going.A quantitative GF-HPLC method was developed to analyze melittin, the typical activeingredient in bee venom. It was also used as a sensitive and reproducible method to determine the content of melittin in bee venom, with a recovery of better than 98% and RSD of 0.55%. Prothrombin time (PT) test was also performed to determine the anti-prothrombin activity of the polypeptides in bee venom. The result showed that at concentrations 0 to 6.0 mg/ml, bee venom prolonged the coagulation times in a dose-dependent manner with a coefficient of 0.9722. The Folin-phenol reagent method was adopted to determine the content of active constituents of bee venom in the preparations. The method was validated and linear response range was 25 to 250 M,g/ml, and the recovery was 100.59% with RSD of 1.25%. The ELISA method was adopted to determine the plasma bee venom concentration validated range from 0.0001-10 fxg/ml (r2=0.9981) in rats with the limit of determination at 0.1 ng/ml and the drug recovery at 100.65%, which indicated this ELISA method was suitable to analyze the samples produced in vitro and in vivo experiments.In this study, the polypeptiedes were concentrated from bee venom through a two-step chromatographic separation method using a hydrophobic HPLC and an ion-exchange HPLC columns. Afterwards, pure melittin (59.0%) was separated using GF-HPLC. A study of physicochemical properties of polypeptides in bee venom showed that the molecular weight were below 3500 with positive charge under physiological conditions. The anti-prothrombin activities of the active constituents in bee venom were independent on physiological pH condition, low temperature and light but sensitive to protease. The chemical structure was stable during the experiment. The conformation of melittin in solution changed with pH value and concentration, and a-helix bound to PC membrane.Utilizing sodium alginate fo...