Research On The Active Antitumor Component Of Periplaneta Americana And The Corresponding Colon-specific Tablet

Periplaneta american has the efficacy of promoting blood circulation to remove blood stasis, detoxicating and eliminating malnutrition, inducing diuresis to alleviate edema, etc. Its body contains proteins, amino acids, peptides, sugars and other active compounts, which have significant curative effect of anticancer, improving the immunity of organs, treating cardiovascular diseases, and repairing skin, among which the significant activity of anti-tumor has caught people’s overall attention in recent years. Based on the study of the documents and the previous works, choosing Periplaneta american as the investigating subject, this dissertation has conducted a system research about:the enriching and purifying of antineoplastic compounds contained in peptides; And according to the tendency of decreasing activity of the active compounds, which is caused by the acidolysis and enzymatic dissociation in circumstance of stomach, the author also developed the preparation of pH dependent oral colon-specific tablet, adopting new techniques and new preparation crafts. The main content is shown below:1. Research on the preparation methods of antineoplastic effective parts of Periplaneta american:with the combination of both the chemical composition test and inhibition experiment of tumor cells in-vitro method, the conditions of preparation process for the effective parts of Periplaneta american were determined: the coarse powder of the crud drug of Periplaneta american, after removeing the grease with aether petrolei, were immersed with10times the amount of90%ethanol for48h, then percolated at a rate of3ml/min/kg; collected and filtrated diacolation fluid; concentrated the fluid to1:1(ml:g)in50℃～60℃under decompression. Through HP20macroporous resin column, elute with2BV70%ethanol elution; collected70%ethanol elution fluid; finally the lyophilized powders were obtained after freezing and drying.2. Research on initial quality standards of the lyophilized powders of Periplaneta american:amino acids and peptides contained in producing were measured with ultraviolet spectrophotometric method; the contents of uracil, hypoxanthine and creatinine were measured with HPLC method; the type and content of amino acid contained in the lyophilized powders were tested with automatic amino acid analyzer; the molecular weight of the peptide in the lyophilized powders was measured with Tricine-SDS-PAGE gel electrophoresis.3. Research on antitumor effect and mechanism of the lyophilized powders of Periplaneta american:based on the previous studies, the in vivo antitumor activity and the immune function of tumor-bearing mice and the effect on SMMOc-7721cell apoptosis are studied. Results show that each dose group of the lyophilized powders of Periplaneta american can inhibite tumor growth as well as improving the immune function of a tumor-bearing organism; can obviously encourage the apoptosis and inhibit the proliferation of SMMC-7721cells, presenting a certain dose-effect relationship, whose mechanism may be related to the reduceing of protein expression of Bcl-2and the increasing of protein expression of Bcl-2/Bax, and the reduceing of Bcl-2/Bax value, thus, which also generally reveal the antitumor mechanism of periplaneta Americana.4. Research on preparation of the colon-specific tablet of the lyophilized powders of Periplaneta american:according to the properties of active antitumor peptides of Periplaneta american, the colon-specific tablet is made from the lyophilized powders with pH sensitive coating material Eudragit S100in case the efficacy might be lost for the acidolysis and enzymatic dissociation in circumstance of stomach. Using the accumulation-releasing-degree of creatinine as the index, the better coating prescription and coating process parameters were determined, base on which the colon-specific tablet of Periplaneta american was made. The release behaviors of three batches sample drugs are similar, which achieve the goal of colon-positioning release. By model fitting of the releasing curve of sample drugs, it is found that the in-vitro releasing behavior of the colon-specific tablet of the lyophilized powders of Periplaneta american conform to the Weibull model.This thesis makes preliminary purification of Periplaneta american antitumor active peptide, establishes the preparation methods of the active parts and the quality standards of lyophilized powders and conducts the in-depth studies of its antitumor mechanisms. The preparation technology of pH dependent oral colon-specific tablet is successful, comfirmed by the in vitro release experiments that localizer can pinpoint to the colon and meet the requirements of drug dissolution,so as to provide a high-efficiency and low-toxicity drug, increasing the drug-using variety in clinical practice.Innovation pointThe Innovative Line of ThoughtA systematic researching pattern of the active compounds of Periplaneta american,"Preparation Process-Chemical Compounds-Pharmacodynamic Effects ", was established, through the works such as the extraction and purification, the preparation of lyophilized powders, the establishment of preliminary quality standard, the discussion of the antitumor effects and mechanisms and the preparation of the colon-specific tablet. This dissertation innovatively provide a new line of thought for the in-depth study of a emerging preparations adapted to animals of TCMs.The Innovative MethodsThis dissertation combines chemical indicators with experiment indicators of pharmacodynamics, investigates the technology of enrichment and purification of the active antitumor compounds of Periplaneta american; Using analysis methods such as western-blot method, Elisa method, Hoechst33342/double staining PI method, Annexin V-FITC/PI double staining method and so on, the in-vitro antitumor effects and mechanisms of the active compound of Periplaneta american were researched. In the preparation of colon-specific table, adopting the creatinine dissolution combined with f2similarity factor method, contrasting the different prescriptions and technological conditions of cumulative release curves, evaluates its similarity of in-vitro drug release, in order to predict whether it reached the colon delivery performance. Provides a new method for the study of colon positioning release of animal medicine Periplaneta american, so the methods of this thesis are innovative. The Innovative ApplicationBased on the research of the in-vivo antitumor activity and the immune function of tumor-bearing mice and the effects on in-vitro cell apoptosis, the antitumor mechanism is preliminarily revealed, thus the foundation of new-drug research and development is established. Moreover, the active antitumor peptide of Periplaneta american, which maintain the tendency of loosing bioactivity and the difficulty of being absorbed in the blood system by the acidolysis and enzymatic dissociation in circumstance of stomach, was prepared as the oral colon-specific tablet. Therefore, the research innovatively extend the application of Periplaneta american in clinical practice.