Purification Of Seal Oil, The Effect Of Prevention And Therapy On Rat Fatty Liver And Benign Prostatic Hyperplasia And Their Mechanism Of Action

Objective: Seal oil was first mentioned in an early traditional Chinese medicine book〈Ben cao gang mu·supplement·9·animal〉but there almost hasn't been mentioning of it in clinic cases. So it is necessary to study purifying methods so that it can have a pharmaceutically useful concentration. There is also a need to set up a determination method for three kinds of ω-3polyunsaturated fatty acids(ω-3PUFAs), whose structures have been confirmed with the use of physical and chemical methods in seal oil, to study the preventional and theraputical effects of fatty liver and benign prostatic hyperplasia and its mechanism of action. The purpose of this study is to provide theoretical basis for clinic use and for the development rare medicinal sources.Methods: 1. RP-HPLC was used to determine three kinds ofω-3polyunsaturated fatty acids(ω-3PUFAs) EPA,DHA and DPA rich in seal oil. The sample used Na-methanol derivation and the temperature of derivation, time of derivation and the stability of Na-methanol are observed for confirmation of the perfomance of derivation and determination of the best derivation method. The chromatographic conditions optimized for separation were tried to find out, which included determination wavelength, concentration, sample amount, chromatographic column, mobile phase, flow rate, column temperature. System suitability test is conducted and validation of this method including linear in range, precision, limit of examine and determination, specialism, robustness, accuracy and repetition were studied.2. Seal oil was purified by molecular distillation. The best conditions of distillation is determined by studying the temperature of distillation, rate of adding material, speed of rotor rotational and the affaction to separating and purition coming from temperture of adding material.3. Physical and chemical methods such as UV,IR,MS,NMR were used to confirme the structures of EPA,DHA and DPA in seal oil.4. In preventing fatty liver experiment,fatty liver models were induced by subcutaneous injecting a low dose of carbon tetrachloride 1.2g/kg one-time on the first day and chronically feeding with high fat diet in rats. At the same time the rats were taken by olive oil in the same volume of seal oil for model group, simvastatin 4 mg ·kg-1 for the control drug group, seal oil 0.5 ,1.6 or 4.8g·kg-1 for low or medial or large dose of seal oil groups by ig ,respectively, once a day ,for 10 weeks. The placebo control group(n＝10) received only normal feed. In therapeutic fatty liver experiment, both subcutaneous injecting a low dose of carbon tetrachloride light injuring liver one-time on the first day and high fat diet chronically feed were given to 52 male Wistar rats for 7 weeks , 2 rats were dissected to assess if fatty liver was induced sccessfully.Then five groups(n＝10 in each) of male Wistar rats received olive oil in the same volume of seal oil per day, simvastatin 4 mg /kg/d ,seal oil 0.5 ,1.6 or 4.8g/kg/d, administered orally for other 8 weeks, respectively. The placebo control group(n＝10) received only normal feed. During the experiment, we observed rats activities, diet, coat and weighed rats once a week. After the last administration, all the rats were put on fast over night and killed. The efficacy of seal oil in both prevention and therapy fatty liver was assessed main based on the evaluation of pathologic including HE dyeing and Sudan Ⅲ dyeing, hepatic weight , hepatic index and serum transaminase ALT, AST and other biochemocal parameters in serum and liver including the contents of triglyceride (TG), total cholesterol (TC), malonyldialdehyde (MDA), free fatty acid (FFA) ,superoxide dismutase(SOD) measured by spectrophotometry; hepatic cytochrome P4502E1(CYP2E1)express detected by immunohistochemistry using CYP2E1 specific antibodies; 6-keto-ProstaglandineF1α(6-keto-PGF1α)and Thromboxane B2 ( TXB2 ) measured by radioimmunoassay. 5. Oral administration of 0.8, 2.4, 7.2g/kg/d or 0.5 ,1.6 ,4.8g/kg/d of seal oil for 7d～9d protected liver...