| Carbonic anhydrase (CA), is widely distributed in tissues and organs of human body, involved in many important physiological function, including acid-base balance, cell respiration, bone resorption, aqueous humor secretion, gluconeogenesis, gastric acid and fat formation. the body will lead to disease if the enzyme is missing or abnormal activity. And may be an important target for a variety of human diseases. Therefore, attempt to develop new CA inhibitors play an important role in treatment of diseases.Now, The clinical applications of carbonic anhydrase inhibitors was sulfonamides and their derivatives, but its more side effects. oral carbonic anhydrase inhibitors may occur a series of systemic reactions, such as metabolic acidosis, electrolyte disturbances, kidney damage, blood disorders, and so on. Have more side effects on the human body. Limiting the application of sulfa carbonic anhydrase inhibitors in cancer, epilepsy and other diseases. So, It’s have a great significance to broth that low side effects of carbonic anhydrase inhibitors from medicinal fungi fermentation.First, optimize the determination of carbonic anhydrase. The value of carbonic anhydrase inhibition activity property was used as the testing index, to optimum the liquid fermentation medium of medicinal fungus Ganoderma fermentation. Separation inhibitor active ingredients from the Ganoderma applanatum fermentation by a variety of means.1.This chapter based on the work of predecessors. The spectrophotometric determination the stability of reagent, reaction intensity of reagent and enzyme, the effect of reagent decomposition rate for enzyme reaction rate. Optimize the determination of carbonic anhydrase.The results showed that the optimal conditions of carbonic anhydrase determination:0.0181g pNPA dissolved in 1ml of anhydrous ethyl alcohol, then this solution and 0.156g diethylmalonic acid in the pH6.4 of 0.2mol/L phosphate buffer, final volume is 100ml.Water bath at 35 C for 30min. This method is suitable for the rapid determination of a large number of samples, and comparison the activity between samples.2.In this study, synthesis of carbonic anhydrase inhibitors use the Ganoderma applanatum fermentation. The value of carbonic anhydrase inhibition activity property was used as the testing index, through a single factor and four factors and three levels of L9(34) orthogonal experiment, the optimal fermentation medium (g/L):20 fructose, sodium 2, potato 173, ammonium sulfate 1.3.Of the fermentation process optimization:The best pH5.5, the best speed 160r/min.Using this medium, liquid culture mycelia of Ganoderma, the fermentation proceeds of carbonic anhydrase inhibition on the ability to reach 91.43%. Significantly higher than the unoptimized medium (69.81%). 3.Used alcohol precipitation the Ganoderma applanatum fermentation, inhibition rate was 80.3% of its filtrate.The inhibition rate of the residue was 20.78%. Identification the chemical composition of its filtrate,The results showed that it contains glycosides and alkaloids, which did not detect the flavonoids and saponin components. And this filtrate was further purified with the M-Bondapak-C18 column and Sephadex LH-20 column to collect the final eluate, then freeze-dried it to obtain white powder. Tested the collected samples using HPLC showed 100% purity.The sample was purified by LC/MS analysis, as positive and negative ions do not correspond and have more ions, can not determine which ion was the main peak.4.Liver glucose consumption experiments of diabetic mice, showed that,the carbonic anhydrase activity was inhibited by inhibitor, affects the product CO2 release of the reaction system.Thus affecting the acetyl CoA pathway of citric acid cycle on liver, further leading to the accumulation of pyruvic acid and lactic acid and to reduce sugar consumption. |
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