Biotransformation Of Baicalin By Human Intestinal Bacteria

The biotransformation of baicalin by human intestinal bacteria system was set upin this thesis, the biotransformation characteristic of human intestinal bacteria systemand method of biotransformation were studied. The structure of bacicalin was changeand its activity was improved.First of all, the human intestinal bacteria system in vitro was established, whichincluded by preparing of thioglycollate medium, collecting of fresh human intestinalbacteria, and homoeothermic cultivating under the anaerobic enviroment for72hours.The HPLC method was used to identify the product. The conditions of HPLC are:the mobile phases consist of methanol-water-acetic acid （65:35:0.2）; the UVwavelength at275nm; flow rate,1.0ml/min, column temperature,28℃. The resultsof HPLC showed that the baicalein peak was clearly appeared at14.149min.The effect of ultrasonic treatment on the transformation of baicalin wasinvestigated. The method that ultrasonic treated medium following cultivation wasdetermined. The results of HPLC showed that the baicalein peak was clearly appearedat14.155min, which deduced that the product was baicalein. Ultrasonic treat couldbreak the cell wall, to release the converting enzyme （β-D-glucuronidase） in the celland improve the conversion of baicalin. The optimal conditions of ultrasonic treatwere40KHz and40minutes.Next, the cultivation conditions of intestinal bacteria were optimized, such astemperature, pH, and metal ions on the growth of intestinal bacteria. The intestinalbacteria will grow well under the following conditions: the pH of medium is5.5;cultivation temperature is30℃; the concentration of Ca²⁺is80mg/ml, Mg²⁺is24mg/ml and Cu²⁺is2.1mg/ml. Meanwhile, adding the vitamin C into medium will beof assistance to the growth of intestinal bacteria.Finally, the enzyme was isolated by alcohol precipitation, and the factor onenzymatic properties was analyzed. As a result, the optimum conditions to the enzymeare pH6.0and55℃. The enzyme will be activated by Ca²⁺、Mg²⁺and Cu²⁺，but inhibited by Fe²⁺; Zn²⁺will play a catalytic role at low concentration，however as aninhibitory factor at high concentration.