Studies On Enzyme-assisted Extraction Of Alkaloids From Rhizoma Coptidis

Objective:To optimize the technological condition of extraction and purification of alkaloids (including jatrorrhizine, palmatine and berberine) in Rhizoma Coptidis and their effect on a-amylase had been studied in this thesis.Methods:1. A method for determining alkaloids in Rhizoma Coptidis and Jinqi Jiangtang Tablet had been established by using RP-HPLC.2. The conventional extraction method of alkaloids in Rhizoma Coptidis was selected by comparing ultrasonic extraction, solvent refluxing extraction and cold-soaked extraction.3. The optimal cold-soaked extraction conditions of alkaloids in Rhizoma Coptidis were established through the factorial and the orthogonal design,4. The pectinase activity was determined by the method of sodium-state fermentation.5. Cellulase and pectinase were used in cold-soaked extraction of alkaloids in Rhizoma Coptidis. Through the factorial and the orthogonal design, the optimal cellulose degradation conditions were obtained.6. Alkaloids were purified by acid sediment in this thesis. Relative factors including acidity, amount of sodium chloride, standing time and drying temperature were studied.7. To study the effect of alkaloids in Rhizoma Coptidis on a-amylase.Results:1. The separation was carried out using the Diamosil C18column (4.6mm×250mm,5μm) with the mobile phase containing acetonitrile-0.1mol·L-1potassium dihydrogen phosphate (30:70) and a detective wavelength at345nm.The flow rate was1.0mL·min-1.2. The results showed that cold-soaked extraction was the optimal method and sulfuric acid aqueous solution was optimal extraction solution. 3. Through the factorial and orthogonal design, the optimal cold-soaked extraction conditions obtained were following:1:8(Weight/Volume) of0.25%sulfuric acid aqueous solution to Rhizoma Coptidis weight ratio, extraction for2times and24h each time, the particle size range was5-7mm.4. The value of pectinase activity was10171U/g.5. Comparing the sampling group and the blank, the result showed that both cellulase and pectinase could increase the extraction rates of berbermine and total alkaloids. The result also showed that the value of extraction rate in cellulose was higher than the value in pectinase. Through the factorial and the orthogonal design, the optimal cellulose degradation conditions obtained were following: Rhizoma Coptidis crushed5～7mm,pH4.0,the enzyme dosage of9mg·g-1,the reaction time of2.5h and the reaction temperature of35℃.6. The optimal purification conditions were following:extraction solvent adjusted to pH1-2by hydrochloride, the dosage of sodium chloride was10%(Weight/Volume), standing time24h, drying temperature60℃.7. It was found that alkaloids could inhibit the activity of a-amylase in this thesis. The inhibiting ratio of alkaloids on a-amylase was33.06%.Conclusion:The determination of alkaloids is simple, accurate and can be used to control the quality of alkaloids in Rhizoma Coptidis and Jinqi Jiangtang Tablet. Extraction and purification of alkaloids are confirmed, which shows high yield and good reproducibility. It is found that alkaloids can inhibit the activity of a-amylase, which is supposed to be caused by alkaloids through altering the molecular conformation of a-amylase. This study has laid a foundation for further pharmacological research on Rhizoma Coptidis.