Study Of The Best Method To Extract Sterols From The Mycelium Of Hirsutella Sinensis By Orthogonal Design

Cordyceps sinensis is a traditional valuable Chinese medicine with many pharmacology effects. Its anamorph is Hirsutella sinensis, which has a broad application prospects. Sterols is one of the main active ingredients of Cordyceps sinensis, ergosterol is Characteristic of Fungi, it has significant activity of anti-cancer. This subject focus on developing the best method to extract sterols from the mycelium of Hirsutella sinensis.H3PO4-H2SO4-FeCl3 and vanillin-glacial acetic acid spectrophotography were studied in their methodology, both of their variety are less than 1% after color reaction, they have good stability; the RSD of their precision are 1.9% and 3.6%; the value of recovery are 126.3±16.5% and 105.4±3.6%; choose the vanillin-glacial acetic acid method to determine the total sterols. Use one-factor experiment to study the factors:extraction time, extraction times, ethanol concentration, the KOH-mycelium ratio and solid-liquid ratio, the result showed that extraction times, ethanol concentration and the KOH-mycelium ratio influence the result remarkably(P< 0.05), extraction time and solid-liquid ratio has little influence on ultrasonic extraction(P>0.05).A 3-factor 3-level orthogonal design was used to optimize the ultrasonic extraction method, the level of KOH-mycelium ratio is 0,0.5 and 1, the level of ethanol concentration is 60%,80% and 95%, the level of extraction times is 1,2 and 3, the optimum conditions is:the KOH-mycelium ratio is 1,95% ethanol, extract 3 times. One-factor experiment showed that the temperature has little influence on thermal reflow extraction. An identical orthogonal design was used to optimize the thermal reflow extraction method, the optimum conditions is:the KOH-mycelium ratio is 0.5,95% ethanol, extract 2 times. Validation experiment showed that the ultrasonic extraction is better than thermal reflow extraction method. Establish a determination method of total ergosterol from the mycelium of Hirsutella sinensis by RP-HPLC with a Hypersil C18 column (250 mm×4.6 mm,5μm) at a temperature of 30℃. The mobile phase is 100% methanol, the flow rate was 1.0 mL min-1, the detecting wavelength was 282 nm, the amount of injection was 20μL. The content of total ergosterol is 0.08%.