Study On The Extraction Of The Active Components Of C.militaris And ItsAnti-tumor Effect

Cordyceps militaris is a kind of pathogens of arthropods. Recent investigations showthat C.militaris is plenty of physiological active components, such as cordycepin,polysaccharides, sterols, which imparts it high medicinal and economic value. Studies alsohave shown its great potential in tumor therapy and adjuvant therapy.This study divided into three parts: determination of concentration of cordycepin,adenosine and polysaccharides and optimization of the extraction conditions of cordycepin,analysis of the inhibitory effect on tumor in vivo and in vitro, and the separation andpurification of the active components. From these three parts, the role of the water-solubleand water-insoluble active componets of C.militaris in anti-tumor was systematicallyinvestigated with a drug development point of view in this study.We firstly determined the content of cordycepin,adenosine and polysaccharide.Thedifference of the content of cordycepin and adenosine was also compared betweencultivated Cordyceps militaris and wild Cordyceps sinensis. By using the technology ofultrasonic extraction at room temperature while water was as a solvent and highperformance liquid chromatograph,we finally determined the content of cordycepin andadenosine.Results showed that the content of cordycepin and adenosine in C.militaris was7.03mg/g and0.59mg/g respectively,while none cordycepin and adenosine was detectedunder the same conditions in natural C.sinensis.With the utilization of phenol-sulfuric acidmethod, the content of polysaccharide was also determined,which was3.10%.To optimize the extraction conditions of cordycepin, one of the main physiologicallyactive components, semi-bionic extraction(SBE) was used to extract C.militaris. Uniformdesign was used to optimize SBE conditions, including the pH value and the extractiontime. Results of the best technologic conditions were as follows: C.militaris was extractedfor3times; the pH values of the water for the1st,2ndand the3rdextractions were2.00,7.00and10.00respectively, while the time for the1st,2ndand the3rdextractions were45min,27min,27min respectively; the whole process lasted99min. we could obtain7.417mg of cordycepin per gram of raw herbs under this condition. This method providedtheoretical reference for the extraction of cordycepin and its industrail production.Then, the inhibitory effect of water extract of C.millitaris on tumor was evaluated in vitro and in vivo respectively. MTT assay was used to analyze the toxicity of the drug to avariety of cell lines.These cell lines include normal somatic cells, HEK293humanembryonic kidney cell and HuVEC human umbilical vein endothelial cell，and also tumorcells, MCF-7breast cancer, MDA-MB-231breast cancer, LoVo colon carcinoma, HLF-K10human hepatoblastoma cancer, HLF-CPN1human hepatoblastoma cancer, HLF-Kollishuman hepatoblastoma cancer, A549human lung adenocarcinoma cell and Lewis lungcancer cell in mice. Water extract showed a dose-dependent inhibitory effect on theproliferation of these cell lines. Results also showed that water extract has unsignificantcytotoxicity to human tumor cell lines while with a strong growth inhibitory effect onLewis lung cancer cells in mice.Using ehrlich ascites tumor (EAC) and mouse ascites hepatoma HepA as object ofstudy, we also investigated the in vivo anti-tumor effect of C.militaris powder,water-soluble and water-insoluble fraction of C.militaris. We found that water extract andwater insoluble fraction all showed a growth inhibitory effect on EAC withcyclophosphamide as a positive control, and the tumor growth inhibition rate of waterinsoluble fraction on EAC was nearly30%, which was statistically significant.We has analyzed and evaluated the toxicity of the water extract and water-insolublefraction with different extract methods in vivo through acute toxicological experiment inmice. Results showed that water-soluble extract and water-insoluble extract was lowtoxicity substances, which indicated that C.millitaris and its water-soluble andwater-insoluble active ingredient was suitable as a new medicinal resource and available inthe value of further development and forthputting.Finally, we isolated two compounds from the water extract with the cytotoxicity of theextracted substances on Lewis lung cancer cells for guidance. By analyzing the spectra dataof ESI-MS, IR and NMR, compounds were finally determined as cordycepin andN6-(2-Hydroxyethyl)adenosine.