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Extraction and Isolation of Cordycepin from the Deserted Solid Medium of Cordyceps Militaris and Antitumor Effects

Author: ZhangLiYan
Tutor: MaoNing
School: Fujian Normal University
Course: Biochemistry and Molecular Biology
Keywords: cordyceps militaris medium cordycepin isolation and purification K562cell H22 cell
CLC: S567.35
Type: Master's thesis
Year: 2010
Downloads: 136
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Abstract


Cordycepin namely 3’-deoxyadenosine, is one of the active components of Cordyceps militaris. Researches of recent 50 years show that cordycepin has the function of antitumor and antifungi activity and enhancing immunity, which has already attracted great attention from scientific research field in many countries. This research chooses cordyceps militaris medium as raw material. A profound and systematical analysis has been done on cordycepin detection method, extraction and purification technique, purity identification of cordycepin and antitumor activity.An HPLC method for determining the contents of cordycepin was established. The standard curve equation was y=64055x-26.599 (r=0.9999),the linear range was 0.01-0.1 mg/mL,the RSD is less than 1%in the precision test and in the stability test. In this way, it is simple and rapid to get a good effect in separating the cordycepin.By studying the isolation of cordycepin from cordyceps militaris medium, the researchers have optimized the progress of water extracting-alcohol precipitating.The optimal extraction technology conditions were as fllows:the temperature was at 40℃, the extraction times were 4 times, the solid-liquid ratio was 1:20 and the extraction time was 2h, extracting solution was concentrated to 2 times of the waste, adding 3 times of acohol, the time of precipitation is 9h.After impurity resmoval process, water extract of cordycepin still have many impurities, which needs further separation. By the study of static adsorption and desorption characteristic of cordycepin on six kinds of cation exchange resins, the optimum one,resin of JK006,was chosen. The adsorption thermodynamics characteristics of JK006 cation exchange resin for cordycepin were researched. The results indicated that the adsorption date fit well into the Freundlich isotherm equation within the temperature range of 298~308 K and investigative concentration range. According to the results of thermodynamics, the△H>0,△G<0 and the adsorption of cordycepin on JK006 resin was endothermic and spontaneous.The influencing factors of JK006 dynamic adsorption and desorption is flow rate, cordycepin concentration, desorption solution concentration. The best purification condition is flow rate 3BV/h, cordycepin concentration 1.69mg/mL, ammonia concentration 0.25mol/L, desorption rate 2BV/h.Purity of cordycepin reaches 96.4% through JK006 column chromatography and 717 anion exchange resin.The cordycepin qualitative and qnantitative analysis by adopting TLC, HPLC, ultraviolet spectrum, infrared spectrum and other method. Comparing with standard, could decided it was cordycepin.The research on the inhibition effect of self-made cordycepin on tumor cells show that cordycepin had significant inhibition effects, in a dose-dependent way, on the proliferation of K562 and H22 cell in vitro, using K562 cell as positive control. By comparing IC50 of K562 and H22, it is proved that K562 is more sensitivity to cordycepin than H22 cell.

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CLC: > Agricultural Sciences > Crop > Economic crops > Medicinal crops > Fungus > Cordyceps sinensis
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