Biosynthesis And Chemical Modification Of Cordycepin

Cordycepin is an important bioactive component of a Chinese traditional fungal drug,Cordyceps militaris(L.) Link,and has many pharmacological actions such as antitumor,anti-leukemia,anti-microbes,immunoregulation,and free radical clearance in vitro,etc.But the high production cost and fast metabolism in vivo have limited its further development and utilization fundamentally.Around these problems,three related studies were carried out and some important results obtained in this dissertation as follows:1.Studies on the biosynthesis of cordycepinThe best strain of cordycepin production screened out through comparative experimental investigation was JF-1,and the optimal growing period for cordycepin production determined by tracking the content of cordycepin in the course of solid fermentation was fruiting bodies emerging.The optimized formula of rice medium for cordycepin production determined by quadratic rotation-orthogonal combination design experiments was as follows:the rate of nutrient water to rice 1.1 mL/g;the dosage of yeast extract,peptone,glucose,KH₂PO₄ and MgSO₄ in nutrient water were 22.6,6.0,25.4,2,0.5 g/L respectively;pH of nutrient water was 6.6.The optimal environmental conditons determined by orthogonal design experiments were as follows:light exposure hours of every day 18h,light intensity 4400 Lux,temperature 22℃.Under the optimal technological conditions and after about 13d of cultivation, the content of cordycepin in solid medium was up to 0.60%,whose production efficiency of producing cordycepin was 15 times higher than that of producing cordycepin from fruiting bodies directly,and the production period could be shortened over 1 times.Compared with the highest level of the world,the content of cordycepin in the solid media by the new method was almost 2 times higher than that in the liquid media by the method of traditional liquid fermentation and the production period could be shortened 3d.2.Studies on the extraction and purification of cordycepinA new method of extracting and purifying cordycepin from rice medium was set up through modifying the traditional process of extracting and purifying cordycepin from fruiting bodies of C.militaris.The new method include 7 steps as follows:Raw material treatment:The rice media were dried at 60-65℃and crushed through 50 mesh sieve.Continuous countercurrent extraction:With 70%ethanol as solvent,and the rate solvent/raw material was 5 mL/g.The raw material was marinated in solvent for 12h at room temperature firstly,then was shaked to extraction for 2h,3 times repeated.Precipitating impurity:Concentrated the extracted liquid and adjusted its pH＝10.0 and 4.0 in sequence,standed for 8h,then filtrated respectively to get loading colum solution(LCS).Column chromatography:Column chromatography was carried out with 732 cation exchange resin,incuding 3 steps:loading colum with LCS,washing with water, and eluting with ammonia.Concentration and removing impurity:Concentrated the elution liquid and resolved with ethanol,filtrated out the impurity,then resolved with distilled water as mothor liquor.Crystallization with water:Adjusted the mother liquor pH＝10,then put it under 4℃and standed for 3d to get crystal,dried the crystal at 60—65℃.Recrystallization with methanol:Resolved the crystal with methanol,then put it under 4℃and standed for 3d to get crystal,dried the crystal at 60—65℃.With the new method to extract and purify cordycepin from rice media,the yield was up to 58.83%,and the product purity was over 98.0%.But when the new method was applied to get cordycepin circularly and industrially,much lost cordycepin in the intermediate steps could be recycled,so the whole yield could exceed 80%.By the new method,not only can cordycepin be obtained from the media of biosynthesis,but also from the waste medium of fruiting bodies production.3.Studies on the synthesis and bioactivities of normal alkyl-amide homologue of cordycepinFor slowing down the metabolic velocity in vivo and improving the bioactivities of cordycepin,with cordycepin as a lead compound,propionyl-,octanoyl-,lauroyl- and stearoyl- chloride as acylating agents,four normal alkyl-amide homologues of cordycepin(C_n-AAC),i.e.propionyl-,octanoyl-,lauroyl- and stearoyl-cordycepin (C₃-AAC,C₈-AAC,C₁2-AAC and C₁8-AAC),were designed and synthesized successfully by acylation reactions,and their bioactivies of metabolism in vivo and antimicrobes were investigated.The results of metabolic experiments in vivo indicated that C_n-AAC could be transformed to cordycepin in the bodies of mice,so it could play a prodrug part of cordycepin.With the increasing of carbon number in the alkyl-acyl chain of C_n-AAC, T_max and t_1/2 of the transformed cordycepin enlongated,and C_max and AUC increased firstly,then decreased gradully.C₈-AAC had highest C_max and AUC of transformed cordycepin,which were 2 and 4 times respectively higher than those of cordycepin control,and its T_max and t_1/2 of transformed cordycepin enlongated 4 times than those of cordycepin control.So,if cordycepin was changed to C_n-AAC,not only could its metabolic velocity in vivo be decreased greatly,but also its bioavailability changed greatly.The results of antimicrobial experiments indicated that the antimicrobial activity would have a regular change when cordycepin was changed to C_n-AAC.C₈-AAC had the highest antimicrobial activity of all C_n-AAC and its MIC to Bacillus subtilis decreased nearly 5 times compared with cordycepin control,and it had antimicrobial activities to Staphylococcus aureus and Candida albicans,to whcih cordycepin had no antimicrobial activities.All the experimental results indicated,when the lipophilia(or hydrophobicity) of C_n-AAC was up to a optimum by chemical modification,its bioactivities of metabolism in vivo and antimicrobes woud be up to the best.So,molecular polarity has an important effect on the bioactivity of such compounds.In general,the high-efficient methods of biosynthesis,extraction and purification of cordycepin were set up through deep investigations in this dissertation.With these new methods of cordycepin production,the production cost of cordycepin could be decreased greatly as compared with the traditional methods,and the production period could be shortened greatly as well as a more simpler production process.When cordycepin was changed chemically to C_n-AAC,both of the metabolic and antimicrobial activities would have a regular change.C₈-AAC had not only the best metabolic activity but also the highest antimicrobial activity.So,it is a new compond having potential value of development and utilization and deserves further investigation.All these new fruits will have significances for improving Chinese study levels of cordycepin in the world,and can promote the developments of caterpillar fungus industry and traditional Chinese medicine.In this thesis,modern biotechnology,mathematical statistics and opimization technology,medicinal chemistry theory were applied to the investigations of natural bioactive components for improving their production level and decreasing their production cost greatly.In addition,with the natural bioactive component as lead compound,by the means of chemical structure modification,new compounds with better bioactivities were found and studied.So,it can give a guide and reference to the study and development of other natural drugs in methodology.