虫草素的生物合成及化学结构修饰
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摘要
虫草素是我国传统中药蛹虫草(Cordyceps militaris(L.) Link)的主要活性成分之一,具有抗肿瘤、抗白血病、抗菌、免疫调节、清除体内自由基等多方面的药理作用。但虫草素目前主要从蛹虫草子实体中获取,生产成本极高;并且虫草素分子结构中的伯氨基在动物体内腺苷脱氨酶(ADA)作用下,容易快速脱氨而成为无生理活性的物质,使虫草素在体内的生物活性严重下降。这两方面的因素从根本上限制了虫草素的进一步研究和开发利用。本论文针对国内外虫草素研究中存在的上述问题展开了如下三方面的深入研究,并取得了一定的研究成果:
1.虫草素高效生物合成技术的研究
论文通过对不同蛹虫草菌株固体培养过程中虫草素产生及变化规律的跟踪测定分析,筛选出了虫草素的高产菌株JF-1,确定收获培养基进行虫草素提取的最佳生育期为现蕾期。通过正交旋转组合试验,确定固体培养生产虫草素的最佳培养基配方为:水料比1.1 mL/g,营养水中酵母膏22.6g/L、蛋白胨6.0g/L、葡萄糖25.4g/L、KH_2PO_4 2g/L、MgSO_4 0.5g/L,营养水pH 6.6。通过正交试验,确定固体发酵生产虫草素的最佳环境因子组合为:每同光照时数18h,光照强度4400Lux.温度22℃。按照上述最佳工艺条件固体发酵生产虫草素,经过大约13d的培养,培养基中虫草素的含量达到0.60%以上。其生产效率比通过生产子实体来获取虫草素提高15倍以上,生产周期缩短1倍以上;和国外液体发酵生产虫草素的最高生产水平相比,本技术所生产的固体培养基中虫草素的含量比液体发酵的培养液中虫草素的含量提高近2倍,生产周期缩短约3d。
2.虫草素高效提取纯化技术的研究
在对传统从蛹虫草子实体中提取纯化虫草素的工艺技术进行改进和优化的基础上,形成了高效地从大米培养基中提取纯化虫草素的新技术。该技术的工艺流程包括如下七步:
原料处理:将大米培养基在60-65℃下烘干,粉碎过50目筛。
连续逆流提取:用70%乙醇进行连续逆流提取。溶剂和原料比为5 mL/g;先将原料在室温下浸泡12h,再在室温下搅拌或震荡提取2h,过滤;反复提取3次。
沉淀去杂:将提取液浓缩到其体积的0.6倍,用氨水调pH＝10.0,静置8h,用100目滤网过滤。再用HAc调滤液pH＝4.0,静置8h,用150目滤网过滤,将滤液作为柱层析的上柱溶液。
柱层析:用732阳离子交换树脂进行柱层析(层析柱所装树脂的体积为Vc)。先用上柱溶液上柱,流速0.02×Vc/min,上柱溶液与树脂的体积比为8:1;再用体积为10×Vc的水沈柱,流速0.02×Vc/min;最后用8×Vc体积的浓度为0.2mol/L的氨水洗脱,流速0.02×Vc/min。
浓缩去杂:将柱层析洗脱液浓缩成稀浸膏状,加入乙醇在60-65℃下充分溶解并定容到浓缩前柱层析洗脱液体积的1/20,趁热过滤。将滤液在70-80℃下浓缩成稀浸膏状,加入蒸馏水在60-65℃下充分溶解并定容到浓缩前乙醇滤液体积,作为结晶母液。
用水结晶:将结晶母液用氨水调其pH＝10.0,均匀分成多份依次在4℃下静置3d进行结晶,将晶体滤出在60—65℃下烘干。
用甲醇重结晶:将干燥晶体用甲醇在60-65℃下充分溶解(所用甲醇体积等于前步结晶所用水的体积),均匀分成多份依次在4℃下静置3d进行结晶,将晶体滤出在60—65℃下烘干得到产品。
采取上述工艺技术从蛹虫草大米培养基中提取纯化虫草素,单次提取纯化的产率达58.83%,所得虫草素产品纯度达98.0%以上。但将本技术用于工业化循环提取纯化虫草素时,多数中间步骤损失的虫草素都可以得到回收进行循环提取,从而把整个工艺过程中虫草素的产率提高到80%以上。利用本工艺技术不仅可以和本论文的虫草素生物合成工艺配套使用高效获取虫草素,而且可以高效地直接从当前蛹虫草子实体生产的废弃培养基中提取纯化虫草素,整个提取纯化过程不需加温操作、不需昂贵的设备和试剂、工艺过程简便。
3.虫草素烷酰胺同系物的合成和生物活性的研究
为减缓虫草素的体内代谢速度和提高虫草素的生物活性,本论文首次成功设计并合成了4个虫草素正构烷酰胺同系物(C_n-AAC),即N-正丙酰胺虫草素(C_3-AAC)、N-正辛酰胺虫草素(C_8-AAC)、N-月桂酰胺虫草素(C_(12)-AAC)和N-硬脂酰胺虫草素(C_(18)-AAC)。论文以虫草素为先导化合物,以丙酰氯、正辛酰氯、月桂酰氯和硬脂酰氯为酰化剂,通过酰化反应,合成并纯化得到4个新化合物。元素分析、紫外和IR光谱分析、~1H NMR谱图分析均证实4个新化合物和目标产物的分子结构相一致。并且,4个新化合物的R_f值、熔点等理化参数呈现的有规律变化,也进一步印证了产物的分子结构。
论文通过动物体内代谢试验,对4个新化合物的体内代谢活性进行了研究,并通过回归分析方法对C_n-AAC分子结构与虫草素生物利用度的关系进行了定量描述。结果表明:通过化学结构修饰,把虫草素改变成C_n-AAC后,C_n-AAC在动物体内可以转变还原成虫草素,故C_n-AAC可以作为虫草素的前药而发挥作用。随着C_n-AAC烷酰基侧链碳原子数目的增加,其在动物体内转变成的虫草素的T_(max)和t_(1/2)显著延长,C_(max)和AUC值则出现先升高、再降低的有规律性的变化。其中C_8-AAC试验组在体内转化成的虫草素的C_(max)和AUC值最高,分别是虫草素对照组的约2倍和4倍,其T_(max)和t_(1/2)也比虫草素对照组延长近4倍。C_n-AAC烷酰基侧链碳原子数目和其在动物体内代谢产生的虫草素的AUC值的关系,可以用一个一元二次方程来进行定量描述。这充分说明,虫草素经化学结构修饰改变成C_n-AAC后,不仅可以达到延缓虫草素体内代谢速度的目的,而且可以极显著地改变其在动物体内的生物利用度。
论文通过体外抗菌试验,对4个新化合物的体外抗菌活性进行了初步研究,结果表明:虫草素改变成C_n-AAC后,其抗菌活性也呈现有规律的变化。其中C_8-AAC的抗菌活性最强,和虫草素相比,其对枯草杆菌(Bacillus subtilis)的MIC降低了近5倍,并可对虫草素不能产生抗菌活性的金黄色葡萄球菌(Staphylococcus aureus)和白色念珠菌(Candida albicans)产生抗菌活性。
以上试验结果均表明,通过化学结构修饰使C_n-AAC的亲脂性(或疏水性)达到一个最佳值时,可以使该化合物的体内代谢活性或抗菌作用达到最大,说明分子极性在很大程度上影响着该类化合物的生物活性。
综上所述,本论文通过深入的研究,形成了高效生物合成虫草素、高效提取纯化虫草素的工艺技术。配套使用此二项技术生产虫草素,比传统从蛹虫草子实体中获取虫草素的生产成本降低15倍以上,并且生产周期大为缩短、生产过程更为简单。虫草素被化学修饰改变成烷酰胺同系物后,无论其体内代谢活性和抗菌活性都会产生有规律的变化。在虫草素烷酰胺同系物中,N-正辛酰胺虫草素不仅具有较好的代谢活性,也具有较高的抗菌活性,是一个值得进一步深入研究和有潜在开发利用价值的新化合物。本项研究对促进虫草素的进一步研究和开发利用、全面提升我国在世界上虫草素方面的研究水平、以及促进我国传统医药技术的发展并走向世界具有重要的意义。
本文成功地将现代生物技术、数理统计优化技术以及药物化学理论应用于天然药物活性成分的研究开发中,全面提升天然药物活性成分的生产水平,大力降低其生产成本,并以天然活性成分为先导化合物、通过化学结构修饰的手段研究开发具有更高药理活性的新型药物,对其它天然药物的研究和开发具有方法学上的指导意义和借鉴作用。
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 cordycepin
The 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,KH_2PO_4 and MgSO_4 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 cordycepin
A 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 cordycepin
For 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 (C_3-AAC,C_8-AAC,C_12-AAC and C_18-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.C_8-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.C_8-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.C_8-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.
1.虫草素高效生物合成技术的研究
论文通过对不同蛹虫草菌株固体培养过程中虫草素产生及变化规律的跟踪测定分析,筛选出了虫草素的高产菌株JF-1,确定收获培养基进行虫草素提取的最佳生育期为现蕾期。通过正交旋转组合试验,确定固体培养生产虫草素的最佳培养基配方为:水料比1.1 mL/g,营养水中酵母膏22.6g/L、蛋白胨6.0g/L、葡萄糖25.4g/L、KH_2PO_4 2g/L、MgSO_4 0.5g/L,营养水pH 6.6。通过正交试验,确定固体发酵生产虫草素的最佳环境因子组合为:每同光照时数18h,光照强度4400Lux.温度22℃。按照上述最佳工艺条件固体发酵生产虫草素,经过大约13d的培养,培养基中虫草素的含量达到0.60%以上。其生产效率比通过生产子实体来获取虫草素提高15倍以上,生产周期缩短1倍以上;和国外液体发酵生产虫草素的最高生产水平相比,本技术所生产的固体培养基中虫草素的含量比液体发酵的培养液中虫草素的含量提高近2倍,生产周期缩短约3d。
2.虫草素高效提取纯化技术的研究
在对传统从蛹虫草子实体中提取纯化虫草素的工艺技术进行改进和优化的基础上,形成了高效地从大米培养基中提取纯化虫草素的新技术。该技术的工艺流程包括如下七步:
原料处理:将大米培养基在60-65℃下烘干,粉碎过50目筛。
连续逆流提取:用70%乙醇进行连续逆流提取。溶剂和原料比为5 mL/g;先将原料在室温下浸泡12h,再在室温下搅拌或震荡提取2h,过滤;反复提取3次。
沉淀去杂:将提取液浓缩到其体积的0.6倍,用氨水调pH＝10.0,静置8h,用100目滤网过滤。再用HAc调滤液pH＝4.0,静置8h,用150目滤网过滤,将滤液作为柱层析的上柱溶液。
柱层析:用732阳离子交换树脂进行柱层析(层析柱所装树脂的体积为Vc)。先用上柱溶液上柱,流速0.02×Vc/min,上柱溶液与树脂的体积比为8:1;再用体积为10×Vc的水沈柱,流速0.02×Vc/min;最后用8×Vc体积的浓度为0.2mol/L的氨水洗脱,流速0.02×Vc/min。
浓缩去杂:将柱层析洗脱液浓缩成稀浸膏状,加入乙醇在60-65℃下充分溶解并定容到浓缩前柱层析洗脱液体积的1/20,趁热过滤。将滤液在70-80℃下浓缩成稀浸膏状,加入蒸馏水在60-65℃下充分溶解并定容到浓缩前乙醇滤液体积,作为结晶母液。
用水结晶:将结晶母液用氨水调其pH＝10.0,均匀分成多份依次在4℃下静置3d进行结晶,将晶体滤出在60—65℃下烘干。
用甲醇重结晶:将干燥晶体用甲醇在60-65℃下充分溶解(所用甲醇体积等于前步结晶所用水的体积),均匀分成多份依次在4℃下静置3d进行结晶,将晶体滤出在60—65℃下烘干得到产品。
采取上述工艺技术从蛹虫草大米培养基中提取纯化虫草素,单次提取纯化的产率达58.83%,所得虫草素产品纯度达98.0%以上。但将本技术用于工业化循环提取纯化虫草素时,多数中间步骤损失的虫草素都可以得到回收进行循环提取,从而把整个工艺过程中虫草素的产率提高到80%以上。利用本工艺技术不仅可以和本论文的虫草素生物合成工艺配套使用高效获取虫草素,而且可以高效地直接从当前蛹虫草子实体生产的废弃培养基中提取纯化虫草素,整个提取纯化过程不需加温操作、不需昂贵的设备和试剂、工艺过程简便。
3.虫草素烷酰胺同系物的合成和生物活性的研究
为减缓虫草素的体内代谢速度和提高虫草素的生物活性,本论文首次成功设计并合成了4个虫草素正构烷酰胺同系物(C_n-AAC),即N-正丙酰胺虫草素(C_3-AAC)、N-正辛酰胺虫草素(C_8-AAC)、N-月桂酰胺虫草素(C_(12)-AAC)和N-硬脂酰胺虫草素(C_(18)-AAC)。论文以虫草素为先导化合物,以丙酰氯、正辛酰氯、月桂酰氯和硬脂酰氯为酰化剂,通过酰化反应,合成并纯化得到4个新化合物。元素分析、紫外和IR光谱分析、~1H NMR谱图分析均证实4个新化合物和目标产物的分子结构相一致。并且,4个新化合物的R_f值、熔点等理化参数呈现的有规律变化,也进一步印证了产物的分子结构。
论文通过动物体内代谢试验,对4个新化合物的体内代谢活性进行了研究,并通过回归分析方法对C_n-AAC分子结构与虫草素生物利用度的关系进行了定量描述。结果表明:通过化学结构修饰,把虫草素改变成C_n-AAC后,C_n-AAC在动物体内可以转变还原成虫草素,故C_n-AAC可以作为虫草素的前药而发挥作用。随着C_n-AAC烷酰基侧链碳原子数目的增加,其在动物体内转变成的虫草素的T_(max)和t_(1/2)显著延长,C_(max)和AUC值则出现先升高、再降低的有规律性的变化。其中C_8-AAC试验组在体内转化成的虫草素的C_(max)和AUC值最高,分别是虫草素对照组的约2倍和4倍,其T_(max)和t_(1/2)也比虫草素对照组延长近4倍。C_n-AAC烷酰基侧链碳原子数目和其在动物体内代谢产生的虫草素的AUC值的关系,可以用一个一元二次方程来进行定量描述。这充分说明,虫草素经化学结构修饰改变成C_n-AAC后,不仅可以达到延缓虫草素体内代谢速度的目的,而且可以极显著地改变其在动物体内的生物利用度。
论文通过体外抗菌试验,对4个新化合物的体外抗菌活性进行了初步研究,结果表明:虫草素改变成C_n-AAC后,其抗菌活性也呈现有规律的变化。其中C_8-AAC的抗菌活性最强,和虫草素相比,其对枯草杆菌(Bacillus subtilis)的MIC降低了近5倍,并可对虫草素不能产生抗菌活性的金黄色葡萄球菌(Staphylococcus aureus)和白色念珠菌(Candida albicans)产生抗菌活性。
以上试验结果均表明,通过化学结构修饰使C_n-AAC的亲脂性(或疏水性)达到一个最佳值时,可以使该化合物的体内代谢活性或抗菌作用达到最大,说明分子极性在很大程度上影响着该类化合物的生物活性。
综上所述,本论文通过深入的研究,形成了高效生物合成虫草素、高效提取纯化虫草素的工艺技术。配套使用此二项技术生产虫草素,比传统从蛹虫草子实体中获取虫草素的生产成本降低15倍以上,并且生产周期大为缩短、生产过程更为简单。虫草素被化学修饰改变成烷酰胺同系物后,无论其体内代谢活性和抗菌活性都会产生有规律的变化。在虫草素烷酰胺同系物中,N-正辛酰胺虫草素不仅具有较好的代谢活性,也具有较高的抗菌活性,是一个值得进一步深入研究和有潜在开发利用价值的新化合物。本项研究对促进虫草素的进一步研究和开发利用、全面提升我国在世界上虫草素方面的研究水平、以及促进我国传统医药技术的发展并走向世界具有重要的意义。
本文成功地将现代生物技术、数理统计优化技术以及药物化学理论应用于天然药物活性成分的研究开发中,全面提升天然药物活性成分的生产水平,大力降低其生产成本,并以天然活性成分为先导化合物、通过化学结构修饰的手段研究开发具有更高药理活性的新型药物,对其它天然药物的研究和开发具有方法学上的指导意义和借鉴作用。
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 cordycepin
The 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,KH_2PO_4 and MgSO_4 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 cordycepin
A 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 cordycepin
For 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 (C_3-AAC,C_8-AAC,C_12-AAC and C_18-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.C_8-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.C_8-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.C_8-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.
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