【作者】 余佳文；

【导师】 莫志宏；

【作者基本信息】 重庆大学 ， 生物医学工程， 2009， 博士

【摘要】 蝉花(Cordyceps cicadae)是我国传统的一种名贵中药材,与冬虫夏草同属虫菌复合体,且具有相近的药用价值,所以医家常将蝉花作为冬虫夏草的代用品。蝉花的主要活性成分之一是多球壳菌素(myriocin,ISP-1),多球壳菌素除具有较强的免疫抑制活性,还具有抗动脉粥样硬化、抗真菌等药理作用。ISP-1在野生和人工蝉花药材的含量很低,这也是产业化的瓶颈。目前人们以期通过蝉花发酵的方法提高其产量。因此,建立多球壳菌素类长链碱的检测和评价方法并优化蝉花菌发酵生产多球壳菌素具有十分重要的现实意义。本论文以蝉花为研究对象,建立了一套多球壳菌素分析技术平台;建立了同时测定蝉花中四种多球壳菌素类长链碱(myriocin-like long-chain bases,MLB)的方法;对蝉花鞘氨醇类长链碱(sphingoid long-chain bases,SLB)进行了代谢轮廓分析。主要内容及结论如下:1、蝉花中多球壳菌素含量低、成分复杂,检测困难。本论文选择固相萃取(solid phase extract,SPE)进行样品的纯化,考察了萃取小柱种类、上样量、洗脱和解析溶剂等因素对ISP-1净化的影响。建立了SPE-HPLC分析蝉花多球壳菌素方法,结果表明该方法简便易行、重现性好,可用于蝉花中ISP-1含量的测定,其中ISP-1的平均回收率为98.95%,RSD为2.22%。同时运用质谱验证了样品中的目标成分与多球壳菌素一致性,并根据碎片荷质比(m/z)推测了ISP-1分子离子的合理裂解方式。2、由于ISP-1没有特征吸收基团,采用紫外检测器检测,选择性和灵敏度较低。为此,探索并建立了一个多球壳菌- 9-氯甲酸芴甲酯(Fmoc-Cl)的反应体系,以吡啶溶解的ISP-1与四氢呋喃为溶剂的Fmoc-Cl在40℃反应10 min为优化的反应条件。运用MS技术对Fmoc-Cl与ISP-1的衍生物的结构进行了分析,推断出ISP-1与Fmoc-Cl发生衍生化反应同时ISP-1的分子内发生了内酯化反应。考察了衍生剂的浓度、反应温度、反应时间等因素对ISP-1衍生物形成的影响及多球壳菌素衍生物的稳定性。以Fmoc-Cl为衍生化试剂,构建了一个多球壳菌素柱前衍生并采用紫外检测的HPLC分析系统。定量限和检测限分别为0.102μg/mL、0.045μg/mL,线性范围为2.0 ~ 500.0μg/mL。采用此方法对天然和人工蝉花中多球壳菌素进行了分析,结果显示天然蝉花中多球壳菌素的含量远远低于人工蝉花。3、ISP-1与鞘氨醇(sphingosine,So)、植物鞘氨醇(phytosphingosine,Phy)和二氢鞘氨醇(sphinganine,Sa)均属MLB,具有重要的生理作用。为此,建立了邻苯二甲醛(o-phthalic aldehyde,OPA)柱前衍生-HPLC-UV同时分析这四种MLB的方法。采用LC-MS技术分析了ISP-1、Phy、So和Sa衍生物的结构,并推测其质谱裂解途径。考察了衍生化反应体系中缓冲盐浓度和pH、OPA量、2-巯基乙醇量、衍生反应时间、衍生反应温度等因素对四种MLB衍生物形成的影响及稳定性。结果发现控制反应温度,2-巯基乙醇能够选择性参与邻苯二甲醛与ISP-1的反应。该方法检测性能指标为:检出限分别为1.7、1.2、1.4和0.9μg/mL;加样回收率都大于96%;日内和日间精密度的RSD小于4.0%。优化的样品水解条件为:水解温度110℃、水解时间16 h、溶剂量4 mL。采用此方法对6种不同碳源的蝉花样品中四种MLB进行了分析,结果显示平均含量最高的ISP-1、So、Sa都分布在蔗糖组(340.19μg/g,124.02μg/g,387.84μg/g),而Phy在麦芽糖组(407.78μg/g)。4、为了阐明蝉花中ISP-1与SLB相关性,利用OPA柱前衍生-HPLC-UV-MS技术,开展蝉花SLB代谢轮廓分析。精密度、重现性、稳定性等3项试验里10个色谱峰的保留时间和峰面积的RSD分别低于1.7%和4.5%。获取24个菌丝体样品的峰面积,经主成分分析模式识别,提取了2个主成分,其累积贡献率分别为79.16%和13.06%。蝉花SLB代谢轮廓分析的结论为:主成分得分图能够区分ISP-1不同含量的6个碳源组;2个主成分与ISP-1的含量曾负相关;所分析的10个变量(色谱峰)在组间的差异有统计学意义。经LC-MS、文献和网上数据库的分子量筛选,8种SLB被推断。更多还原

【Abstract】 Cordyceps cicadae, a caterpillar-shaped Chinese traditional medicinal mushroom, is one of the entomopathogenic fungal, which belongs to the class Ascomycetes and DongChongXiaCao group in Chinese herbs. In addition to a strong immunosuppressive activity, myriocin has anti-atherosclerotic, anti-fungal and other pharmacological effects.However, both scarce resources of wild medicinal materials and lower production from wild and natruanl C. cicadae, have limited its industrial output and become a bottleneck. It is imperative to establish more efficient and effective detection methods for myriocin and carring out metabolomics studies for C. cicadae. Facing to low content of myriocin and serious interference, this paper has developed new methods of detection for myriocin and other three sphingoid bases by pre-column derivatization and HPLC. By 6 different carbon sources for fermentation model, preliminary study about metabolomics of sphingoid bases in C. cicadae was carried out. The main contents and conclusions are as follows:1. To establish a sensitive method for the determination of the content of myriocin in samples collected from submerged cultivation in C. cicadae, sample was extracted with methanol and solid phase extraction, and identified by MS. Myriocin in C. cicadae was separated on a ODS column.The mobile phase was acetonitrile -water (65∶35) at a flow rate of 1.0 mL/min. The UV detection wavelength was 203 nm. Myriocin in sample solution was well separated. The average recovery was 98.95% with RSD 2.22% ( n = 6) and the linear range was 0.15– 7.54μg. The method is convenient, rapid, accurate, and sensitive, thereby leading to a wide and effective analysis for the determination of the myriocin-like immunoregulational ingredients in C. cicadae.2. A more sensitive method, based on the pre-column derivatization with 9-fluorenylmethyl chloroformate, was developed for the determination of myriocin. The derivatization reaction was performed in organic solvents of pyridine and tetrahydrofuan at 40oC. Several factors influencing the derivative yield were investigated and optimized. The formed derivative was stable for more than 24 h at room temperature. The detection wavelength was 262 nm. The system offered the following analytical parameters: the limit of detection was 0.045μg/ mL, the linear correlation coefficient was 0.9963 and the linear range response was from 2.0 to 500.0 μg ml-1. The precision of the method was < 2.0%. As a preliminary application, the method has been successfully applied to the determination of myriocin in natural and cultured C. cicadae.3. ISP-1, is potent inhibitor of the de novo sphingolipid biosynthesis via inhibiting the key enzyme serine palmitoyltransferase synthase. To study the cellular accumulation of ISP-1, phytosphingosine (Phy), sphingosine (So), and sphinganine (Sa), we developed a method of simultaneous determination for ISP-1, Phy, So and Sa. Several factors influencing the derivative yield were investigated and optimized. The formed derivative was stable for more than 8 h at 4 oC in refrigerator. The detection wavelength was 230 nm. The detection limits of 1.7μg/ml for ISP-1(signal-to-noise ratio S/N =3:1), 1.2μg/mL for Phy, 1.4μg/ml for So and 0.9μg/ml for Sa were established.The average recovery for ISP-1, Phy, So and Sa was higher than 96%. As a preliminary application, the method has been successfully applied to the determination of 4 sphingoid bases in C. cicadae from 6 different carbon sources.4. According to the result of C. cicadae in the HPLC-UV-MS, 10 components were identified. SPSS software was used to process the data obtained with different carbon sources as a model. Principal component analysis (PCA) and metabolic profile analysis of sphingolipid metabolism were carried out, respectively. Results of metabolic profiling analysis of sphingoid long-chain bases show that the score plot of PCA had the ability to distinguish six carbon groups, that two principal components had a negative correlation with the ISP-1 levels, and that the analysis of 10 variables (peaks) differences between the groups was statistically significant. By LC-MS analysis and the molecular weight contrast, eight metabolites have been proved.更多还原