| 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.
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