| Cordyceps militaris is also known as North Cordyceps sinensis. Its effectiveness is similar to the wild Cordyceps sinensis. It has good effect to lung and kidney, could stop bleeding and dissipate phlegm, and has anti-fatigue and anti-anoxia effect. It also can regulate the immune and support anti-tumor. Because of its low requirement of the natural environment for growth, its artificial cultivation is successful and it could become a substitute for C. sinensis. Today there are a lot of cultured C. militaris on the market, but without a standardized method for quality control. And there is no standard for quality control of C. militaris in the Chinese Pharmacopoeia. In order to apply cultured C. militaris rationally and effectively and evaluate the quality of it with specific quantitative indicators, this paper established a systematical quality standard including using fingerprint as a quality control tool. This could draw on the ideas and methods for the quality control of cultured C. militaris, and provide corresponding basis for its future quality assessment.According to the relevant regulations in the 2005 edition Chinese Pharmacopoeia, this paper systematically studied various indicators for the quality control of cultured C. militaris, including characters, identification, examination and content determination. The optimal extracting condition of adenosine and cordycepin was optimized by orthogonal design method. The preparation method was determined as follows: 20% methanol 30 times, ultrasonic extraction 10min. The content of adenosine and cordycepin was determined simultaneously by HPLC. The adenosine presented a good linear correlation in the range of 23.2ng～464ng(r = 0.9999). The average recovery was 98.92%(RSD=1.17%). The cordycepin presented a good linear correlation in the range of 30ng～600ng(r = 0.9999). The average recovery was 99.37%(RSD=1.42%). It was showed that the extracting and determination method was simple, reproducible and the result was accurate and reliable. It could provide basis for evaluating the quality of cultured C. militaris.According to the related regulations in the provisional technical requirements of injection fingerprint study of TCM, the experiment developed fingerprint of cultured C. militaris by HPLC/DAD method. The separation was performed on Agilent TC–18(5μm, 4.6×250mm) column with methanol-0.02mol/LKH2PO4 in 60min as mobile phase, gradient elution. The detection wavelength was at 260nm, and the flow rate was 1.0mL/min with column temperature at 30℃. It was proved that the method was accurate and reliable. Fingerprints of 12 batches of cultured C. militaris were collected and 15 common peaks were identified. And the peaks of uridine, adenosine and cordycepin were identified. Similarity was calculated by similarity evaluation system for chromatographic fingerprint of TCM. The results showed that the similarity of 12 batches of cultured C. militaris were above 0.90 in contrast with controlled fingerprint. There was a good consistency between different batches of cultured C. militaris.HPLC fingerprint analysis was applied to analyze 15 batches of cultured C. militaris from different areas. Systematic hierarchical cluster analysis was carried out by using characteristic data obtained. 15 batches of cultured C. militaris were divided into two grades. Among them the grade I was commendatory, the grade II was non-commendatory. The experiment established Canonical and Bayes discriminant function by using stepwise discriminant analysis method, and then identified the effect of discriminant function with cross-validation method. It was indicated that the discriminant result corresponded with the fact very well. The establishment of two types of discriminant function provided an accurate, efficient tool for the identification of unknown samples.The study established a system for quality assessment of cultured Cordyceps militaris. And it had laid a theoretical foundation for the comprehensive, scientific and effective quality control of cultured Cordyceps militaris.
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