Cordyceps sinensis, an entomogenous fungus also known as Dongcong Xiacao (winter worm, summer grass) in Chinese, is one of the most valued medicinal herbs in traditional Chinese medicine (TCM). With the appearances of cultured or alternative species Cordyceps, it became an important problem for authentication and quality control of Cordyceps. Nucleosides are believed to be one of the most important constitutes in Cordyceps for their pharmacological activities. And amino acids are considered to be the key regulators of nutrient metabolism in cells. And they may also provide tonic activities and act as the nutrition monitoring indexes in both food and health care products, meanwhile, amino acids usually contribute the unique flavors to fungus. Therefore, detecting of nucleosides and amino acids is required. The samples were prepared through a heating reflux extraction. And in order to obtain more information about the differences between micromolecule (nucleosides and amino acids) and macromolecule (nucleotide, protein and etc.), this experiment also determined the compounds hydrolyzed from macromolecule using the microwave-assisted hydrolysis. The chromatographic separations were performed using an Agilent Zorbax SB-C18 column (5 μm, 4.6 mm×250 mm) with an Agilent Zorbax SB-C18 guard column (5 μm, 4.6 mm×12.5 mm) at a constant temperature of 30 ºC. The standards and samples were injected with 10 μL and separated using a gradient mobile phase consisting of 4‰ TFA in water (A) and acetonitrile (B). At a flow rate of 0.6 mL/min, the gradient condition is : 0-15 min, 0% B; 15-20 min, 5% B; 20-25 min, 5% B; 25-35 min, 7% B; 35-40 min,7% B; 40-45 min, 7-40% B; 45-55 min, 40% B; 55-60 min, 40-0% B; while recondition the column with 0% B for 15 min. The column was washed every five runs. The nucleosides and nucleobases were monitored at 254 nm, and the ELSD was used for detection of amino acids. Under this chromatographic condition, seven nucleosides and nucleobases (uracil, hypoxanthine, uridine, adenine, inosine, adenosine, cordycepin), and eight amino acids (alanine, histidine, valine, tyrosine, isoleucine, leucine, phenylalanine, tryptophane) were baseline separated. And the method revealed good linear relationships, high repeatabilities and recoveries. The results suggested that the contents of nearly all the investigated compounds would increase after hydrolysis. Among the cultured ones, only the contents of cordycepin and adenosine decreased with hydrolysis. And for the natural group, except adenine and uridine were almost disappeared, the others all showed a smaller growth with hydrolysis. On the other side, the data of amino acids suggested that there were a larger increase in cultured Cordyceps after hydrolysis. According the analysis of SPSS software, some of the same species could be classified into one group through the data of nucleosides and nucleobases, and with only the hydrolysis data, the samples could be generally divided into natural and cultured ones. This suggested that there may be some significant difference in macromolecule between natural and cultured Cordyceps. To sum up, seven nucleosides and nucleobases (uracil, hypoxanthine, uridine, adenine, inosine, adenosine, cordycepin), and eight amino acids (alanine, histidine, valine, tyrosine, isoleucine, leucine, phenylalanine, tryptophane) have been selected as markers for analysis. And a rapid qualitative and quantitative analysis HPLC method was established for natural and cultured Cordyceps with good selectivity of detection and high repeatability. Key words: Cordyceps; HPLC-DAD-ELSD; Nucleosides; Amino acids; Acid hydrolysis; Microwave-assisted;

冬蟲夏草是我國的名貴中藥，傳統中醫藥理論認為其有補腎益肺，止血化痰的功效， 用於治療腎虛精虧，陽痿遺精，腰膝酸痛，久咳虛喘，勞嗽咯血等症。現代藥理研究表明， 冬蟲夏草具有增強人體免疫功能和抗腫瘤等活性。隨著蟲草天然替代品的發掘及人工發酵 培養技術的發展，加之市場上的一些混亂現象，蟲草的質量顯得越來越重要。核苷類成份 是蟲草非常重要的活性成分之一，同時，蟲草作為藥食兩用的藥材，氨基酸被認為是其活 性成分及營養評價指標，此外它還是真菌類獨特風味的形成因素之一。故本實驗選擇了以 核苷類及氨基酸類物質為蟲草的質控指標。另一方面，本實驗還測定了水解后核苷及氨基 酸的含量，用以從一定程度上反應蟲草中核苷酸，蛋白質等大分子物質的情況。 本實驗採用平行迴流提取，微波輔助酸水解的方法，通過HPLC-DAD-ELSD對14種天 然及人工蟲草樣品分析，完成了對7種核苷（尿嘧啶，次黃嘌呤，尿苷，腺嘌呤，肌苷，腺 苷，蟲草素）和8種氨基酸（丙氨酸，組氨酸，纈氨酸，絡氨酸，異亮氨酸，亮氨酸，苯丙 氨酸，色氨酸）水解前後的含量測定與比較。方法如下：色譜柱Agilent Zorbax SB-C18 column (5 μm, 4.6 mm×250 mm) ；預柱Agilent Zorbax SB-C18 guard column (5 μm, 4.6 mm×12.5 mm)； 柱溫30 ºC；進樣量10 μL。流動相：4‰ TFA(A)–乙腈(B)；流速：0.6 mL/min；梯度洗脫： 0-15 min，0% B；15-20 min，5% B；20-25 min，5% B；25-35 min，7% B；35-40 min，7% B；40-45 min，7-40% B；45-55 min，40% B；55-60 min，40-0% B；回到初始流動相平衡 15 min。檢測波長；254 nm；ELSD參數：溫度為50 ºC，Gain值為8。本方法線性關係良好， 加樣回收率與重複性基本在要求內 。 結果表明，天然蟲草水解前後的核苷與氨基酸含量普遍低於人工蟲草。核苷類成份水 解后，人工蟲草中除蟲草素與腺苷的含量下降，其他皆呈上升趨勢。天然蟲草中大多數核 苷類成份也有相同趨勢但增幅較之減小很多，同時，部份成份如腺嘌呤與尿苷，幾乎完全 消失；氨基酸類成分中，水解后，人工蟲草的增幅遠大於天然蟲草。最後，通過SPSS分析 可得，利用蟲草遊離的核苷類成份數據能較好的區分其物種關係。水解后，則可將天然與 人工蟲草兩個大類大致區分出來。此結果可能預示著人工蟲草在核苷類的大分子上與天然 者有著較大差異。 此方法可以用於比較天然與人工蟲草在小分子（核苷類及氨基酸）和大分子（核苷酸 及蛋白質等）上的差異。結果表明，本方法穩定可靠，耗時較短，為蟲草的質量控制做出 了一定的參考。 關鍵字：蟲草；核苷；氨基酸；HPLC-DAD-ELSD；微波輔助酸水解