Abstract Cordyceps sinensis (Berk.) Sacc., one of the well-known and valuable traditional Chinese medicines (TCM), is a composite consisting of the stromata of the fungus and the dead caterpillar. It has been traditionally used in the treatment of chronic bronchitis, especially in elderly patients, and asthma, chronic obstructive pulmonary disease (COPD), tuberculosis, cor pulmonale, and other disea ses of the respiratory system in China. However, the yield of the wild C. sinensis is decreasing because of the deterioration of ecosystem for Cordyceps due to over exploration. Therefore, isolation of mycelia strain from Cordyceps is a trend of many scientists to achieve a large scale production of Cordyceps. Besides cultivation of Cordyceps, much effort has also been focus on the discovery of alternative species, and Cordyceps militaris is the most popular one. Nowadays, several products of cultured mycel ia of С. sinensis, C. militaris and other species of Cordyceps were used and sold in the market. Therefore, it is very important to evaluate the difference among different kinds of Cordyceps (natural Cordyceps spp., cultured Cordyceps mycelia and Cordyceps militaris), which is beneficial to the quality control. Sample preparation is the first and key step in TCM analysis. Choosing a proper sample preparation method is very important for the quality control. Based on previous study, a significant change of the contents of nucleosides was found in natural C. sinensis with different sample preparation methods, but not in commercial Cordyceps mycelium. However the reason of this phenomenon need to be further studied. On the other hand, Cordyceps mainly contains nucleosides, steroids, carbohydrates, amino acids, fatty acids, protein and polysaccharides, etc. However, the marker for quality control of Cordyceps sinensis recorded in China Pharmacopeia (2005) was adenosine alone, which can not effectively control the quality of Cordyceps. In order to ensure the safety and efficacy of C. sinensis, the simultaneous quantification of multiple components in Cordyceps is required. The dissertation consists of three chapters. Chapter 1 reviewed the chemical components in C. sinensis and the quality control methods. Chapter 2 showed the preliminary research on the relationship of different sample preparation and nucleosides quantification in natural and cultured Cordyceps. Ambient temperature water extraction was chosen as the extraction method to compare the content of nucleosides in natural and cultured Cordyceps using different sample pre-treatment. It is suggested that some enzymes, which were sensitive to high temperature, might be contained in natural and cultured Cordyceps. Chapter 3 focused on simultaneously qualitative and quantitative determination of nucleosides (uracil, cordycepin, adenine, adenosine, uridine, hypoxanthine, inosine and guanosine), carbohydrates (mannitol, glucose and trehalose) and atypic amino acid, myriocin, in natural Cordyceps (including Cordyceps sinensis, Cordyceps liangshanensis and Cordyceps gunnii) and cultured Cordyceps (including different kinds of Cordyceps sinensis mycelia, the fruiting body of Cordyceps militaris and the mycelium of Cordyceps militaris) using PLE extraction and high performance liquid chromatography- evaporative light scattering detection. SPSS hierarchical clustering analysis based on the contents of 12 investigated compounds showed that 29 tested samples can be grouped into four main clusters, which is in accordance with the clustering result derived from the contents of three carbohydrates, i. e. mannitol, glucose and trehalose. It is suggested that the characteristics of carbohydrates can be used as markers for quality control of Cordyceps. Key words: Cordyceps, nucleosides, carbohydrates, myriocin, HPLC-DAD-ELSD, sample preparation VI

冬蟲夏草為麥角菌科真菌冬蟲夏草菌 Cordyceps sinensis (Berk.) Sacc.寄生 在蝙蝠 科昆蟲幼蟲上的子座及幼蟲屍體的複合體,是我國傳統的名貴中藥, 具有補肺益腎,止血化痰等功效,常用於久咳虚喘,勞嗽咯血,陽痿遺精,腰 膝酸痛等症。由於冬蟲夏草生長環境苛刻,野生資源被過度採挖使其生長環境 遭到嚴重破壞,導致天然蟲草資源稀缺。近年來,研究人員從天然蟲草中分離 得到多個菌株並用於大工業發酵生產,產品廣銷亞洲保健品市場。此外,一些 天然蟲草替代品,如 蟲草及非藥典品種天然蟲草,也常被用於臨床治療,而 蟲草偽品的出現則使得藥材市場更加混亂。為了保證蟲草臨床用藥的安全有 效,冬蟲夏草、蟲草替代品及其製品的化學成分評價顯得尤為重要。 樣品處理是中藥分析過程關鍵的第一步,選擇合適的樣品處理方法對分析 結果至關重要。前期研究發現不同的樣品提取方法會導致天然蟲草的核苷含量 發生變化,而商品人工蟲草菌粉卻無變化。這個現象是源於蟲草種類的不同還 是取決於樣品處理中某個步驟,尚且無法定論。另外,目前認為蟲草中主要含 有核苷及核苷鹼基、醇、糖、糖醇、氨基酸、脂肪酸、多糖及蛋白質等活性 成分,而現有蟲草質量控制標準--中國藥典(2005 年版)僅以腺苷為指標,評 價標準過於單一,無法反映蟲草藥材的實際質量。因此,對蟲草中具有藥理活 性的多種成分進行同時定量分析的方法有利於提高蟲草質量控制水平。 全文共分三章,第一章綜述了冬蟲夏草中主要活性成分及其藥效關係、蟲 草化學分析現狀與問題,並提出對策。第二章針對天然蟲草的核苷類含量隨樣 品處理方法的不同而改變但商品人工蟲草菌粉沒有變化的現象,選擇室溫水浸 泡的提取方法,初步探討了兩種不同樣品處理方法(高溫高濕模式和室溫模式) 與天然及人工蟲草核苷含量的關係,發現高溫高濕條件會將天然蟲草和冷凍乾 燥人工蟲草菌絲體中潜在酶類成分滅活,從而阻止了核苷類成分的轉化;而室 溫冷水浸泡提取的核苷成分則因潜在酶類成分的作用而發生量的改變。同時通 過商品人工蟲草菌粉在兩種樣品處理方法下無核苷量變化的現象解釋了其原因 可能是工業生產中高溫高濕滅菌過程對該成分的破壞。第三章是本研究的重 點建立了高效液相色譜-紫外-蒸發光散射檢測器聯用技術(HPLC-DAD-ELSD) 澳門大學碩士學位論文 同時定性、定量分析天然和人工蟲草中多指標成分,即8個核苷類成分(尿 啶、蟲草素、腺苷、腺 吟、尿苷、次黃 吟、肌苷和鳥 )、3個糖及糖醇類 成分(D-甘露醇、D-葡萄糖和 D-海藻糖)及1個非典型氨基酸-多球殼菌素的 方法,並採用 SPSS 軟件對所測定的29個蟲草樣品進行聚類分析,結果聚合成 四組,每組分別以天然冬蟲夏草、非藥典品種天然蟲草、低糖含量人工蟲草菌 絲體、高糖含量人工蟲草菌絲體為代表。當以糖及糖醇類作為變量仍可以得到 與上述相同的聚類結果,提示糖類成分有利於蟲草的定性鑒別。 關鍵字:蟲草,核苷,糖,多球殼菌素,HPLC-DAD-ELSD,樣品處理