I.中藥材何首烏之輻射滅菌劑量研究
II.台灣蒲公英及其誤用品之分子鑑定研究

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Title:	I.中藥材何首烏之輻射滅菌劑量研究 II.台灣蒲公英及其誤用品之分子鑑定研究 I.Studies on the Gamma Radiation Antiseptic Dose of Polygoni Multiflori Radix II.Studies on Molecular Identification of Taraxacum formosanum and Distinguishing from Its Adulterants
Authors:	江瑩真
Contributors:	中國藥學暨中藥資源學系博士班
Keywords:	輻射滅菌;何首烏;台灣蒲公英;分子鑑定 Gamma radiation;Polygoni Multiflori Radix;Molecular Identification;Taraxacum formosanum
Date:	2011-07-25
Issue Date:	2011-10-17 16:00:32 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	中藥材何首烏之輻射滅菌劑量研究本研究目的為探討何首烏中藥材輻射照射滅菌最適條件及分析輻射照射後中藥材抗氧化活性、抗氧化成分及指標成分變化情形，找出達到最佳輻射滅菌效果的最小吸收劑量，建立何首烏中藥材輻射照射滅菌的最適劑量。何首烏中藥材分別以 2 kGy、4 kGy、6 kGy、8 kGy 和 10 kGy 照射之實驗結果顯示，輻射照射2 kGy即可消滅腸內菌，4 kGy能抑制真菌及酵母菌的生長，在6 kGy的輻射照射後黴菌和真菌即完全滅菌；菌數方面，隨著輻射劑量的增加而減少，8 kGy 照射可達到徹底滅菌的效果。在抗氧化活性實驗及指標成分比較之實驗方面，何首烏藥材分別照射5 kGy、10 kGy 和 15 kGy的結果顯示，以照射 5 kGy 有最好的抗氧化活性及最低對DPPH自由基清除率達50%時的樣品濃度（即IC50值）。抗氧化成分實驗結果顯示，所含總多酚類、總類黃酮以照射5 kGy為最高，照射15 kGy最低，而總黃酮醇之含量並無明顯差異。因此，8 kGy的輻射照射，可以完全滅菌，可作為何首烏中藥材滅菌最適合之劑量，來延長藥材的架儲期，確保何首烏中藥材的品質。台灣蒲公英及其誤用品之分子鑑定研究藥用植物的基原鑑定是品管的重要環節，本研究藉由比對GenBank資料庫上台灣蒲公英與其易混用藥材間的ITS2基因序列，找出其中差異較大的區域，作為分子鑑定引子設計的依據。以設計好的一對引子進行台灣蒲公英與其易混用藥材的聚合酶連鎖反應，可擴增到250 bp大小的ITS2片段反應產物，將產物定序並分析比對DNA序列，成功的鑑別出台灣蒲公英與其易混用的5種藥材。此外，運用DNA分子標誌，針對台灣蒲公英與其混用藥材之ITS2基因組，以引子開拓者V3軟體設計一組高特異性的恆溫圈環式核酸擴增法鑑定(LAMP)之引子，並找出其最佳化反應條件、分析方法的靈敏度和特異性檢測，評估台灣蒲公英正品及混用藥材的LAMP檢測效果。本研究設計之LMAP引子可專一地於65℃的溫度，45分鐘內放大含台灣蒲公英模板DNA的反應溶液，且於核酸濃度1 pg下即可進行LAMP之反應，其敏感性遠勝過傳統PCR法，成功地建立LAMP做為台灣蒲公英中藥材基因體鑑別的檢驗系統。 Studies on the Gamma Radiation Antiseptic Dose of Polygoni Multiflori Radix Gamma radiation is a physical process commonly used for the eradication of microorganisms distributed in food ingredients, medicinal plants and other bioresearches. The aim of this study was to investigate the effect of radiation dosage on the microbial load, chemical compounds and antioxidative characteristics of Polygoni Multiflori Radix (POMU). Ten commercial POMUs were purchased from different herbal markets and treated with 2 kGy, 4 kGy, 6 kGy, 8 kGy and 10 kGy gamma radiation doses to evaluate the microbial burdens of irradiated and unirradiated POMUs. Our results confirmed that 2 kGy was sufficient for the inactivation of enterobacteria; at 4 kGy, mold and yeast counts were obviously reduced; and at 6 kGy, neither yeasts nor fungi were observed any longer. The antioxidative effects and major antioxidant components of 0 kGy, 5 kGy, 10 kGy and 15 kGy irradiated POMU samples were also examined. Our results confirmed that 5 kGy irradiated POMU had both the highest antioxidative activity and lowest value in IC50 of DPPH radical-scavenging activity. The content of total phenols had no statistically significant changes. Therefore gamma irradiation at 8 kGy could be a potential method for decontaminate the microbial load of POMU to prolong shelf life and to improve hygienic quality. Studies on Molecular Identification of Taraxacum formosanum and Distinguishing from Its Adulterants Original identification of medicinal plants is important for quality control. In this study, the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA served as a DNA barcode and was amplified by allele-specific sequence-primed PCR; this approach was exploited to differentiate Taraxacum formosanum from five related adulterants. Using a set of designed PCR primers, a highly specific 250 bp PCR product of Taraxacum formosanum was successfully amplified by PCR. However, no product was amplified from any of the adulterants. This indicates that our allele specific primers have high specificity and can accurately discriminate Taraxacum formosanum from other 5 adulterant plants. We also established a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating Taraxacum formosanum. A set of four specific LAMP primers was designed ba sed on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when genomic of TF was added in the LAMP reaction under isothermal condition (65℃) within 45 min. This specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the minimum concentration limit of the LAMP assay.
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