運用巢式PCR 與DNA 定序方法區別中藥製劑中的黃耆與紅耆藥材   全文下載

Discriminating Astragali Radix from Hedysarum Radix in Chinese Medicine Preparations Using Nested PCR and DNA Sequencing Methods

黃耆的基原為膜莢黃耆與蒙古黃耆，但在臺灣，紅耆經常被用來當做替代品，特別在中藥製劑中，所用的黃耆是否為真品很難被確認。在本篇研究中，巢式PCR 與DNA 定序方法被用來鑑別製劑中所含的藥材，並設計兩組引子能夠專一地偵測製劑中的黃耆及紅耆成份。利用有機溶液及套組抽取純化製劑樣品的DNA，再利用巢式PCR 擴增ITS 片段並加以定序。對比樣品ITS 序列與標準序列，以確認樣品中所含的黃耆和紅耆藥材。結果顯示，30 件製劑檢體中有20 件有黃耆與紅耆摻混使用的情形，5件是誤用紅耆入藥，僅有5 件是單純使用黃耆。另外也嘗試結合巢式PCR與限制片段多型性方法，用以快速區別膜莢黃耆、蒙古黃耆與其雜交種。而本研究之方法確認可以用以鑑別中藥製劑中的黃耆與紅耆藥材。

Astragali Radix are originated from Astragalus membranaceus and A. membranaceus var. mongholicus. However, Hedysarum Radix, the dried root of Hedysarum polybotrys, is often used as a substitute in Taiwan. In Chinese medicinal preparations, authenticity of Astragali Radix is difficult to be certified. The nested PCR and DNA sequencing method was used to identify Astragali Radix and Hedysarum Radix in preparation samples. Primer sets were designed for detecting Astragali Radix and Hedysarum Radix. The total DNA of all samples was extracted. Internal transcribed spacer (ITS) regions of the DNA samples were amplified by nested PCR and the sequences received from an auto-sequencer. We compared ITS sequences of the preparation samples with the standard to check whether Astragali Radix or Hedysarum Radix was in the preparation samples. Results indicated that 20 out of 30 samples contained a mixture of Astragali Radix and Hedysarum Radix, five Hedysarum Radix as substitute and five Astragali Radix. The combination of nested PCR and restriction fragment lengths polymorphism (RFLP) was employed to rapidly differentiate Astragalus membranaceus, A. membranaceus var. mongholicus and hybrid. The nested PCR and DNA sequencing method successfully identified Astragali Radix and Hedysarum Radix in Chinese medicine preparations.