Discrimination of three Scrophularia plants utilizing ‘Scrophularia Radix’ by DNA markers based on internal transcribed spacer (ITS) sequences

Abstract

One of the medicinal materials produced by plants belonging to the genus Scrophularia, ‘Scrophularia Radix’ (SR), has been prescribed for many centuries to treat diseases such as inflammation, abscesses of carbuncles and constipation. In China, the dried root of S. ningoensis Hemsley is the source of SR. In contrast, the root of S. buergeriana is generally prescribed as SR in Korea. Studies conducted to identify the bioactive compounds in these two Scrophularia plants have revealed marked differences in the contents and concentration of the compounds they contain. However, S. ningpoensis has been indiscriminately prescribed in Korea along with S. buergeriana as SR. Furthermore, S. koraiensis has long been used in lieu of S. buergeriana as SR in Korea. Therefore, a standard or method to reliably distinguish these three species of Scrophularia is needed. Recently, we found that the differences in the nucleotide sequences of the internal transcribed spacer (ITS) of Scrophularia plants could be applied to develop DNA markers to discriminate each plant. In this study, ITS sequences of 22 samples including three types of Scrophularia plants were amplified, determined and analyzed. Based on the results of these analyses, we designed the following primer sets: Ni F (5′-TTAACCATATAGGGGCCTCG-3′) / Ni R (5′-C CCCTCTCTGTATCCCAA-3′) to amplify a 379 bp DNA marker for the identification of S. ningpoensis; Bu F (5′-TTAACC ATATCGGGGCCAAG-3′) / Bu R (5′-ATCACGACAGCAC GCGA-3′) to amplify a 491 bp DNA marker for S. buergeriana; and Ko F (5′-ATAACCATATCGGGGCCTC-3′) / Ko R (5′-TCAAGAAACGCACTATCCC-3′) to amplify a 167 bp DNA marker of S. koraiensis. Using these primer sets, we were able to efficiently identify Scrophularia plants sold as SR in the herbal market in dried and sliced states after processing as medicinal materials.