Molecular authentication of Thai medicinal plant Khamin khruea by PCR-RFLP analysis

Abstract

Khamin khruea is a traditional medicine referred to the woody climber with yellow wood and root. Its stem has been widely used in Thai traditional medicine for treatment of stomachic and inflammatory-related symptoms such as cold, fever, and arthritis. In Thailand, Khamin khruea is the local name of three species in the family Menispermaceae including Arcangelisia flava (L.) Merr., Fibraurea tinctoria Lour. and Copscinium fenestratum (Gaertn.) Colebr [1]. These three plants have likely macroscopic and microscopic characteristics. Since neuro-toxicity of C. fenestratum has been reported [2], the precise authentication of C. fenestratum is necessary for the quality control purpose. Based on the18s rDNA sequences of the 3 species, a distinctive site recognized by a restriction enzyme Xma I in C. fenestratum was found and then PCR-restriction fragment length polymorphism (RFLP) analysis was established. A pair of new primers, 18SF404 and 18SR842, was designed based on the sequence of C. fenestratum to amplify a smaller fragment of 439 bp in length from genomic DNAs of the leaves of these three species and of crude drug samples. The PCR fragment of 18S (partial) rDNA region from C. fenestratum was cleaved into two fragments of 112 bp and 327 bp while the other species remained undigested. This method provides an effective and accurate identification of C. fenestratum.