PCR and PCR–RFLP of the 5S-rRNA-NTS region and salvinorin A analyses for the rapid and unequivocal determination of Salvia divinorum

Abstract

Salvia divinorum Epling & Játiva-M. is a perennial herb belonging to the Lamiaceae family; its active ingredient, the neoclerodane diterpene salvinorin A, is a psychotropic molecule that produces hallucinations. A comparative evaluation of S. divinorum fresh and dried leaves, S. officinalis fresh leaves, and dried powdered leaves claimed to be S. divinorum was done. HPLC–MS data confirmed the presence of salvinorin A in both S. divinorun leaf extracts and the powdered leaves, whereas no salvinorin A was found in S. officinalis. The non-transcribed spacer (NTS) in the 5S-rRNA gene of all leaf samples and the dried powdered leaves was amplified by PCR using a pair of primers located at the 3′ and 5′ ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 500 bp for S. divinorum and 300 bp for S. officinalis) were gel purified, subcloned into pGEM ®-T Easy vector and sequenced. By aligning the isolated nucleotide sequences, great diversities were found in the spacer region of the two species. Specific S. divinorum primers were designed on the sequence of the 5S-rRNA gene spacer region. In addition, a PCR–restriction fragment length polymorphism (PCR–RFLP) method was applied using NdeI and TaqI restriction enzymes. An NdeI site, absent in S. officinalis, was found in S. divinorum NTS region at 428–433 bp. For TaqI, multiple sites (161–164, 170–173, and 217–220 bp) were found in S. officinalis, whereas a unique site was found in S. divinorum (235–238 bp). The results of this work show that the combined use of analytical chemical (HPLC–MS) and molecular (DNA fingerprinting) methods lead to the precise and unequivocal identification of S. divinorum.