Molecular Identification of Kava-Kava (Piper methysticum G. Forst.) Using the Internal Transcribed Spacer (ITS2) Region

Background: Piper is one of the largest genera in the family Piperaceae, with approximately 2100 species. Most Piper species are used as spices or as medicinal plants. Piper methysticum G. Forst., popularly known as kava-kava (or kava), is widely used to treat anxiety disorders. Due to similar morphological features, P. auritum Kunth (known as “false kava”) is sometimes mistakenly or intentionally used as an alternative botanical source for “kava” extracts. The false kava extracts do not contain active kavalactones but contain safrole, which is hepatotoxic. It is important to verify the component botanical materials in order to evaluate the quality and safety attributes of a potential botanical drug. Some studies have evaluated genetic variation in Piper sp. using the chloroplast regions matK, rbcL, rpoC1 and trnH-psbA and the nuclear ITS2 markers. However, none has focused on the identification of P. methysticum using DNA barcodes. In the present investigation, the ITS2 DNA barcode region from the nuclear genome was tested to confirm the identification and authentication of kava-kava samples. Methods: Seven P. methysticum samples were collected from three different geographic lo-cations and two P. auritum samples were collected and the ITS2 region from the nuclear genome, was amplified, sequenced and aligned to determine their genetic distances. Results: The ITS2 locus showed high amplification and sequence output with a discriminating barcode gap. A distance-based phylogenetic tree and BLAST confirmation (using blastn) revealed the ITS2 locus as a diagnostic DNA barcode for the accurate identification of kava-kava species. Discussion: In conclusion, the ITS2 region proves to be an effective and reliable DNA barcode for distinguishing P. methysticum from closely related species such as P. auritum. Its application can significantly improve the safety, quality, and traceability of kava-containing products, addressing a critical need in the standardization of botanical drugs.