Identification of <i>Nigella sativa</i> Seed and Its Adulterants Using DNA Barcode Marker

Abstract

Adulteration, misidentification, and substitution are the biggest challenges in maintaining safety and therapeutic efficacy of medicinal herbs. <i>Nigella sativa</i> seed, which is well known medicinal herb susceptible to adulteration or substitution due to its great therapeutic value. Adulteration and substitution by morphologically similar seeds are the primary concern in commercially available <i>Nigella sativa</i> seed. In this study, we have used DNA barcode marker to find out adulteration, misidentification, and substitution of <i>Nigella sativa</i> seed sold in various markets. We collected 10 samples, which were labelled as Black seed/<i>Nigella sativa</i> seed from open markets in India (1 No.), Pakistan (1 No.), Saudi Arabia(1 No.), Egypt (2 No.), Turkey (1 No.), Syria (1 No.), Tunisia(2 No.) and Oman (1 No.). All samples collected from different geographies were studied morphologically. Although few samples were quickly identified as <i>Nigella sativa</i> seeds, few were tough to detect and differentiate accurately. This is where DNA barcode marker proved to be useful. Plant DNA were obtained from seed coat cells of samples, was amplified by PCR with forward and reverse rbcl and matK primers as recommended by CBOL (The Consortium for the Barcode of Life). PCR amplification of plastid genome with matK was not very successful, while PCR amplification with rbcl primers was quite successful. We used rbcl sequences for alignment and further analysis. PCR products obtained were subjected to electrophoresis on 1.5% agarose plate. PCR products were sent to Macrogen (Seoul, South Korea) for DNA sequencing. DNA reads obtained with rbcl sequences were aligned and analyzed for nucleotide composition, conserved sites, variable sites, singleton sites and parsimony-informative sites, genetic distance and phylogenetic tree using MEGA 7. The phylogenetic tree was constructed using UPGMA method. NCBI Blast along with phylogenic tree and nucleotide characteristic were used to identify <i>Nigella sativa</i> seeds from different geographies and discriminate two adulterants as <i>Allium cepa</i> seed and <i>Clitoria guianensis</i> seed. Both of these adulterants are different regarding their active medicinal contents and therapeutic utility from <i>Nigella sativa</i> seed. This study proved the utility of DNA marker, especially rbcl loci in accurately identifying medicinal herb and its adulterants.