Standardization of Euphorbia tithymaloides (L.) Poit. (Root) by Conventional and DNA Barcoding Methods

[Image: see text] Adulteration and substitution of medicinal plants have become a matter of great concern in recent years. Euphorbia tithymaloides is one such medicinal plant that has gained importance but is often confused with other plants of the same species. In order to address this issue, this study aimed to conduct a conventional and molecular pharmacognostic study for the identification of the root of E. tithymaloides. The root of the plant was studied for the macroscopic observations, and then, the root was ground into coarse powder for microscopic studies and to determine the physiochemical properties. The powder was subjected to extraction with solvents such as ethanol, ethanol/water (1:1), hexane, and ethyl acetate. The extracts were then used for qualitative and quantitative (phenol, alkaloids, and flavonoids) phytochemical analysis. The molecular study was performed with the DNA barcoding technique. The DNA was extracted from the root of the plant, and its purity was examined by gel electrophoresis (1% w/v). The DNA was then amplified using an Applied Biosystems 2720 thermal cycler for the rbcL, matK, and ITS primers. The amplified primers were sequenced with a 3130 Genetic Analyzer, and the generated sequences were searched for similarity in the GenBank Database using the nucleotide BLAST analysis. The micro- and macroscopic studies revealed the morphological and organoleptic characters as well as the presence of medullary rays, fiber, cork, sclereids, parenchymal cells, and scalariform vessels. The physiochemical properties were found within the limit. The phytochemical analysis revealed the presence of terpenoids, flavonoids, saponins, and alkaloids. In addition, the alkaloidal content was high in the ethanol extract (63.04 ± 3.08 mg At E/g), while the phenol content was high in the hexane extract (10.26667 ± 1.77 mg At E/g), and the flavonoid content was high in the ethyl acetate extract (41.458 ± 1.33 mg At E/g). After the BLAST analysis from the GenBank database, the rbcL, ITS, and matK primers showed a similarity percentage of 99.83, 99.84, and 100. The phylogenetic tree for the species closest to each primer was generated using the MEGA 6 software. The matK loci had the highest percentage similar to the rbcL and ITS loci, indicating that the matK loci can be used to identify the root of E. tithymaloides as a standalone. The results from this study can be used to establish a quality standard for E. tithymaloides that will ensure its quality and purity.