Unveiling Adulteration in Herbal Markets: MassARRAY iPLEX Assay for Accurate Identification of Plumbago indica L

Abstract

The root of Plumbago indica L. is commercially available in herbal markets in both crude and powdered forms. P. indica root is a key ingredient in numerous polyherbal formulations. However, P. indica has two closely related species, P. zeylanica L. and P. auriculata Lam. Since only P. indica is traditionally used in Thai polyherbal products, adulteration with other species could potentially compromise the therapeutic efficacy and overall effectiveness of these formulations. To address this issue, a MassARRAY iPLEX assay was developed to accurately identify and differentiate P. indica from its closely related species. Five single nucleotide polymorphism (SNP) sites-positions 18, 112, 577, 623, and 652-within the internal transcribed spacer (ITS) region were selected as genetic markers for species identification. The assay demonstrated high accuracy in identifying P. indica and was capable of detecting the species at DNA concentrations as low as 0.01 ng/µL. Additionally, the assay successfully identified P. zeylanica in commercial crude drug samples, highlighting potential instances of adulteration. Furthermore, it was able to distinguish P. indica in mixed samples containing P. indica, along with either P. zeylanica or P. auriculata. The developed MassARRAY iPLEX assay proves to be a reliable and effective molecular tool for authenticating P. indica raw materials. Its application holds significant potential for ensuring the integrity of herbal products by preventing misidentification and adulteration.

Keywords: DNA barcode; MassARRAY; Plumbago; identification; leadwort; raw material; single nucleotide polymorphism (SNP).

Figure 1

Morphological Characteristics and Utilization of Plumbago indica L.: (A). habit, (B). flowers, (C). roots, (D). Jettamoon Pleung Daeng crude drugs derived from dried P. indica roots, (E). Ya Benchakun formulation.

Figure 2

Mass spectra of authentic Plumbago species. The colors represent the mass profiles of the unextended primers (UEPs) and their corresponding base extension products: yellow for P1#1, green for P1#2, blue for P2#1, purple for P2#2, and red for P2#3.

Figure 3

MALDI-TOF mass spectral fingerprint of Plumbago-mixed samples containing P. indica (PI), P. zeylanica (PZ), and P. auriculata (PA): (A) P. indica and P. auriculata, (B) P. auriculata and P. zeylanica, (C) P. indica and P. zeylanica, (D) P. indica, P. auriculata, and P. zeylanica. The colors represent the mass profiles of the unextended primers (UEPs) and their corresponding base extension products: yellow for P1#1, green for P1#2, blue for P2#1, purple for P2#2, and red for P2#3.

Figure 4

Molecular Principles and Workflow of the MassARRAY Method and Primer Design Concept for Differentiating Three Plumbago Species. The colors represent the arrows and mass profiles of the unextended primers (UEPs) and their corresponding base extension products: green for P1#1, red for P1#2, purple for P2#1, yellow for P2#2, and blue for P2#3. Created using BioRender (Intharuksa, A., 2025). Available online: https://BioRender.com/nu2pugl (accessed on 5 June 2025).