Integrative Study of Dipsaci Radix and Phlomidis Radix: Nomenclature, Morphology, DNA-Based Authentication, and Comparative Effects on Osteoclastogenesis

Abstract

Background/Objectives: Dipsaci Radix (Dipsacus asper) and Phlomidis Radix (Phlomoides umbrosa) are both traditional medicines used in Korea and China for various bone-associated diseases. However, the two are misused due to similarities in name and appearance. Additionally, D. japonicus root frequently contaminates Dipsaci Radix in Korean herbal markets. Methods: We examined morphological plant traits and performed a DNA barcoding analysis using ITS2 and matK sequences to differentiate between these three species. The effects of root extracts on bone resorption and osteoclast differentiation, measured as tartrate-resistant acid phosphatase (TRAP)-positive cell formation, were evaluated using mouse (5 weeks male ICR mice) bone marrow-derived macrophages. Cytotoxicity assays were conducted to assess extract safety. Results: Phlomoides umbrosa is easily distinguished by its verticillaster inflorescences and 2-labiate corollas. Dipsacus asper and D. japonicus, which share globose inflorescences, are distinguishable by flower color and leaf lobation. The ITS2 and matK sequences clearly differentiated the three species, with haplotype analysis supporting their genetic distinctiveness, enabling robust species discrimination. All three extracts decreased osteoclastic bone resorption and inhibited TRAP-positive cell formations in a dose-dependent manner. Only the D. japonicus extract demonstrated toxicity. Conclusions: This integrative study provides the current scientific names of the original species and proposes their use in the Korean Herbal Pharmacopoeia. Moreover, a reasonable molecular method for authenticating medicinal materials is suggested. Dipsacus japonicus shows promise as an additional origin species in the Korean Pharmacopoeia. However, processing methods that reduce toxicity must be discovered.

Keywords: DNA barcoding; Dipsaci Radix; Dipsacus japonicus; Phlomidis Radix; bone resorption; nomenclature; osteoblasts.

Figure 1

Inflorescence (A–C) and leaf (D–F) morphologies of Dipsacus asper (A,D), D. japonicus (B,E), and Phlomoides umbrosa (C,F).

Figure 2

Median-Joining haplotype networks of D. asper (H1), D. japonicus (H2), and P. umbrosa (H3). The networks are based on the (A) ITS2 and (B) matK DNA sequences. Black hatch marks indicate mutations, and black dots represent inferred haplotypes.

Figure 3

Effects of D. asper, D. japonicus, and P. umbrosa extracts on bone resorption by mature osteoclasts. (A) Hydroxypatite-adherent cells on hydroxyapatite-coated plates were imaged under a light microscope after exposure to cultures containing mature osteoclasts (control) or mature osteoclasts and one of the three extracts, all scale bars = 200 μm and (B) The proportion of resorbed area was quantified. Data are presented as the mean ± SD. Statistical significance levels for comparisons between the treatments and the control are represented by asterisks: *** p < 0.001 vs. control (DMSO).

Figure 4

Effects on osteoclast differentiation of the ethanol extracts of D. asper, D. japonicus, and P. umbrosa at concentrations of 50, 100, and 200 µg/mL. (A) Tartrate-resistant acid phosphatase (TRAP)-positive cells photographed at 100× magnification after bone marrow macrophages were cultured with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand alone (control [dimethyl sulfoxide]) or in the presence of one of the three extracts. All scale bars = 200 μm. (B) TRAP-positive cells were counted as osteoclasts. (C) Cell viability was affected by the D. asper, D. japonicus, and P. umbrosa extracts. Statistical significance levels for comparisons between the treatments and the control are represented by asterisks: **, p < 0.01, and ***, p < 0.001 vs. control (DMSO).