Unmasking Nature's Double: a Morphological, Chromatographic and Spectroscopic Approach to the Validation of the Roots of Morinda lucida and Nauclea latifolia

Background and objectives: The substitution or misidentification of medicinal plant materials compromises herbal medicine quality. Morinda lucida and Nauclea latifolia, of the family Rubiaceae, possess yellow roots that become indistinguishable when processed, thereby fueling substitution practices or misidentification and compromising efficacy. This study aimed to develop methods to differentiate between the roots of Morinda lucida and Nauclea latifolia using multiple analytical approaches. Methods: Fresh roots of both species were subjected to standardized morphological (macro and micromorphological) examination, phytochemical screening, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), Fourier-transform infrared (FTIR) spectroscopy, and UV-visible spectrophotometry. Results: Macromorphological analysis revealed that Nauclea latifolia roots are fibrous and heavily fissured with coarse texture, while Morinda lucida roots are compact, dense, and cylindrical when chopped. Microscopic examination showed distinct differences in calcium oxalate crystal distribution, predominantly acicular in Morinda and prismatic in Nauclea. TLC analysis using chloroform extracts revealed distinctive spot patterns under UV light at 254 nm and 365 nm. HPLC fingerprinting at 235 nm and 275 nm showed significant differences in the elution of compounds, with Morinda lucida eluting more compounds than Nauclea latifolia. FTIR spectroscopy revealed similar functional groups but different transmittance patterns, while UV-visible analysis was able to discriminate between the two plants due to the pH sensitive spectra shift in various solvents. Conclusion: This research successfully established reliable, cost-effective authentication methods for distinguishing Morinda lucida and Nauclea latifolia roots. The developed protocols provide standardized quality control parameters that can be implemented in resource-limited settings by researchers, manufacturers and regulatory bodies.