Comparative Analysis of Morphological, Molecular, and Physicochemical Markers to Evaluate Trollius ledebouri Rchb. as a Potential Alternative Source to Trollius chinensis Bunge for High-Quality Flos Trollii Supplements

<i>Trollius chinensis Bunge</i> (TCB), a perennial Ranunculaceae herb, produces Flos Trollii-dried flowers with medicinal properties including heat clearing, detoxification, and relieving oral/throat discomfort, eye pain, and cold-induced fever. TCB is mainly cultivated in northern China, while <i>Trollius ledebouri</i> Rchb. (TLR), distributed in Heilongjiang's Great Xing'an Mountains, is morphologically similar to TCB. However, their regulatory statuses are inconsistent, and comprehensive comparative studies are lacking. This study adopted morphological assessment, microscopy, DNA barcoding, and physicochemical analysis to explore whether TLR could be a potential alternative source of Flos Trollii. Key differences were identified: TLR's sepals are shorter than petals, whereas TCB's sepals and petals are nearly equal in length; TLR has brown secretory structures absent in TCB. Genetic distance analysis showed high conservation in <i>ITS</i>2 and <i>trnL-trnF</i> sequences between the two species, but <i>psbA-trnH</i> sequence divergence exceeded the 0.05 threshold. HPLC quantification revealed that TLR contained slightly higher levels of orientin and vitexin than TCB. HPLC quantification revealed that TLR contained slightly higher levels of orientin (5.370-5.377 mg/g) and vitexin (1.954-2.053 mg/g) compared to TCB (orientin: 4.493-4.620 mg/g; vitexin: 1.361-1.451 mg/g). Collectively, TLR exhibits comparable flavonoid content and holds potential as an alternative Flos Trollii source. Given the limited bioactive compounds analyzed, future research should conduct comprehensive metabolomic profiling to fully evaluate its phytochemical composition and medicinal value. These data establish chemotaxonomic markers for Trollius authentication in herbal medicine.