An integrated study of Violae Herba (Viola philippica) and five adulterants by morphology, chemical compositions and chloroplast genomes: insights into its certified plant origin

Abstract

Background Viola philippica Cav. is the only original plant for Violae Herba, as described in the Chinese Pharmacopoeia. The quality of this crude drug is affected by several adulterants from congeneric Viola species, and the authentic plant origin of Violae Herba is still controversial. Genome-based identification offers abundant genetic information and potential molecular markers that can be used for the authentication of closely related species. This study aims to investigate the certified origin of Violae Herba and to develop more effective markers for these easily confused species at the genetic level. Methods We compared the morphology and chemical composition of 18 batches of commercial samples and six widespread medicinal Viola plants used as Violae Herba or its substitutes by TLC and HPLC-Triple-TOF–MS/MS analyses. The complete chloroplast genomes of these species were sequenced and analyzed, including the general features, repeat sequences, mutational hotspots and phylogeny. The complete chloroplast genomes used as superbarcodes and some specific barcodes screened from mutational hotspots were tested for their ability to distinguish Viola species. Results A comparative study showed that Violae Herba is a multi-origin traditional Chinese medicine. Commercial decoction pieces and the standard reference drug were mainly derived from V. prionantha, clashing with the record in the Chinese Pharmacopoeia. Chloroplast genome analyses of V. philippica and five adulterants indicated that sequence divergence was relatively low within Viola species. By tree-based approaches, the complete chloroplast genomes showed a better discrimination ability and phylogenetic resolution for each Viola species. These results indicate that the whole chloroplast genomes can be used as superbarcodes to differentiate Viola medicinal plants. More specific DNA barcodes could be further developed from the Viola chloroplast genomes for more efficient and rapid identification of commercial Violae Herba and its adulterants. Conclusions This study has implications for chloroplast genome-based phylogenetic analysis and the authentication of multiple Viola species used as Violae Herba. The legal origin recorded in the Chinese Pharmacopoeia should be further revised to V. prionantha, in line with the commercial Violae Herba in the TCM markets.

Complete chloroplast genomes of the hemiparasitic genus Cymbaria: Insights into comparative analysis, development of molecular markers, and phylogenetic relationships

“…Distinguishing between these two Cymbaria species is exceedingly difficult because they only differ in anther morphology (Zhang et al., 2013 ). Some previous studies have suggested that specific barcodes are superior in performance to universal barcodes for distinguishing between morphologically similar species (Fang et al., 2023 ; Lu et al., 2022 ). Therefore, several divergence hotspot regions were first identified through sequence divergence and nucleotide variability, Then, we developed and validated specific DNA barcodes to distinguish between these two species.…”

Complete chloroplast genomes of 11 Sabia samples: Genomic features, comparative analysis, and phylogenetic relationship

“…across regions and trans-splicing of rps12, were ubiquitous in most plants (Wang et al, 2018;Thode and Lohmann, 2019;Lu et al, 2022), and no specific structural variation has been found in this genus.…”

Systematic evaluation of Rutongshu oral liquid based on chromatographic fingerprinting and nontargeted metabolomic analysis

“…In recent years, the quality of herbal medicine as well as their related products have been evaluated adopting chromatographic fingerprinting which emphasizes the characteristics of overall sample composition [8][9][10]. Among the many techniques used to characterize chemical profiles including Fourier-transform infrared (FT-IR) [11][12][13], mass spectrometry (MS) [14][15][16], nuclear magnetic resonance (NMR) [17][18][19], gas chromatography (GC) [20][21][22], high performance thin layer chromatography (HPTLC) [23][24][25] and thin-layer chromatography (TLC) [26][27][28], ultra-high performance liquid chromatography (UHPLC) [29][30][31] and high-performance liquid chromatography (HPLC) [32][33][34] are the most popularly used approaches used for fingerprinting due to its high sensitivity, high selectivity and low cost [35,36].…”