Background: Tanshinone IIA (Tan IIA) is a lipophilic active constituent derived from the rhizomes and roots of Salvia miltiorrhiza Bunge (Danshen), a common Chinese medicinal herb. However, clinical applications of Tan IIA are limited due to its poor solubility in water. Methods: To overcome this limitation, we developed a calcium alginate hydrogel (CA) as a hydrophilic carrier for Tan IIA, which significantly improved its solubility. We also prepared nanoparticles with pH-responsive properties to explore their potential for controlled drug delivery. The physicochemical properties of Tan IIA/CA nanoparticles were evaluated, including their size, stability, and release profile. We also utilized RNA sequencing to further investigate the underlying anticancer mechanisms of Tan IIA/CA nanoparticles. Results: The Tan IIA/CA nanoparticles demonstrated enhanced solubility and exhibited potent anticancer activity in vitro. Additionally, the nanoparticles showed promising pH-responsive behavior, which is beneficial for controlled release applications. Further investigation into the molecular mechanisms revealed that the anticancer effects of Tan IIA/CA were mediated through apoptosis, ferroptosis, and autophagy pathways. Conclusions: This study confirms the anticancer potential and mechanisms of Tan IIA, while also presenting an innovative approach to enhance the solubility of this poorly soluble compound. The use of CA-based nanoparticles could be a valuable strategy for improving the therapeutic efficacy of Tan IIA in cancer treatment.

Nicosulfuron, an acetolactate synthase (ALS) inhibitor herbicide, is a broad-spectrum and highly effective post-emergence herbicide. Glycosyltransferases (GTs) are widely found in organisms and transfer sugar molecules from donors to acceptors to form glycosides or sugar esters, thereby altering the physicochemical properties of the acceptor molecule, such as participating in detoxification. In this study, nine glycosyltransferases in group D of the apple glycosyltransferase family I were predicted to possibly be involved in the detoxification metabolism of ALS-inhibiting herbicides based on gene chip data published online. In order to confirm this, we analysed whether the expression of the nine glycosyltransferase genes in group D was induced by the previously reported ALS-inhibiting herbicides by real-time PCR (polymerase chain reaction). It was found that the ALS-inhibiting herbicide nicosulfuron significantly increased the expression of the MdUGT73CG22 gene in group D. Further investigation of the mechanism of action revealed that the apple glycosyltransferase MdUGT73CG22 glycosylated and modified nicosulfuron both in vivo and ex vivo to form nicosulfuron glycosides, which were involved in detoxification metabolism. In conclusion, a new glycosyltransferase, MdUGT73CG22, was identified for the first time in this study, which can glycosylate modifications of the ALS-inhibiting herbicide nicosulfuron and may be involved in the detoxification process in plants, which can help to further improve the knowledge of the non-targeted mechanism of herbicides.

Sulcotrione is a member of triketone herbicides, a class of HPPD (4-hydroxyphenylpyruvate dioxygenase) inhibitors with broad-spectrum herbicidal activity. Modifications of glycosylation mediated by glycosyltransferases (GT) are involved in plant detoxification. In this study, we analyzed chip data published online and found that eight glycosyltransferases from group A of the apple glycosyltransferase family 1 may be involved in the metabolic mechanism of detoxification of triketone herbicides. To verify this prediction, we induced apple seedlings with six types of triketone herbicides, and then detected the expression levels of eight glycosyltransferase genes through real-time PCR. We found that triketone herbicides induced up-regulation of eight glycosyltransferase genes to varying degrees, with MdUGT91AJ2 being the most significantly up-regulated by sulcotrione-induced glycosyltransferase gene expression. Then, through in vitro enzymatic reactions and HPLC identification of glycoside substrates, it was found that the glycosyltransferase MdUGT91AJ2 had the highest specific enzyme activity against the triketone herbicide sulcotrione. Furthermore, the in vivo mechanism of the glycosyltransferase MdUGT91AJ2 in the detoxification metabolism of sulcotrione was further validated by overexpressing the strain in the plant. HPLC analysis showed that the content of sulcotrione glycosides in the overexpressing strain of MdUGT91AJ2 was significantly higher than that in the wild type. This result indicated that the apple glycosyltransferase MdUGT91AJ2 can still glycosylate and modify sulfotrione in plants, and participate in its detoxification metabolism. In summary, this study identified for the first time a novel apple glycosyltransferase MdUGT91AJ2 and elucidated its mechanism of action in the detoxification and metabolism of the triketone herbicide sulfotriene.

Rutaceae encompasses numerous medicinal plants with unclear genetic relationships and mitochondrial genome (mitogenome) characteristics, which hinders understanding of their evolutionary adaptation and medicinal trait development. Here, we aimed to investigate the conserved and divergent features of mitogenomes among closely related medicinal genera of Rutaceae, and to explore how mitogenomic variations correlate with their phylogenetic positions. Citrus aurantium, Toddalia asiatica, and Zanthoxylum nitidum (Roxb.) DC are medicinally valuable but understudied Rutaceae species belonging to different genera. We sequenced, assembled, and annotated their complete mitogenomes: C. aurantium (504,387 bp, 45.17% GC content) contained 62 genes (36 protein-coding genes (PCGs), 3 rRNAs, and 23 tRNAs), T. asiatica (566,784 bp, 45.37% GC content) harbored 58 genes (35 PCGs, 3 rRNAs, and 20 tRNAs), and Z. nitidum (539,013 bp, 45.48% GC content) possessed 61 genes (34 PCGs, 3 rRNAs, and 24 tRNAs). Comparative analyses with 12 published Rutaceae mitogenomes showed that the three species exhibited the highest number of RNA editing sites in PCGs among the examined species. Evaluation of selective pressure and gene clusters revealed fewer differences between C. aurantium and C. maxima cultivar Hirado Buntan than with the other two species, while C. sinensis displayed greater dissimilarities with other Citrus species (notably, C. maxima and C. maxima cultivar Hirado Buntan were conspecific and should have the least divergence). Phylogenetic trees constructed using 24 conserved mitochondrial PCGs clarified evolutionary relationships: C. maxima was closest to C. sinensis, followed by C. maxima cultivar Hirado Buntan and C. aurantium formed an unexpected separate clade, highlighting the need for more Rutaceae species' mitogenome in future phylogenetic analyses. This study expands the resource pool in Rutaceae mitogenomic resources, provides insights into features that may be associated with ecological adaptation, and offers valuable genomic data for future phylogenetic and comparative studies of Rutaceae medicinal plants.