Limited research has been conducted on the metabolites present in the essential oils (Es) of Phlomoides rotata (Benth. ex Hook. f.) Mathiesen (PR, syn. Lamiophlomis rotata (Benth.) Kudô), and their potential pharmacological activities, such as antioxidant and anticancer properties, are often overlooked. We conducted a metabolomics-based screening to identify the characteristic metabolites (CMs) present in the Es extracted from PR. Furthermore, we evaluated their in vitro antioxidant activities (AOAs). The Es obtained through hydro-distillation are characterized by a light yellow color and a fresh, elegant fragrance. Cryoprecipitation was used to separate the crystals (Cs) from the Es, resulting in the extraction of crystal-free essential oils (CFs). A total of 107 components belonging to 8 categories were identified and quantified in the Es, Cs, and CFs using GC-MS and GC-FID techniques. Among these components, 76 were reported for the first time in the Es of PR. The major compounds identified include long-chain fatty acids (LCFAs) and their esters, such as palmitic acid (PA), myristic acid (MA), linoleic acid (LA), oleic acid (OA), and methyl palmitate. Additionally, seven C13-norisoprenoids, with trans-β-damascenone being particularly notable for its flavor, were also identified. A sum of nine CMs were identified in the Es, including PA, MA, LA, OA, methyl palmitate, hexahydrofarnesyl acetone, phytol, trans-β-damascenone, and hexanal. The main metabolic pathway in the Es is the biosynthesis of FAs and terpenoids revealed by MetaboAnalyst 6.0 and KEGG analyses. Furthermore, the in vitro AOAs of the Es, Cs, CFs, and four selected CMs (PA, MA, LA, and OA) were evaluated. Generally, PA and MA exhibited pro-oxidant activities (POAs) or weak AOAs. LA and OA demonstrated POAs or weak AOAs at lower concentrations, but at higher concentrations, they displayed medium to strong AOAs. Importantly, the CFs exhibited stronger AOAs compared to the corresponding Es, which may be attributed to their different PA content. Overall, this study provides valuable insights into the potential utilization of Es from PR.

Due to the low content, few studies are focused on the volatile compounds of Phlomoides rotata (Benth. ex Hook. f.) Mathiesen (PR, syn. Lamiophlomis rotata (Benth.) Kudô). However, such volatile compounds may have important pharmacological activities such as antioxidant, anticancer. In order to identify the chemical markers (CMs) in the essential oils (Es) and explore the potential bioactivities, we first conduct a thoroughly investigation on the volatile compounds and evaluate their anti-oxidant activities (AOAs), to the best of our knowledge. Light yellow Es with a fresh and elegant smell are obtained by hydro-distillation with an average yield of 0.11% (mL/g). After separation through cryoprecipitation, the crystals (Cs) and EOs removed crystals (RCs) were gotten, respectively. A total of 81 components are qualified and quantified in the Es, Cs and RCs, in which 44 ones are first reported from PR. As for content, the main compounds are long-chain fatty acids (LCFAs) and their esters. Among them, palmitic acid (PA), myristic acid (MA), linoleic acid (LA), oleic acid (OA) and methyl palmitate are highlighted, Besides, hexahydrofarnesyl acetone and phytol are also prominent Four kinds of C13-norisoprenoids are first reported from PR, in which, trans-β-damascenone is potent in flavour. In sum, these eight representative compounds can be chosen as the CMs. The AOAs of Es, Cs, RCs and four CMs such as PA, MA, LA and OA are evaluated by in vitro assays. In general, PA and MA present pro-oxidant activities (POAs) or weak AOAs. LA and OA show POAs or weak AOAs in lower concentration group, whereas demonstrate medium or strong AOAs in higher concentration group. Usually, the RCs demonstrate stronger AOAs and the Cs present weaker AOAs compared with those of the corresponding Es, which should be related to their different content of PA. This study can give some hints on the utilization of such Es which are abundant in LCFAs.

Ginkgolic acids (GA) have been reported to exhibit anticancer properties, however, the mechanisms remain unclear. This study aims to investigate the mechanisms of GA C13:0 that was isolated from Ginkgo biloba exocarp (GBE) for anti-proliferation, pro-apoptosis and anti-migration effects in human MCF-7 and mouse 4T-1 breast cancer cells. The cytotoxic effect, apoptosis induction and migration inhibition were measured using MTT, TUNEL and Wound healing assays. The expression of mRNA and protein were determined using qPCR and Western blot. Our results showed that no cytotoxicity was found at concentrations of C13:0 below 100µM. The effects of GA C13:0 was further demonstrated by up-regulation of the Bax/Bcl-2 apoptosis pathway and the expression of Apaf-1 protein in the mitochondria. In addition, GA C13:0 also suppressed cell migration and epithelial to mesenchymal transition (EMT) with the increase of E-cadherin expression accompanied by the decrease of Snail, MMP-2, MMP-9 and Vimentin expression. Moreover, GA C13:0 induced cytochrome P450 (CYP) 1B1 expression in aryl hydrocarbon receptor (AhR) pathway. Notably, the up-regulation of CYP1B1 also might play a pivotal regulatory role in mitochondrial and EMT pathways in MCF-7 and 4T-1 cells. Our results may have implications for the development of anticancer agents containing GA as functional additives.