The genus Bletilla is a small genus of only five species distributed across Asia, including B. chartacea, B. foliosa, B. formosana, B. ochracea and B. striata, which is of great medicinal importance. Furthermore, this genus is a member of the key tribe Arethuseae (Orchidaceae), harboring an extremely complicated taxonomic history. Recently, the monophyletic status of Bletilla has been challenged, and the phylogenetic relationships within this genus are still unclear. The plastome, which is rich in both sequence and structural variation, has emerged as a powerful tool for understanding plant evolution. Along with four new plastomes, this work is committed to exploring plastomic markers to elucidate the phylogeny of Bletilla. Our results reveal considerable plastomic differences between B. sinensis and the other three taxa in many aspects. Most importantly, the specific features of the IR junction patterns, novel pttRNA structures and codon aversion motifs can serve as useful molecular markers for Bletilla phylogeny. Moreover, based on maximum likelihood and Bayesian inference methods, our phylogenetic analyses based on two datasets of Arethuseae strongly imply that Bletilla is non-monophyletic. Accordingly, our findings from this study provide novel potential markers for species identification, and shed light on the evolution of Bletilla and Arethuseae.

The polyphenolic extract of Ilex latifolia (PEIL) exhibits a variety of biological activities. An evaluation of the parameters influencing the ultrasonic extraction process and the assessment of PEIL antioxidant activity are presented herein. Response surface methodology (RSM) was used to optimize the experimental conditions for the polyphenols ultrasonic-assisted extraction (UAE) from the leaves of Ilex latifolia. We identified the following optimal conditions of PEIL: ethanol concentration of 53%, extraction temperature of 60 °C, extraction time of 26 min and liquid–solid ratio of 60 mL/g. Using these parameters, the UAE had a yield of 35.77 ± 0.26 mg GAE/g, similar to the value we predicted using RSM (35.864 mg GAE/g). The antioxidant activity of PEIL was assessed in vitro, using various assays, as well as in vivo. We tested the effects of various doses of PEIL on D-galactose induced aging. Vitamin C (Vc) was used as positive control. After 21 days of administration, we measured superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, malondialdehyde (MDA) levels in mouse serum and liver tissue. The results demonstrated that the PEIL exhibits potent radical scavenging activity against 1,1-diphenyl-2-picrythydrazyl (DPPH∙), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS⁺), and hydroxyl (∙OH) radicals. The serum concentrations of SOD and GSH-Px were higher, and MDA levels were lower, in the medium- and high-dose PEIL-treated groups than those in the aging group (p < 0.01), and the activity of MDA was lower than those of the model group (p < 0.01). The liver concentrations of SOD and GSH-Px were higher (p < 0.05), and MDA levels were lower, in the medium- and high-dose PEIL-treated groups than those in the aging control group (p < 0.01). These results suggest that optimizing the conditions of UAE using RSM could significantly increase the yield of PEIL extraction. PEIL possesses strong antioxidant activity and use as a medicine or functional food could be further investigated.