Integrated Analysis of Basic Helix Loop Helix Transcription Factor Family and Targeted Terpenoids Reveals Candidate AarbHLH Genes Involved in Terpenoid Biosynthesis in Artemisia argyi

Artemisia argyi is a valuable traditional medicinal plant in Asia. The essential oil from its leaves is rich in terpenoids and has been used to enhance health and well-being. In China, the market scale of industries related to A. argyi has attained tens of billions of Chinese Yuan. The basic helix-loop-helix (bHLH) family is one of the largest transcription factors families in plants that plays crucial roles in diverse biological processes and is an essential regulatory component of terpenoid biosynthesis. However, the bHLH TFs and their regulatory roles in A. argyi remain unknown. Here, 53 AarbHLH genes were identified from the transcriptome of A. argyi and were classified into 15 subfamilies based on the classification of bHLH proteins in Arabidopsis thaliana. The MEME analysis showed that the conserved motif 1 and motif 2 constituted the most conserved bHLH domain and distributed in most AarbHLH proteins. Additionally, integrated analysis of the expression profiles of AarbHLH genes and the contents of targeted terpenoids in different tissues group and JA-treated group were performed. Eleven up-regulated AarbHLHs and one down-regulated AarbHLH were screened as candidate genes that may participate in the regulation of terpenoid biosynthesis (TPS-AarbHLHs). Correlation analysis between gene expression and terpenoid contents indicated that the gene expression of these 12 TPS-AarbHLHs was significantly correlated with the content changes of 1,8-cineole or β-caryophyllene. Protein–protein interaction networks further illustrated that these TPS-AarbHLHs might be involved in terpenoid biosynthesis in A. argyi. This finding provides a basis to further investigate the regulation mechanism of AarbHLH genes in terpenoid biosynthesis, and will be helpful to improve the quality of A. argyi.

Caffeic Acid, an Allelochemical in Artemisia argyi, Inhibits Weed Growth via Suppression of Mitogen-Activated Protein Kinase Signaling Pathway and the Biosynthesis of Gibberellin and Phytoalexin

Artemisia argyi is widely distributed in Asia, and it often becomes the dominant population in the field because of its strong ecological niche competitiveness. Allelochemicals secreted by plants are generally considered an important reason for their dominance in ecological competition. In this study, the allelochemicals in A. argyi were screened by a series of experiments and their mechanisms were explored via transcriptomics. First, the inhibitory effects of A. argyi on Echinochloa crusgalli, Setaria viridis, Portulaca oleracea and Amaranthus retroflexus were evaluated. Then, we carried out a qualitative and quantitative analysis of the chemical composition of the aqueous extract of A. argyi to screen for potential allelochemicals that can inhibit weed growth. Four potential allelochemicals were quantified: neochlorogenic acid (5-CQA), chlorogenic acid (3-CQA), cryptochlorogenic acid (4-CQA), and caffeic acid (CA). Coincidentally, their allelopathic effects on weeds seemed to be identical to their content, in the order CA>4−CQA>5−CQA>3-CQA. These findings suggested that CA might be the main allelopathic compound in the aqueous extract of A. argyi. Subsequently, the allelopathic effect and molecular mechanism of CA on S. viridis leaves were investigated. The physiological results showed that CA significantly induced reactive oxygen species (ROS) production, led to malondialdehyde (MDA) accumulation, and disrupted enzyme activities (POD, SOD, CAT) in S. viridis leaves. Moreover, transcriptome results revealed that CA inhibited S. viridis growth by downregulating multiple genes involved in gibberellin (GA) and phytoalexin biosynthesis and Mitogen-activated protein kinase (MAPK) signaling pathways. In addition, differentially expressed genes (DEGs) related to the biosynthesis and signaling pathways of phytohormones were verified by Quantitative Real-Time PCR (RT-qPCR). Taken together, this study may be the first to identify allelochemicals and explore their molecular mechanism about A. argyi. Importantly, the ecological advantages of A. argyi could be applied to ecological regulation and the development of botanical herbicides.

Acid-Catalyzed Water Extraction of Two Polysaccharides from Artemisia argyi and Their Physicochemical Properties and Antioxidant Activities

In this study, two purified polysaccharide fractions, Artp1 and Artp2, were obtained using acid-catalyzed water extraction, and then purified by DEAE-52 cellulose and Sephadex G-200 column chromatography from the crude polysaccharides of Artemisia argyi. Their physicochemical properties were investigated by gel permeation chromatography (GPC), high-performance anion exchange chromatography (HPAEC), Fourier transform infrared (FT-IR), scanning electron microscope (SEM), thermal analysis, and methylation analysis. The average molecular weight (Mw) of Artp1 and Artp2 were estimated to be 42.17 kDa and 175.22 kDa, respectively. Monosaccharide composition analysis revealed that the Rha, Gal, and GalA occupied main proportion in Artp1 with the molar ratio of 25.1:24.7:40.4, while the Rha, Gal, Xly, and GalA occupied the main proportion in Artp2 with the molar ratio of 16.7:13.5:12.8:38.7. Due to the high yield and the relatively high carbohydrate content, the Artp1 was determined by the methylation analysis and NMR. The results of Artp1 indicated that 1,4-GalpA and 1,2,4-Rhap formed the backbone with some 1,2-Rhap, 1,3-Galp, and 1,6-Galp in the backbone or the side chains. Artp1 and Artp2 exhibited effective antioxidant activities by DPPH radical scavenging assay and hydroxyl radical scavenging assay in a dose-dependent manner. These investigations of the polysaccharides from A. argyi. provide a scientific basis for the uses of Artp1 and Artp2 as ingredients in functional foods and medicines.

Preparation and Antibacterial Activity of Thermo-Responsive Nanohydrogels from Qiai Essential Oil and Pluronic F108

Essential oils (EOs) have been used in cosmetics and food due to their antimicrobial and antiviral effects. However, the applications of EOs are compromised because of their poor aqueous solubility and high volatility. Qiai (Artemisia argyi Levl. et Van. var. argyi cv. Qiai) is a traditional Chinese herb and possesses strong antibacterial activity. Herein, we report an innovative formulation of EO as nanohydrogels, which were prepared through co-assembly of Qiai EO (QEO) and Pluronic F108 (PEG-b-PPG-b-PEG, or PF108) in aqueous solution. QEO was efficiently loaded in the PF108 micelles and formed nanohydrogels by heating the QEO/PF108 mixture solution to 37 °C, by the innate thermo-responsive property of PF108. The encapsulation efficiency and loading capacity of QEO reached 80.2% and 6.8%, respectively. QEO nanohydrogels were more stable than the free QEO with respect to volatilization. Sustained QEO release was achieved at body temperature using the QEO nanohydrogels, with the cumulative release rate reaching 95% in 35 h. In vitro antibacterial test indicated that the QEO nanohydrogels showed stronger antimicrobial activity against S. aureus and E. coli than the free QEO due to the enhanced stability and sustained-release characteristics. It has been attested that thermo-responsive QEO nanohydrogels have good potential as antibacterial cosmetics.

The Essential Oil of Artemisia argyi H.Lév. and Vaniot Attenuates NLRP3 Inflammasome Activation in THP-1 Cells

Artemisia argyi H. Lév. and Vaniot is a traditional medical herb that has been used for a long time in China and other Asian counties. Essential oil is the main active fraction of Artemisia argyi H. Lév. and Vaniot, and its anti-inflammatory potential has been observed in vitro and in vivo. Here, we found that the essential oil of Artemisia argyi H. Lév. and Vaniot (EOAA) inhibited monosodium urate (MSU)- and nigericin-induced NLRP3 inflammasome activation. EOAA suppressed caspase-1 and IL-1β processing and pyroptosis. NF-κB p65 phosphorylation and translocation were also inhibited. In addition, EOAA suppressed nigericin-induced NLRP3 inflammasome activation without blocking ASC oligomerization, suggesting that it may inhibit NLRP3 inflammasome activation by preventing caspase-1 processing. Our study thus indicates that EOAA inhibits NLRP3 inflammasome activation and has therapeutic potential against NLRP3-driven diseases.

Artemisia argyi Essential Oil Inhibits Hepatocellular Carcinoma Metastasis via Suppression of DEPDC1 Dependent Wnt/β-Catenin Signaling Pathway

The tumor metastasis is the major hurdle for the treatment of advanced hepatocellular carcinoma (HCC), due in part to the lack of effective systemic treatments. DEPDC1, a novel oncoantigen upregulated in HCC, is thought to be a molecular-target for novel therapeutic drugs. Artemisia argyi is a traditional Chinese medicine with anti-inflammatory and anti-tumor activities. This study investigated the potential therapeutic benefits of Artemisia argyi essential oil (AAEO) in suppressing metastasis of HCC by targeting DEPDC1. Assessment of AAEO cytotoxicity was performed by MTT assay. Anti-metastatic effects of AAEO were investigated in vitro using wound healing and transwell assays. The HepG2 cells were transduced with lentiviral vector containing luciferase (Luc). A metastasis model of nude mice was established by tail vein injection of HepG2-Luc cells. The nude mice were treated with AAEO (57.5, 115, and 230 mg/kg) or sorafenib (40 mg/kg). Metastasis of HCC cells was monitored via in vivo bioluminescence imaging. After treatment for 21 days, tissues were collected for histological examination and immunohistochemistry analysis. Gene and protein levels were determined by real-time quantitative PCR and western blotting. The results revealed that AAEO significantly inhibits the migration and invasion in vitro in a concentration-dependent manner. In vivo assays further confirmed that AAEO markedly inhibits HCC metastasis into lung, brain, and femur tissues and exhibits low toxicity. Our results suggested that AAEO significantly downregulates the mRNA and protein expression of DEPDC1. Also, AAEO attenuated Wnt/β-catenin signaling through reduction of Wnt1 and β-catenin production. Moreover, AAEO prevented epithelial-mesenchymal transition (EMT) by downregulation of vimentin and upregulation of E-cadherin. Furthermore, we found that DEPDC1 promoted HCC migration and invasion via Wnt/β-catenin signaling pathway and EMT. These results demonstrate that AAEO effectively inhibits HCC metastasis via attenuating Wnt/β-catenin signaling and inhibiting EMT by suppressing DEPDC1 expression. Thus, AAEO likely acts as a novel inhibitor of the DEPDC1 dependent Wnt/β-catenin signaling pathway.