Bioassay-guided isolation of antibacterial and anti-inflammatory components from Atractylodes lancea.

A bioassay-guided isolation from Atractylodes lancea (Thunb.) DC. obtained 22 compounds, including eight previously undescribed sesquiterpenoids and polyacetylenes (1, 3 and 12-17), as well as fourteen known analogues, and their structures were confirmed by extensive spectroscopic methods. This study evaluated their antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA) for the first time, as well as anti-inflammatory activity. Most of them, including new compounds, showed varying degrees of antibacterial activity against S. aureus and MRSA. Notably, compound 21 exhibited significant antibacterial activity against four different bacteria (MIC 6.25-20.00 µg/mL). This suggested that 21 may have the potential to be developed into a broad-spectrum antibacterial agent. Moreover, except for 9 and 11, most compounds exhibited great anti-inflammatory activity (IC50 1.92-37.91 µM), and iNOS might be a potential target of these compounds according to the molecular docking analysis.

Elucidation of the anti-colorectal cancer mechanism of Atractylodes lancea by network pharmacology and experimental verification.

Atractylodes lancea which was listed in "Shennong's Materia Medica" and could be used to treat gastrointestinal-associated diseases. However, its roles, core and active ingredients, and mechanisms in treatment of colorectal cancer (CRC) were still unknown. Therefore, network pharmacology and experimental validation were used to clarify the effects, core active ingredients and molecular mechanisms of Atractylodes lancea. We found that Atractylodes lancea has 28 effective active components and 213 potential targets. Seventy-three genes which demonstrate interaction between the Atractylodes lancea and CRC were confirmed. Enrichment analysis showed that 2033 GO biological process items and 144 KEGG pathways. Survival and molecular docking analysis revealed that luteolin as the core component interacted with these genes (Matrix metalloproteinase 3 (MMP3), Matrix metalloproteinase 9 (MMP9), Tissue inhibitor of metalloproteinases 1 (TIMP1), Vascular endothelial growth factor A (VEGFA)) with the lowest binding energy, and these genes were involved in building a prognostic model for CRC. Cellular phenotyping experiments showed that luteolin could inhibit the proliferation and migration of CRC cells and downregulate the expression of MMP3, MMP9, TIMP1, VEGFA probably by Phosphoinositide 3-kinase/ serine/threonine kinase Akt (PI3K/AKT) pathway. To conclude, Atractylodes lancea could inhibit proliferation and migration of CRC cells through its core active ingredient (luteolin) to suppress the expression of MMP3, MMP9, TIMP1, VEGFA probably by PI3K/AKT pathway.

[Research progress on biological characteristics and propagation technology of Atractylodes lancea].

Atractylodes lancea is a perennial herb of the Asteraceae family and is one of the well-known traditional Chinese medicine(TCM). Several studies have documented polyene alkyne and sesquiterpenoid compounds as the main bioactive compounds of A. lancea, especially atractylodin, atractylon, ß-eudesmol, and hinesol in its rhizomes, which possess anti-virus, anti-inflammation, hypoglycemic, anti-hypoxia, liver protection, and diuresis activities. In parallel with the recent advancements in biotechnology, important achievements have been made in the study of biological characteristics and propagation technology of A. lancea. This study reviews the research progress on morphological features, cytogenetics, ecological planting, effective ingredients, and tissue culture techniques of A. lancea from the biology perspective, so as to provide a theoretical basis for reasonable development of A. lancea resources.

Atractylodes lancea (Thunb.) DC. [Asteraceae] rhizome-derived exosome-like nanoparticles suppress lipopolysaccharide-induced inflammation in murine microglial cells.

Background: Exosome-like nanoparticles (ELNs) mediate interspecies intercellular communications and modulate gene expression. Hypothesis/Purpose: In this study, we isolated and purified ELNs from the dried rhizome of Atractylodes lancea (Thunb.) DC. [Asteraceae] (ALR-ELNs), a traditional natural medicine, and investigated their potential as neuroinflammatory therapeutic agents. Methods: ALR-ELN samples were isolated and purified using differential centrifugation, and their physical features and microRNA contents were analyzed through transmission electron microscopy and RNA sequencing, respectively. BV-2 microglial murine cells and primary mouse microglial cells were cultured in vitro, and their ability to uptake ALR-ELNs was explored using fluorescence microscopy. The capacity of ALR-ELNs to modulate the anti-inflammatory responses of these cells to lipopolysaccharide (LPS) exposure was assessed through mRNA and protein expression analyses. Results: Overall, BV-2 cells were found to internalize ALR-ELNs, which comprised three microRNAs (ath-miR166f, ath-miR162a-5p, and ath-miR162b-5p) that could have anti-inflammatory activity. Pretreatment of BV-2 cells with ALR-ELN prevented the pro-inflammatory effects of LPS stimulation by significantly reducing the levels of nitric oxide, interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Notably, the mRNA levels of Il1b, Il6, iNos, ccl2, and cxcl10 in BV-2 cells, which increased upon LPS exposure, were significantly reduced following ALR-ELN treatment. Moreover, the mRNA levels of heme oxygenase 1, Irf7, ccl12, and Irg1 also increased significantly following ALR-ELN treatment. In addition, pretreatment of primary mouse microglial cells with ALR-ELN prevented the pro-inflammatory effects of LPS stimulation by significantly reducing the levels of nitric oxide. Conclusion: Our findings indicate that ALR-ELNs exhibit anti-inflammatory effects on murine microglial cells. Further validation may prove ALR-ELNs as a promising neuroinflammatory therapeutic agent.

Allochthonous Trichoderma Isolates Boost Atractylodes lancea Herb Quality at the Cost of Rhizome Growth.

Atractylodes lancea is a perennial herb whose rhizome (AR) is a valuable traditional Chinese medicine with immense market demand. The cultivation of Atractylodes lancea faces outbreaks of root rot and deterioration in herb quality due to complex causes. Here, we investigated the effects of Trichoderma spp., well-known biocontrol agents and plant-growth-promoters, on ARs. We isolated Trichoderma strains from healthy ARs collected in different habitats and selected three T. harzianum strains (Th2, Th3 and Th4) with the strongest antagonizing effects on root rot pathogens (Fusarium spp.). We inoculated geo-authentic A. lancea plantlets with Th2, Th3 and Th4 and measured the biomass and quality of 70-day-old ARs. Th2 and Th3 promoted root rot resistance of A. lancea. Th2, Th3 and Th4 all boosted AR quality: the concentration of the four major medicinal compounds in ARs (atractylon, atractylodin, hinesol and ß-eudesmol) each increased 1.6- to 18.2-fold. Meanwhile, however, the yield of ARs decreased by 0.58- to 0.27-fold. Overall, Th3 dramatically increased the quality of ARs at a relatively low cost, namely lower yield, showing great potential for practical application. Our results showed selectivity between A. lancea and allochthonous Trichoderma isolates, indicating the importance of selecting specific microbial patches for herb cultivation.

First Detection of Atractylodes Mild Mottle Virus in Atractylodes lancea (Thunb.) DC. in Hubei Province of China.

The cultivated aromatic medicinal herb Atractylodes lancea (Thunb.) DC. is widely used in the pharmaceuticals, nutraceuticals, and cosmetics industries (Na-Bangchang et al. 2014; Zhan et al. 2023). Huanggang in Hubei Province is a major production area for A. lancea (Huang et al. 2022; Wang et al. 2023). In April 2023, more than two-thirds of the surveyed plant leaves in this region exhibited virus-like symptoms, such as curling and mosaic patterns. To identify the underlying cause, 80 symptomatic plant leaf samples were collected from four fields (20 leaves per field) in this region and pooled for virome analysis. Total RNA, including ribosomal RNA, was extracted from the pooled samples using the Plant RNA Extraction Mini Kit (Onrew Biotech, Guangdong, China), for sequencing library construction. The Illumina NovaSeq 6000 platform was used to sequence the library and generate 150 bp paired-end reads. After processing the raw data with Trimmomatic software, a total of 44,354,650 high-quality clean reads were obtained. The clean reads were aligned against ribosomal RNA using BWA software (v0.7.17) to avoid interference and eliminate corresponding sequences. After removing potential contamination, contig assembly of the clean reads was performed using Megahit software (v1.2.9). The resulting contigs were compared with the virus NT database using the BLASTn program. Sequence pairwise comparison revealed 8 contigs (574 nt to 2243 nt) with identities ranging from 81.88% to 90.77% with Atractylodes mild mottle virus (AMMV, NC_027924.1, Lim et al., 2015). Additionally, contigs mapped to Carlavirus, Pelarspovirus, and other plant viruses in our virome dataset had low coverage and pairwise identity (less than 70%), which need to be further investigated. The presence of AMMV was confirmed by aligning the clean reads to the reference sequence (NC_027924.1) using BWA and SAMtools software, resulting in a consensus sequence (8024 nt) with gaps. DNA extraction from the pooled samples was performed using the Rapid Universal Genomic DNA Extraction Kit (Simgen, Zhejiang, China). Two pairs of specific primers, 3399F (5'-AAAGAAGAACCTCCTGATACGG-3')/5924R (5'-TGAACCTGATTCTCTTGGC-3') and 1830F (5'- CTCAGGAAATCCCAATGC -3')/3640R(5'-TTTCCCAATGTTCTTCGGG-3'), were designed to amplify the complete gene sequences of polymerase and coat protein (CP), based on the consensus sequence. The PCR products with the lengths of 2521 bp and 1814 bp were cloned into the pMD18-T vector (Takara Biotech, Dalian, China) for sequencing. The BLASTn analysis showed that the polymerase and CP gene sequences shared an identity of 94.51% (1929/2041 nt) and 88.41% (1419/1605 nt) with the AMMV isolate (NC_027924.1), respectively. The sequences have been deposited in GenBank under the accession numbers OR544810 and OR544811. We collected leaves from 32 A. lancea plants (16 symptomatic and 16 asymptomatic) in the fields. RT-PCR was conducted using CPF (5'-CTGCGAATATGAAAGTGC-3') and CPR (5'-GGTGAGCTTGTCTGTTAGG-3') primers, which were designed targeting a 527bp fragment of the CP gene (OR544811). Amplicons of the expected size (527bp) were detected in 24 plants (11 symptomatic and 13 asymptomatic), three of which were sequenced by Sanger sequencing, showing a 100% match to OR544811. These findings indicate that AMMV is prevalent in the major production area of A. lancea. Further research is needed to better characterize the potential risks of AMMV to A. lancea cultivation in China as well as other countries.

The investigation of thermal stability and GC-MS analysis of Acorus tatarinowii and Atractylodes lancea volatile oils treated by ß cyclodextrin inclusion and Pickering emulsion technologies.

Objective: To investigate the stability of Acorus tatarinowii and Atractylodes lancea essential oils (ATaAL-EO) under a hot environment at 60 °C, and to analyze the differences in component, quantity, and quality changes, as well as variations in the main components, under different treatment methods of crude oil, ß-cyclodextrin inclusion of ATaAL-EO, and Pickering emulsion, to improve the stability and quality of ATaAL-EO. Methods: The stability of the ATaAL-EO group, the ß-cyclodextrin inclusion ATaAL-EO group, and the Pickering emulsion group were investigated under a 60 °C heat environment. Volatile oil retention rate and peroxide value were collected and measured. The volatile oil components of each group were determined by GC-MS, and t-tests were used to screen for differential components. PCA plots for each group were constructed using the OmicShare online platform. Line plots were generated using the Rmisc and reshape2 packages. Upset Venn diagrams under different hot environments were created using the OmicShare online platform to identify quantitative and qualitative changing components and heat map stack plots for newly generated compounds and connected line plots for disappearing compounds were produced for each group. Boxplots for the main component compounds under different hot environments were generated using the reshape2 and ggplot2 packages. Results: In a hot environment of 60 °C, the ß-cyclodextrin inclusion ATaAL-EO and Pickering emulsion group with 1, 3, and 8 h of placement showed higher retention and lower oxidation degree compared to the stability of the ATaAL-EO group. GC-MS analysis results showed that the stability of volatile components in the Pickering emulsion group and ß-cyclodextrin inclusion ATaAL-EO group was significantly improved compared to the crude oil group. Conclusion: ß-cyclodextrin inclusion complexes with ATaAL-EO, as well as Pickering emulsions, can significantly enhance the stability and quality of ATaAL-EO. Pickering emulsions have more advantages.

Structure of crude polysaccharides from Atractylodes lancea rhizome and treatment of diarrhea owing to spleen deficiency through intestinal flora.

To optimize the extraction process of crude polysaccharides from Atractylodes and elaborate the mechanism of Atractylodes polysaccharides in treating diarrhea owing to spleen deficiency, so as to lay a foundation for further development and utilization of Atractylodes lancea, we used an orthogonal test to optimize the extraction process and established a model of spleen deficiency. It was further combined with histopathology and intestinal flora to elaborate the mechanism of Atractylodes polysaccharides in the treatment of spleen-deficiency diarrhea. The optimized extraction conditions were as follows: the ratio of material to liquid was 1:25; the rotational speed was 150 rpm; the extraction temperature was 60°C; the extraction time was 2 h; and the extraction rate was about 23%. The therapeutic effect of Atractylodes polysaccharides on a spleen-deficiency diarrhea model in mice showed that the water content of stools and diarrhea grade in the treatment group were alleviated, and the levels of gastrin, motilin and d-xylose were improved. The analysis results based on gut microbiota showed that the model group had a higher diversity of gut microbiota than the normal group and treatment group, and the treatment group could correct the diversity of gut microbiota in model mice. Analysis based on the level of phylum and genus showed that the treatment group could inhibit the abundance of Helicobacter pylori genus and increase beneficial bacteria genera. The conclusion was that the optimized extraction process of Atractylodes polysaccharides was reasonable and feasible, and had a good therapeutic effect on spleen deficiency diarrhea.

Comparative analysis of the rhizosphere microbiome and medicinally active ingredients of Atractylodes lancea from different geographical origins.

This study aimed to explore the important role of the rhizosphere microbiome in the quality of Atractylodes lancea (Thunb.) DC. (A. lancea). The rhizosphere microbial community of A. lancea at two sampling sites was studied using metagenomic technology. The results of α-diversity analysis showed that the rhizosphere microbial richness and diversity were higher in the Maoshan area. The higher abundance of core microorganisms of the rhizosphere, especially Penicillium and Streptomyces, in the Maoshan area compared with those in the Yingshan area might be an important factor affecting the yield of A. lancea. Redundancy analysis illustrated that the available phosphorus had a significant effect on the rhizosphere microbial community structure of A. lancea. We also showed that the plant-microbe and microbe-microbe interactions were closer in the Maoshan area than in the Yingshan area, and Streptomyces were the main contributors to the potential functional difference between the two regions. A. lancea in the Maoshan area had a high content of atractylodin and atractylon, which might be related to the enhanced abundance of Streptomyces, Candidatus-Solibacter, and Frankia. Taken together, this study provided theoretical insights into the interaction between medicinal plants and the rhizosphere microbiome and provides a valuable reference for studying beneficial microbes of A. lancea.

The roles of CYP2C19 and CYP3A4 in the in vitro metabolism of ß-eudesmol in human liver: Reaction phenotyping and enzyme kinetics.

ß-eudesmol is a major bioactive component of Atractylodes lancea (AL). AL has been developed as the capsule formulation of standardized AL extract for treating cholangiocarcinoma (CCA). However, the complex constituents of herbal products increase the risk of adverse drug interactions. ß-eudesmol has demonstrated inhibitory effects on rCYP2C19 and rCYP3A4 in the previous research. This study aimed to identify the cytochrome P450 (CYP) isoforms responsible for the metabolism of ß-eudesmol and determine the enzyme kinetic parameters and the metabolic stability of ß-eudesmol metabolism in the microsomal system. Reaction phenotyping using human recombinant CYPs (rCYPs) and selective chemical inhibitors of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was performed, and enzyme kinetics and metabolic stability were investigated using human liver microsome (HLM). The results suggest that CYP2C19 and CYP3A4 play significant roles in ß-eudesmol metabolism. The disappearance half-life (t1/2 ) and intrinsic clearance (CLint ) of ß-eudesmol were 17.09 min and 0.20 mL/min·mg protein, respectively. Enzyme kinetic analysis revealed the Michaelis-Menten constant (Km ) and maximum velocity (Vmax ) of 16.76 µM and 3.35 nmol/min·mg protein, respectively. As a component of AL, ß-eudesmol, as a substrate and inhibitor of CYP2C19 and CYP3A4, has a high potential for drug-drug interactions when AL is co-administered with other herbs or conventional medicines.