Unusual cadinane-involved sesquiterpenoid dimers from Artemisia annua and their antihepatoma effect.

Artemisia annua L. ("Qinghao" in Chinese) is a famous traditional Chinese medicinal herb and has been used to treat malaria and various tumors. Our preliminary screening indicated that the EtOAc extract of A. annua manifested activity against HepG2, Huh7, and SK-Hep-1 cell lines with inhibitory ratios of 53.2%, 52.1%, and 59.6% at 200 µg/mL, respectively. Bioassay-guided isolation of A. annua afforded 14 unusual cadinane-involved sesquiterpenoid dimers, artemannuins A‒N (1-14), of which the structures were elucidated by extensive spectral analyses, ECD calculations, and single-crystal X-ray diffraction. Structurally, these compounds were classified into five different types based on the coupled modes of two monomeric sesquiterpenoids. Among them, compounds 1-9 represented the first examples of sesquiterpenoid dimers formed via the C-3‒C-3' single bond of two 5(4 â†’ 3)-abeo-cadinane sesquiterpenoid monomers, while compounds 13 and 14 were dimers fused by cadinane and humulane sesquiterpenoids via an ester bond. Methylated derivatives of 1, 4, 6, and 8 showed antihepatoma activity against HepG2, Huh7, and SK-Hep-1 cell lines with IC50 values ranging from 30.5 to 57.2 µM.

The AaBBX21-AaHY5 module mediates light-regulated artemisinin biosynthesis in Artemisia annua L.

The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood (Artemisia annua L.). Light was previously shown to promote artemisinin production, but the underlying regulatory mechanism remains elusive. In this study, we demonstrate that ELONGATED HYPOCOTYL 5 (HY5), a central transcription factor in the light signaling pathway, cannot promote artemisinin biosynthesis on its own, as the binding of AaHY5 to the promoters of artemisinin biosynthetic genes failed to activate their transcription. Transcriptome analysis and yeast two-hybrid screening revealed the B-box transcription factor AaBBX21 as a potential interactor with AaHY5. AaBBX21 showed a trichome-specific expression pattern. Additionally, the AaBBX21-AaHY5 complex cooperatively activated transcription from the promoters of the downstream genes AaGSW1, AaMYB108, and AaORA, encoding positive regulators of artemisinin biosynthesis. Moreover, AaHY5 and AaBBX21 physically interacted with the A. annua E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). In the dark, AaCOP1 decreased the accumulation of AaHY5 and AaBBX21 and repressed the activation of genes downstream of the AaHY5-AaBBX21 complex, explaining the enhanced production of artemisinin upon light exposure. Our study provides insights into the central regulatory mechanism by which light governs terpenoid biosynthesis in the plant kingdom.

In Vitro and In Silico Anti-Glioblastoma Activity of Hydroalcoholic Extracts of Artemisia annua L. and Artemisia vulgaris L.

Glioblastoma, the most aggressive and challenging brain tumor, is a key focus in neuro-oncology due to its rapid growth and poor prognosis. The C6 glioma cell line is often used as a glioblastoma model due to its close simulation of human glioma characteristics, including rapid expansion and invasiveness. Alongside, herbal medicine, particularly Artemisia spp., is gaining attention for its anticancer potential, offering mechanisms like apoptosis induction, cell cycle arrest, and the inhibition of angiogenesis. In this study, we optimized extraction conditions of polyphenols from Artemisia annua L. and Artemisia vulgaris L. herbs and investigated their anticancer effects in silico and in vitro. Molecular docking of the main phenolic compounds of A. annua and A. vulgaris and potential target proteins, including programmed cell death (apoptosis) pathway proteins proapoptotic Bax (PDB ID 6EB6), anti-apoptotic Bcl-2 (PDB ID G5M), and the necroptosis pathway protein (PDB ID 7MON), mixed lineage kinase domain-like protein (MLKL), in complex with receptor-interacting serine/threonine-protein kinase 3 (RIPK3), revealed the high probability of their interactions, highlighting the possible influence of chlorogenic acid in modulating necroptosis processes. The cell viability of rat C6 glioma cell line was assessed using a nuclear fluorescent double-staining assay with Hoechst 33342 and propidium iodide. The extracts from A. annua and A. vulgaris have demonstrated anticancer activity in the glioblastoma model, with the synergistic effects of their combined compounds surpassing the efficacy of any single compound. Our results suggest the potential of these extracts as a basis for developing more effective glioblastoma treatments, emphasizing the importance of further research into their mechanisms of action and therapeutic applications.

Artemisia annua L. polysaccharide improves the growth performance and intestinal barrier function of broilers challenged with Escherichia coli.

With the high intensification of poultry breeding, a series of diseases caused by pathogenic bacteria threaten the health of poultry and human. Among them, poultry diseases induced by Escherichia coli cause significant economic loss every year. The aim of this study was to investigate the effects of dietary supplementation with Artemisia annua L. polysaccharide (AAP) on the growth performance and intestinal barrier function of broilers with Escherichia coli (E. coli) challenge. A total of 256 one-day-old chicks were randomly assigned to four treatment groups: control group (fed basal diet), AAP group (fed basal diet supplemented with AAP), E. coli group (fed basal diet and orally administered E. coli), AAP + E. coli group (fed basal diet supplemented with AAP and orally administered E. coli). Dietary AAP supplementation elevated the BW, ADG and ADFI in non-challenged broilers. AAP also increased the apparent metabolic rate of EE and Ca in E. coli-challenged broilers. Moreover, AAP not only enhanced the serum IgA content but also decreased the serum and jejunum content of IL-6, as well as the jejunum level of IL-1ß in non-challenged broilers. AAP also down-regulates the mRNA level of inflammatory factors (IL-1ß, IL-6, and TNF-α) by inhibiting the mRNA expression of TLR4 and MyD88 in intestinal NF-κB signaling pathway of E. coli-challenged broilers. Meanwhile, AAP up-regulates the activity and mRNA level CAT by down-regulating the mRNA level of Keap1 in intestinal Nrf2 signaling pathway of E. coli-challenged broilers, and decreased serum MDA concentration. AAP significantly elevated the mRNA level of CAT, SOD and Nrf2 in jejunal of non-challenged broilers. Interestingly, AAP can improve intestinal physical barrier by down-regulating serum ET content, increasing the jejunal villus height/crypt depth (VH/CD) and ZO-1 mRNA level in broilers challenged by E. coli. AAP also elevated the VH/CD and the mRNA level of Occludin, ZO-1, Mucin-2 in non-challenged broilers. Importantly, AAP reshaped the balance of jejunum microbiota in E. coli-challenged broilers by altering α diversity and community composition. In summary, AAP ameliorated the loss of growth performance in broilers challenged with E. coli, probably by regulating the intestinal permeability and mucosa morphology, immune function, antioxidant ability, and microbiota.

Anticancer Effect by Combined Treatment of Artemisia annua L. Polyphenols and Docetaxel in DU145 Prostate Cancer Cells and HCT116 Colorectal Cancer Cells.

Docetaxel (DTX), a semi-synthetic analogue of paclitaxel (taxol), is known to exert potent anticancer activity in various cancer cells by suppressing normal microtubule dynamics. In this study, we examined how the anticancer effect of DTX is regulated by polyphenols extracted from Korean Artemisia annua L. (pKAL) in DU145 prostate cancer cells (mutant p53) and HCT116 colorectal cancer cells (wild-type p53). Here, we show that the anticancer effect of DTX was enhanced more significantly by pKAL in HCT116 cells than in DU145 cells via phase-contrast microscopy, CCK-8 assay, Western blot, and flow cytometric analysis of annexin V/propidium iodide-stained cells. Notably, mutant p53 was slightly downregulated by single treatment of pKAL or DTX in DU145 cells, whereas wild-type p53 was significantly upregulated by pKAL or DTX in HCT116 cells. Moreover, the enhanced anticancer effect of DTX by pKAL in HCT116 cells was significantly associated with the suppression of DTX-induced p53 upregulation, increase of DTX-induced phospho-p38, and decrease of DTX-regulated cyclin A, cyclin B1, AKT, caspase-8, PARP1, GM130, NF-κB p65, and LDHA, leading to the increased apoptotic cell death and plasma membrane permeability. Our results suggest that pKAL could effectively improve the anticancer effect of DTX-containing chemotherapy used to treat various cancers expressing wild-type p53.

Targeted screening of the synergistic components in Artemisia annua L. leading to enhanced antiplasmodial potency of artemisinin based on a "top down" PD-PK approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisinin (ART) showed enhanced antimalarial potency in the herb Artemisia annua L. (A. annua), from which ART is isolated. Increased absorption of ART with inhibited metabolism in the plant matrix is an underlying mechanism. Several synergistic components have been reported based on a "bottom-up" approach, i.e., traditional isolation followed by pharmacokinetic and/or pharmacodynamic evaluation. AIM OF THE STUDY: In this study, we employed a "top-down" approach based on in vivo antimalarial and pharmacokinetic studies to identify synergistic components in A. annua. MATERIALS AND METHODS: Two A. annua extracts in different chemical composition were obtained by extraction using ethyl acetate (EA) and petroleum ether (PE). The synergistic antimalarial activity of ART in two extracts was compared both in vitro (Plasmodium falciparum) and in vivo (murine Plasmodium yoelii). For the PD-PK correlation analysis, the pharmacokinetic profiles of ART and its major metabolite (ART-M) were investigated in healthy rats after a single oral administration of pure ART (20 mg/kg) or equivalent ART in each A. annua extract. A liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS)-based analytical strategy was then applied for efficient component classification and structural characterization of the differential components in the targeted extract with a higher antimalarial potency. Major components isolated from the targeted extract were then evaluated for their synergistic effect in the same proportion. RESULTS: Compared with pure ART (ED50, 5.6 mg/kg), ART showed enhanced antimalarial potency in two extracts in vivo (ED50 of EA, 2.9 mg/kg; ED50 of PE, 1.6 mg/kg), but not in vitro (IC50, 15.0-20.0 nM). A significant increase (1.7-fold) in ART absorption (AUC0-t) was found in rats after a single oral dose of equivalent ART in PE but not in EA; however, no significant change in the metabolic capability (AUCART-M/AUCART) was found for ART in either extract. The differential component analysis of the two extracts showed a higher composition of sesquiterpene compounds, especially component AB (3.0% in PE vs. 0.9% in EA) and component AA (14.1% in PE vs. 5.1% in EA). Two target sesquiterpenes were isolated and identified as arteannuin B (AB) and artemisinic acid (AA). The synergism between ART and AB/AA in the same proportion with PE extract (20:1.6:7.6, mg/kg) was verified by a pharmacokinetic study in rats. CONCLUSIONS: A "top-down" strategy based on PD-PK studies was successfully employed to identify synergistic components for ART in A. annua. Two sesquiterpene compounds (arteannuin B and artemisinic acid) could enhance the antimalarial potency of ART by increasing its absorption.

Artemisia annua L. Polyphenols Enhance the Anticancer Effect of ß-Lapachone in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells.

Recent studies suggest that the anticancer activity of ß-lapachone (ß-Lap) could be improved by different types of bioactive phytochemicals. The aim of this study was to elucidate how the anticancer effect of ß-Lap is regulated by polyphenols extracted from Korean Artemisia annua L. (pKAL) in parental HCT116 and oxaliplatin-resistant (OxPt-R) HCT116 colorectal cancer cells. Here, we show that the anticancer effect of ß-Lap is more enhanced by pKAL in HCT116-OxPt-R cells than in HCT116 cells via a CCK-8 assay, Western blot, and phase-contrast microscopy analysis of hematoxylin-stained cells. This phenomenon was associated with the suppression of OxPt-R-related upregulated proteins including p53 and ß-catenin, the downregulation of cell survival proteins including TERT, CD44, and EGFR, and the upregulation of cleaved HSP90, γ-H2AX, and LC3B-I/II. A bioinformatics analysis of 21 proteins regulated by combined treatment of pKAL and ß-Lap in HCT116-OxPt-R cells showed that the enhanced anticancer effect of ß-Lap by pKAL was related to the inhibition of negative regulation of apoptotic process and the induction of DNA damage through TERT, CD44, and EGFR-mediated multiple signaling networks. Our results suggest that the combination of pKAL and ß-Lap could be used as a new therapy with low toxicity to overcome the OxPt-R that occurred in various OxPt-containing cancer treatments.

Oxidative stress in liver of streptozotocin-induced diabetic mice fed a high-fat diet: A treatment role of Artemisia annua L.

Objective. The aim of this study was the investigation of a treatment role of Artemisia annua L. (AA) on liver dysfunction and oxidative stress in high-fat diet/streptozotocin-induced diabetic (HFD/STZ) mice. Methods. Sixty mice were divided into 12 groups including control, untreated diabetic, and treated diabetic ones with metformin (250 mg/kg), and doses of 100, 200, and 400 mg/kg of water (hot and cold) and alcoholic (methanol) extracts of AA. Type 2 diabetes mellitus (T2DM) was induced in mice by high-fat diet for 8 weeks and STZ injection in experimental animals. After treatment with doses of 100, 200 or 400 mg/kg of AA extracts in HFD/STZ diabetic mice for 4 weeks, oxidative stress markers such as malondialdehyde (MDA), glutathione (GSH), and free radicals (ROS) were determined in the liver tissue in all groups. Results. Diabetic mice treated with metformin and AA extracts showed a significant decrease in ROS and MDA concentrations and a notable increase in GSH level in the liver. Effectiveness of higher doses of AA extracts (200 and 400 mg/kg), especially in hot-water and alcoholic ones, were similar to and/or even more effective than metformin. Conclusion. Therapeutic effects of AA on liver dysfunction showed that antioxidant activity of hot-water and alcoholic AA extracts were similar or higher than of metformin.

Mechanism of Artemisia annua L. in the treatment of acute myocardial infarction: network pharmacology, molecular docking and in vivo validation.

This study was to evaluate the potential mechanism of action of Artemisia annua L. (A. annua) in the treatment of acute myocardial infarction (AMI) using network pharmacology, molecular docking and in vivo experiments. 22 active chemical compounds and 193 drug targets of A. annua were screened using the Traditional Chinese Medicine System Pharmacological (TCMSP) database. 3876 disease targets were also collected. Then 158 intersection targets between AMI and A. annua were obtained using R 4.2.0 software. String database was used to construct the protein-protein interaction (PPI) network and 6 core targets (MAPK1, TP53, HSP90AA1, RELA, AKT1, and MYC) were screened. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the R package. GO enrichment results were mainly related to cell responses to chemical stress and cell membrane microregions. KEGG pathways were mainly involved in lipids, atherosclerosis and fluid shear stress. In addition, molecular docking between A. annua active compounds and core targets showed high binding activity. As for in vivo validation, A. annua extract showed significant effects on improving post-infarction ventricular function, delaying ventricular remodeling, and reducing myocardial fibrosis and apoptosis. This study has revealed the potential components and molecular mechanisms of A. annua in the treatment of AMI. Our work also showed that A. annua has great effect on reducing myocardial fibrosis and scar area after infarction.

Effects of Artemisia annua L. Water Extract on Growth Performance and Intestinal Related Indicators in Broilers.

Artemisia annua L. is a natural herb with a variety of bioactive substances, which can play a variety of biological functions such as anti-inflammatory, antioxidant, antibacterial and antiviral, and can be used as a potential feed additive. The purpose of this study was to investigate the effects of different doses of Artemisia annua L. water extract (AAWE) on growth performance and intestinal related indicators in broilers. A total of 200 one-day-old Arbor Acre broilers were selected and randomly divided into five treatment groups, with five replicates in each group and eight birds per replicate. The control group was fed a basal diet, whereas the other groups were fed a basal diet supplemented with 0.5, 1.0, 1.5, or 2.0 g/kg AAWE. On d 21, with the increase in AAWE dose, final body weight and feed efficiency showed a quadratic increase effect, whereas feed intake showed a linear reduction effect; however, the apparent metabolic rate of dry matter, crude protein, and ether extract increased quadratically on d 42. In addition, the activity of duodenal chymotrypsin and trypsin, and of jejunal lipase quadratically increased, whereas the intestine crypt depth linearly decreased on d 42. The number of total anaerobic bacteria increased quadratically, whereas the number of Escherichia coli decreased quadratically. The number of Lactobacillus increased linearly, whereas H2S emission linearly decreased on d 21; moreover, NH3 emission (24 h) quadratically decreased on d 42. In conclusion, AAWE promoted the growth performance and intestinal related indicators of broilers.

Five Different Artemisia L. Species Ethanol Extracts' Phytochemical Composition and Their Antimicrobial and Nematocide Activity.

Among the plants that exhibit significant or established pharmacological activity, the genus Artemisia L. deserves special attention. This genus comprises over 500 species belonging to the largest Asteraceae family. Our study aimed at providing a comprehensive evaluation of the phytochemical composition of the ethanol extracts of five different Artemisia L. species (collected from the southwest of the Russian Federation) and their antimicrobial and nematocide activity as follows: A. annua cv. Novichok., A. dracunculus cv. Smaragd, A. santonica cv. Citral, A. abrotanum cv. Euxin, and A. scoparia cv. Tavrida. The study of the ethanol extracts of the five different Artemisia L. species using the methods of gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-MS/MS) allowed establishing their phytochemical profile. The obtained data on the of five different Artemisia L. species ethanol extracts' phytochemical composition were used to predict the antibacterial and antifungal activity against phytopathogenic microorganisms and nematocidal activity against the free-living soil nematode Caenorhabditis elegans. The major compounds found in the composition of the Artemisia L. ethanol extracts were monoterpenes, sesquiterpenes, flavonoids, flavonoid glycosides, coumarins, and phenolic acids. The antibacterial and antifungal activity of the extracts began to manifest at a concentration of 150 µg/mL. The A. dracunculus cv. Smaragd extract had a selective effect against Gram-positive R. iranicus and B. subtilis bacteria, whereas the A. scoparia cv. Tavrida extract had a selective effect against Gram-negative A. tumefaciens and X. arboricola bacteria and A. solani, R. solani and F. graminearum fungi. The A. annua cv. Novichok, A. dracunculus cv. Smaragd, and A. santonica cv. Citral extracts in the concentration range of 31.3-1000 µg/mL caused the death of nematodes. It was established that A. annua cv. Novichok affects the UNC-63 protein, the molecular target of which is the nicotine receptor of the N-subtype.

Artemeriosides A-F, the first examples of natural sesquiterpenoids substituted by a 6'-O-crontonyl ß-glucopyranoside from Artemisia annua.

Artemeriosides A-F (1-6), six novel sesquiterpenoids containing a 6'-O-crontonyl ß-glucopyranoside, were isolated from Artemisia annua L. Their structures were determined by spectral data including HRESIMS, IR, UV, 1D and 2D NMR, and ECD calculations. Compounds 1-6 represented the first examples of natural sesquiterpenoid substituted by 6'-O-crontonyl ß-glucopyranoside. By antihepatoma assay, compounds 1 and 2 demonstrated inhibitory effect against both HepG2 and SK-Hep-1 cells with inhibitory ratios of 77.0%, 88.8%, and 86.8%, 83.9% at 200.0 µM, and compound 1 showed inhibitory activity against Huh7 cells with inhibitory ratio of 56.8%.

AaWRKY6 contributes to artemisinin accumulation during growth in Artemisia annua.

Artemisinin, which is extracted from the plant Artemisia annua L., is a crucial drug for curing malaria and has potential applications for treating cancer, diabetes, pulmonary tuberculosis, and other conditions. Demand for artemisinin is therefore high, and enhancing its yield is important. Artemisinin dynamics change during the growth cycle of A. annua; however, the regulatory networks underlying these changes are poorly understood. Here, we collected A. annua leaves at different growth stages and identified target genes from transcriptome data. We determined that WRKY6 binds to the promoters of the artemisinin biosynthesis gene artemisinic aldehyde Δ11(13) reductase (DBR2). In agreement, overexpression of WRKY6 in A. annua resulted in higher expression levels of genes in the artemisinin biosynthesis pathway and greater artemisinin contents than in the wild type. When expression of WRKY6 was down-regulated, artemisinin biosynthesis pathway genes were also down-regulated and the content of artemisinin was lower. WRKY6 mediates the transcriptional activation of artemisinin biosynthesis by binding to the promoter of DBR2, making it a key regulator for modulating the dynamics of artemisinin changes during the A. annua growth cycle.

Antimicrobial and Antioxidant Properties of Chemically Analyzed Essential Oil of Artemisia annua L. (Asteraceae) Native to Mediterranean Area.

Artemisia annua (AA) is an aromatic plant belonging to the Asteraceae family, which has long been known for its several medicinal virtues. In addition, essential oils (EOs) extracted from AA have a wide range of therapeutic properties. Therefore, this study aimed to investigate the phytochemical composition, anti-microbial, and anti-oxidant properties of Artemisia annua essential oil (EOAA). EO was extracted, and its chemical constituents were ascertained by the use of GC-MS analysis. EOAA shows remarkable antioxidant capacities of DPPH free radical scavenging with an IC50 value of 29 ± 5.3 µg/mL and ferric reducing antioxidant power with an EC50 value of 9.21 ± 0.3 µg/mL, and it also has a good total antioxidant capacity of 911.59 ± 115.71 milligrams of ascorbic acid equivalence per gram of EO (mg AAE/g EO). Moreover, the in vitro antimicrobial screening results indicate that EOAA has shown promising antibacterial activity, especially against the Escherichia coli strain, and it also shows significant antifungal activity against Fusarium oxysporum and Candida albicans yeasts. Taken together, our findings highlight the importance of EOAA as a source of strong antioxidant and antimicrobial agents, which could be used as an alternative form to control free radicals and combat drug-resistant microbes.

Antibacterial and Phytochemical Screening of Artemisia Species.

Taking into account the increasing number of antibiotic-resistant bacteria, actual research focused on plant extracts is vital. The aim of our study was to investigate leaf and stem ethanolic extracts of Artemisia absinthium L. and Artemisia annua L. in order to explore their antioxidant and antibacterial activities. Total phenolic content (TPC) was evaluated spectrophotometrically. Antioxidant activity was evaluated by DPPH and ABTS. The antibacterial activity of wormwood extracts was assessed by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enteritidis cultures, and by zone of inhibition in Klebsiella carbapenem-resistant enterobacteriaceae (CRE) and Escherichia coli extended-spectrum ß-lactamases cultures (ESBL). The Artemisia annua L. leaf extract (AnL) exhibited the highest TPC (518.09 mg/mL) and the highest expression of sinapic acid (285.69 ± 0.002 µg/mL). Nevertheless, the highest antioxidant capacity (1360.51 ± 0.04 µM Trolox/g DW by ABTS and 735.77 ± 0.02 µM Trolox/g DW by DPPH) was found in Artemisia absinthium L. leaf from the second year of vegetation (AbL2). AnL extract exhibited the lowest MIC and MBC for all tested bacteria and the maximal zone of inhibition for Klebsiella CRE and Escherichia coli ESBL. Our study revealed that AbL2 exhibited the best antioxidant potential, while AnL extract had the strongest antibacterial effect.

Genetic analysis reveals the inconsistency of amorpha-4,11-diene synthase, a key enzyme in the artemisinin synthesis pathway, in asteraceae.

BACKGROUND: Amorpha-4,11-diene synthase (ADS) is a key enzyme in the artemisinin biosynthetic pathway. ADS promotes the first step of artemisinin synthesis by cyclizing faresyl pyrophosphate to synthesize the sesquiterpene product amorpha-4,11-diene. Thanks to the continuous improvement of genomic information, its evolutionary trace can be analyzed in a genome view. METHODS: Phylogenetic analysis was used to identify ADS-like genes in other Asteraceae. Gene structure and motif analysis was used to analyze the structural similarity of these identified genes. Heterologous expression and GC-MS analysis were performed to determine whether the functions of ADS and Cna4666 are consistent. Validation of ADS genes evolutionary trajectories was achieved by selective pressure and synteny analysis. RESULT: In this study, we extracted 8 ADS genes from the Artemisia annua L. genome annotation and 121 ADS similar genes from the genomes of Artemisia annua L. and other plants in the Asteraceae, and further exploring their evolutionary relationship. Phylogenetic analysis showed that the genes most closely related to ADS genes were found in the genome of Chrysanthemum nankingense. Among them, the gene structure and motif composition of Cna4666 is very similar to ADS, we wondered whether it has the potential to synthesize amorpha-4,11-diene. Therefore, we extracted the products of recombinant p0_ADS.1 and Cna4666 proteins by HS-SPME combined with GC-MS analysis, the results indicate that Cna4666 is an α-bisabolol synthase, which cannot synthesize amorpha-4,11-diene. Through synteny analysis, we did not find collinear blocks of ADS genes in the Helianthus annuus and C. nankingense genomes. Furthermore, Ka/Ks ratios indicated that the evolution of ADS genes from their similar genes principally underwent purifying selection, and there was a strong positive selection between ADS genes. CONCLUSIONS: This study proved that ADS is a multi-copy gene in Artemisia annua L., and they are not widely distributed in Asteraceae. The data will increase our understanding of the evolutionary selection pressure on ADS genes. The results suggest that ADS genes are subject to strong positive selection internally, and it is possible that they are a recently evolved gene in the Artemisia.

Artemisia annua L. Extracts Irreversibly Inhibit the Activity of CYP2B6 and CYP3A4 Enzymes.

Artemisia annua L. has long been known for its medicinal properties and isolation of ingredients whose derivatives are used for therapeutic purposes. The CYP2B6 and CYP3A4 enzymes belong to a large family of cytochrome P450 enzymes. These enzymes are involved in the metabolism of drugs and other xeonobiotics. It is known that various compounds can induce or inhibit the activity of these enzymes. The aim of this study was to investigate the nature of the inhibitory effect of Artemisia annua extract on CYP2B6 and CYP3A4 enzymes, as well as the type of inhibition, the presence of reversible or pseudo-irreversible inhibition, and the possible heme destruction. The methanolic extract of Artemisia annua showed an inhibitory effect on CYP2B6 (by almost 90%) and CYP3A4 enzymes (by almost 70%). A significant decrease in heme concentration by 46.8% and 38.2% was observed in different assays. These results clearly indicate that the studied plant extracts significantly inhibited the activity of CYP2B6 and CYP3A4 enzymes. Moreover, they showed irreversible inhibition, which is even more important for possible interactions with drugs and dietary supplements.

Mechanism of Artemisia annua L. in GIOP with kidney-yin deficiency based on network pharmacology / 药学实践杂志

Objective To predict and preliminarily verify the potential targets and related signaling pathways of Artemisia annua L. in treating glucocorticoid-induced osteoporosis (GIOP) with kidney-yin deficiency by network pharmacology and in vitro experiments. Methods The pharmacological targets of Artemisia annua L. were obtained from TCMSP database and were converted to gene names through Uniprot database. The target genes of GIOP with kidney-yin deficiency were obtained from GeneCards database, OMIM database and Drugbank database, and the common target genes were obtained by cross analysis with drug target gene. Protein-protein interaction (PPI) network was constructed by String database, and visualization analysis and core targets screening were performed by Cytoscape 3.9.0. All common targets were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis through Metascape database. Finally, the prediction results were verified by in vitro experiments. Results Ninety-eight targets of Artemisia annua L. to GIOP with kidney-yin deficiency were screened, including 17 core genes. The results of GO and KEGG functional enrichment analysis indicated that Artemisia annua L. treating GIOP with kidney-yin deficiency was related to biological processes such as hormonal response, positive regulation of cell death and extracellular stimulation response, et al, as well as signaling pathways such as PI3K/AKT, AGE/RAGE, MAPK and IL-17 et al. The number of genes enriched in PI3K/AKT signaling pathway was the largest. In vitro experiment results showed that Artemisia annua L. promoted the proliferation of osteoblasts damaged by dexamethasone (DEX), increased alkaline phosphatase activity, activated PI3K/AKT pathway, and promoted the phosphorylation of AKT. Conclusion Artemisia annua L. treating GIOP with kidney-yin deficiency has the characteristics of multi-targets and multi-pathway, which could promote the proliferation and differentiation of osteoblasts through multiple pathways. The PI3K/AKT signaling pathway is an important pathway. Artemisia annua L. treating GIOP with kidney-yin deficiency might be related to its ability to promote the PI3K/AKT signaling pathway and promote the phosphorylation of AKT.

Flavonoid glycosides from the water extract of Artemisia annua L / 药学学报

Twenty one flavonoid glycosides were isolated and purified from n-butanol portion of the water extract of A. annua by various chromatographic techniques such as HP-20 macroporous adsorption resin, silica gel, ODS, Sephadex LH-20 gel column chromatography and preparative high performance liquid chromatography. Their structures were identified by analysis of physicochemical properties and spectral data, and determined as axillarin-7-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside (1), orientin (2), apigenin-6-C-β-D-glucopyranosyl-8-C-β-L-arabinopyranoside (3), apigenin-6-C-β-D-galactopyranosyl-8-C-β-L-arabinopyranoside (4), apigenin-6-C-β-L-arabinopyranosyl-8-C-β-D-glucopyranoside (5), apigenin-6-C-α-L-arabinofuranosyl-8-C-β-D-glucopyranoside (6), quercetin-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (7), apigenin-6-C-α-L-arabinopyranosyl-8-C-β-D-glucopyranoside (8), vicenin-2 (9), patuletin-7-O-β-D-glucopyranoside (10), luteolin-6-C-glucopyranoside (11), vitexin (12), kaempferol-3-O-β-galactopyranosyl-(1→2)-β-glucopyranoside (13), quercetin-7-O-β-D-glucopyranoside (14), patuletin-3-O-β-D-glucopyranoside (15), 7-O-methyl-quercetagetin-6-O-β-D-glucopyranoside (16), quercetin-3-O-β-D-glucopyranoside (17), nepitrin (18), rutin (19), kaempferol-3-O-β-sophoroside (20), and patuletin-3-O-rutinoside (21). Compound 1 is a new compound, compounds 2, 4, 6, 7, 10, 11, 13, 15, 16, 18, 20 and 21 are isolated from A. annua for the first time. In the anti-inflammatory assay, compound 1 inhibited the release of IL-6 from LPS-induced RAW264.7 cells to significantly degrees with the high (100 μmol·L-1), medium (50 μmol·L-1), low (25 μmol·L-1) concentration.

A Discovery-Based Metabolomic Approach Using UPLC-Q-TOF-MS/MS Reveals Potential Antimalarial Compounds Present in Artemisia annua L.

In 1972, Nobel laureate Youyou Tu's research team conducted clinical trials on the dried material of Artemisia annua L. from Beijing extracted by ether and then treated with alkali (called "ether neutral dry"), which showed that artemisinin was not the only antimalarial component contained. The biosynthesis of sesquiterpenoids in A. annua has increased exponentially after unremitting cultivation efforts, and the plant resources are now quite different from those in the 1970s. In consideration of emerging artemisinin resistance, it is of great theoretical and practical value to further study the antimalarial activity of A. annua and explore its causes. The purpose of this study is to clarify scientific questions, such as "What ingredients are synergistic with artemisinin in A. annua?", and "Are there non-artemisinin antimalarial ingredients in A. annua?". In this paper, Beijing wild A. annua was used as a control and two representative cultivation species of A. annua were selected to evaluate the antimalarial activity of the herbal medicine. The antimalarial activity of different extracts on mice was studied using the Peters' four-day suppressive test. UPLC-Q-TOF-MS was used to obtain mass spectrum data for all samples, and a UNIFI platform was used for identification. A multivariate statistical method was used to screen the different compounds with positive correlations. The antimalarial activity of different components from the ether extract and alkali treatments was determined and antimalarial components other than artemisinin were obtained. A total of 24 flavonoids, 68 sesquiterpenoids and 21 other compounds were identified. Compounds associated with differential antimalarial activity were identified. The material basis for the antimalarial activity of A. annua was clarified. The antimalarial components of A. annua include two categories: first, artemisinin and non-artemisinin antimalarial active components, of which the non-artemisinin antimalarial active components may include 5α-hydroperoxy-eudesma-4(15),11-diene; second, several antimalarial synergistic ingredients in A. annua, including arteanniun B, arteanniun B analogues and polymethoxy flavonoids.