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.

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.

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%.

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.

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.

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.

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.

Through network pharmacology and molecular docking to explore the underlying mechanism of Artemisia annua L. treating in abdominal aortic aneurysm.

Background: Abdominal aortic aneurysm (AAA) is a degenerative disease that causes health problems in humans. However, there are no effective drugs for the treatment of AAA. Artemisia annua L. (A. annua) is a traditional herbal that has been widely used in cardiovascular disease. Based on network pharmacology and molecular docking technology, this study predicted the practical components and potential mechanisms of A. annua inhibiting the occurrence and development of AAA. Methods: The main active ingredients and targets of A. annua were screened through the TCMSP database; the GeneCards, OMIM, PharmGkb, and TTD databases were used to search for the targeted genes of AAA and map them to the targets of the active ingredients to obtain the active ingredient therapy of A. annua. The targets of AAA were to construct a protein interaction network through the STRING platform. R software was used to carry out the enrichment analysis of GO and KEGG for relevant targets, and Cytoscape was used to construct the active ingredient-target network prediction model of A. annua. Finally, AutoDock Vina was used to verify the results of the active ingredients and critical targets. Results: The main active ingredients obtained from A. annua for the treatment of AAA include quercetin, luteolin, kaempferol, isorhamnetin, and artemetin, as well as 117 effective targets, including RELA, MAPK14, CCND1, MAPK1, AKT1, MYC, MAPK8, TP53, ESR1, FOS, and JUN. The 11 targeted genes might play a key role in disease treatment. Enriched in 2115 GO biological processes, 159 molecular functions, 56 cellular components, and 156 KEGG pathways, inferred that its mechanism of action might be related to PI3K-Akt signaling pathway, fluid shear stress, atherosclerosis, and AGE-RAGE signaling pathway. Molecular docking results showed that the top five active components of A. annua had a good affinity for core disease targets and played a central role in treating AAA. The low binding energy molecular docking results provided valuable information for the development of drugs to treat AAA. Conclusion: Therefore, A. annua may have multiple components, multiple targets, and multiple signaling pathways to play a role in treating AAA. A. annua may have the potential to treat AAA.

A high-efficiency trichome collection system by laser capture microdissection.

Trichomes, which are classified as glandular or non-glandular, are hair-like epidermal structures that are present on aerial parts of most plant species. Glandular secretory trichomes (GSTs) have the capacity to secrete and store specialized metabolites, which are widely used as natural pesticides, food additives, fragrance ingredients or pharmaceuticals. Isolating individual trichomes is an essential way for identifying trichome-specific gene functions and discovering novel metabolites. However, the isolation of trichomes is difficult and time-consuming. Here, we report a method to isolate the GSTs from leaf epidermis dispense with fixation using laser capture microdissection (LCM). In this study, 150 GSTs were captured efficiently from Artemisia annua leaves and enriched for artemisinin measurement. UPLC analysis of microdissected samples indicated specific accumulation of secondary metabolites could be detected from a small number of GSTs. In addition, qRT-PCR revealed that the GST-specific structural genes involved in artemisinin biosynthesis pathway were highly expressed in GSTs. Taken together, we developed an efficient method to collect comparatively pure GSTs from unfixed leaved, so that the metabolites were relatively obtained intact. This method can be implemented in metabolomics research of purely specific plant cell populations and has the potential to discover novel secondary metabolites.

Artemisia annua L. Aqueous Extract Promotes Intestine Immunity and Antioxidant Function in Broilers.

This study was conducted to investigate the effects of Artemisia annua L. aqueous extract (AAE) on intestinal immune and antioxidative function of broilers. A total of 200 one-day-old Arbor Acre broilers were randomly allotted into five dietary treatment groups, with five replicates per treatment and eight broilers per replicate. The five treatment diets were formulated by adding, respectively, 0 (control group), 0.5, 1.0, 1.5, and 2.0 g/kg AAE in the basal diet. The results showed that dietary inclusion of AAE quadratically decreased interleukin (IL)-1ß content, linearly decreased IL-6 content in the small intestine through regulating the nuclear factor-kappa B signal pathway, and quadratically increased immunoglobulin (Ig)M and sIgA content in ileum and jejunum. Besides, there was a quadratic decrease in the gene expression of IL-1ß, IL-6, and toll like receptor 4 (TLR4) in ileum on day 21, and the gene expression of IL-6 and TLR4 in duodenum on day 42, thereby improving small intestinal immune function in broilers. Additionally, dietary inclusion of AAE improves antioxidative function through the nuclear factor-erythroid 2-related factor 2 (Nrf2) signal pathway in the small intestinal mucosa of broilers, especially, quadratically increased catalase (CAT) and superoxidase dismutase activity in ileum, and total antioxidant capacity and glutathione peroxidase activity in duodenum, and quadratically decreased malondialdehyde concentration in ileum, besides, linearly increased heme oxygenase-1 and Nrf2 gene expression in jejunum and ileum on day 42, quadratically increased CAT gene expression in the small intestine. Furthermore, regression analyses of the above parameters showed that the optimal dose range of AAE in the diet of broilers was 1.12-1.38 g/kg.

Dietary Supplementation of Enzymatically Treated Artemisia annua L. Improves Lactation Performance, Alleviates Inflammatory Response of Sows Reared Under Heat Stress, and Promotes Gut Development in Preweaning Offspring.

Artemisia annua L., which is known for its antimalarial compound artemisinin, has commonly been used for its anti-inflammatory and antibacterial functions. Enzymatically treating Artemisia annua L. can improve its bioavailability. The purpose of this study was to investigate the effects of dietary enzymatically treated Artemisia annua L. (EA) supplementation in late gestation and lactation diets on sow performance, serum hormone, inflammatory cytokines, and immunoglobulin level of heat-stressed sows. A total of 135 multiparous sows (Large White × Landrace) on day 85 of gestation were selected and randomly distributed into 3 groups with 45 replicates per group. The control group was reared under standard conditions (temperature: 27.12 ± 0.18°C, THI (temperature-humidity index): 70.90 ± 0.80) and fed with basal diet. The heat stress (HS) and HS + EA groups were raised in heat-stressed conditions (temperature: 30.11 ± 0.16°C, THI: 72.70 ± 0.60) and fed with basal diets supplemented with 0 or 1.0 g/kg EA, respectively. This trial lasted for 50 consecutive days until day 21 of lactation. Compared with the control group, HS increased the concentrations of serum endotoxin and heat shock protein 70 (HSP-70), and inflammatory cytokines in serum, colostrum, and 14 day milk of sows. Meanwhile, the EA supplementation decreased levels of serum endotoxin, HSP-70, and inflammatory cytokines in both sows and offspring and increased serum triiodothyronine (T3) level and average daily feed intake (ADFI) of sows. In addition, EA significantly improved average daily gain (ADG) and altered intestinal morphology with an increased villus height in the duodenum and ileum of piglets. Collectively, EA supplementation at 1.0 g/kg in late gestation and lactation diets alleviated the adverse effects of HS, which were reflected by enhancing ADFI and decreasing endotoxin as well as inflammatory cytokine levels in the serum and colostrum of heat-stressed sows, while promoting ADG and gut development of their offspring.

Casticin and chrysosplenol D from Artemisia annua L. induce apoptosis by inhibiting topoisomerase IIα in human non-small-cell lung cancer cells.

BACKGROUND: Artemisia annua L. (A. annua) and its active components exhibit antitumour effects in many cancer cells. However, the biological processes and mechanisms involved are not well understood, especially for the treatment of non-small-cell lung cancer (NSCLC). PURPOSE: This study aimed to comprehensively explore the biological processes of A. annua and its active components in NSCLC cells and to identify the mechanism by which these compounds induce apoptosis. STUDY DESIGNS/METHODS: Cell viability and flow cytometry assays were used to evaluate the cytotoxicity of A. annua active components casticin (CAS) and chrysosplenol D (CHD) in A. annua in NSCLC cells. After treatment with CAS and CHD, A549 cells were subjected to RNA sequencing (RNA-seq) analysis, differentially expressed genes (DEGs) were screened and subjected to functional enrichment analysis (KEGG and GO analysis) as well as protein interaction network analysis. The key targets associated with apoptosis induction in A549 cells were screened by Cytoscape, and the screened DEGs were validated by qRT-PCR. Immunoblotting, immunofluorescence, and molecular docking assays were used to determine whether CAS and/or CHD could induce apoptosis in NSCLC cells by inducing DNA damage through down-regulation of topoisomerase IIα (topo IIα) expression. The same experiments were verified again in the H1299 lung cancer cell line. RESULTS: CAS and CHD inhibited NSCLC cells proliferation in a time- and dose-dependent manner, and significantly induced apoptosis. A total of 115 co-upregulated DEGs and 277 co-downregulated DEGs were identified in A549 cells following treatment with CAS and CHD. Comprehensive and systematic data about biological processes and mechanisms were obtained. DNA damage pathways and topo IIα targets were screened to study the apoptosis effects of CAS and CHD on NSCLC cells. CAS and CHD may be able to induce DNA damage by binding to topo IIα-DNA and reducing topo IIα activity. CONCLUSION: This study suggested that CAS and CHD may reduce topo IIα activity by binding to topo IIα-DNA, affecting the replication of DNA, triggering DNA damage, and inducing apoptosis. It described a novel mechanism associated with topo IIα inhibition to reveal a novel role for CAS and CHD in A. annua as potential anticancer agents and/or adjuvants in NSCLC cells.

Genome-Wide Identification, Characterization and Expression Analysis of Lipoxygenase Gene Family in Artemisia annua L.

Lipoxygenase (LOX) is a ubiquitous oxygenase found in animals and plants and plays a pivotal role in diverse biological processes, including defense and development. Artemisinin, which can only be obtained from Artemisia annua L., is the most effective therapeutic drug for malaria without serious side effects. This study identified and analyzed LOX gene family members in the A. annua genome at the chromosomal level. Twenty LOX genes with various molecular weights, isoelectric points, and amino acid numbers were identified and named AaLOX, which were located in the cytoplasm or chloroplast. The average protein length of all AaLOX was 850 aa. Phylogenetic tree analysis revealed that the AaLOX was divided into two major groups, 9-LOX and 13-LOX. The exon numbers ranged from 1 to 12, indicating that different AaLOX genes have different functions. The secondary structure was mainly composed of alpha helix and random coil, and the tertiary structure was similar for most AaLOX. Upstream promoter region analysis revealed that a large number of cis-acting elements were closely related to plant growth and development, light response, hormone, and other stress responses. Transcriptome data analysis of different tissues suggested that the gene family was differently expressed in the roots, stems, leaves, and flowers of two A. annua strains HAN1 and LQ9. qRT-PCR confirmed that AaLOX5 and AaLOX17 had the highest expression in flowers and leaves. This study provides a theoretical basis for the further functional analysis of the AaLOX gene family.