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.

The effect of Artemisia annua L. aqueous and methanolic extracts on insulin signaling in liver of HFD/STZ diabetic mice.

OBJECTIVES: Many studies have shown the anti-diabetic effects of medicinal plants. But their molecular mechanism has been less studied. Understanding of these mechanisms can help to better manage the treatment of diabetes by using these plants. So, this research examined the effect of Artemisia annua extract on PI3K (phosphatidylinositol 3-kinase)/AKt (serine/threonine kinase protein B) signaling pathway in liver of high-fat diet (HFD)/Streptozotocin (STZ)-induced type 2 diabetic mice. METHODS: Groups of mice were control, untreated diabetic mice, diabetic mice treated with various doses (400, 200, 100 mg/kg) of methanolic and aqueous extract of A. annua and metformin for four weeks. Type 2 diabetes was produced by feeding high-fat diet following injection of low dose of STZ. After experiment duration all mice were sacrificed and blood glucose, insulin, homeostasis model assessment of insulin resistance index (HOMA-IR), index of insulin sensitivity index (ISI) were detected and liver tissues were isolated for to detect m-RNA expression of PI3K and Akt. RESULTS: Extracts of aqueous and methanolic this plant markedly reduced hyperglycemia, hyperinsulinemia, HOMA-IR and elevated ISI in diabetic group in comparison with un-treated diabetic mice. In addition, they could enhance the expression of AKt and PI3K m-RNA in liver tissues in diabetic mice. CONCLUSIONS: Artemisia annua extract ameliorated insulin resistance and improved insulin action in liver via the high activity of PI3K/AKt signaling pathway. So, it can be a suitable alternative treatment to synthetic antidiabetic drugs to improve insulin action in condition of type 2 diabetes.

Effects of dietary Artemisia annua supplementation on growth performance, antioxidant capacity, immune function, and gut microbiota of geese.

This experiment aimed to study the effect of 1% Artemisia annua added to the diet on growth performance, antioxidant capacity, immunity and intestinal morphology, and gut microbiota of geese. Seventy-two 35-day-old male geese (Zi goose) with similar body weight were selected and randomly divided into 2 groups. Each treatment group of 36 geese was divided into 6 subgroups, each having 6 male geese. The experiment lasted for 21 d. Control group (CON) was fed a basal diet and the experimental group (AAL) was fed a basal diet + 1% Artemisia annua. BW, ADG, and ADFI of the AAL group increased (p < 0.05) and the FCR decreased (p < 0.05) compared with the CON group. The addition of Artemisia annua to the diet increased catalase (CAT), glutathione peroxidase (GSH-px), and superoxide dismutase (SOD) enzyme activities, increased total antioxidant capacity (T-AOC), and decreased malondialdehyde (MDA) content in serum and jejunum of geese (p < 0.05). Meanwhile, serum IgA, IgG, IgM, and lysozyme (LZM), increased at different time points in the AAL group compared to the CON group (p < 0.05), and decrease in the content of interferon-γ (IFN-γ) , IL-6 (p < 0.05), but no effect on complement C3 and C4. Morphological observation of the small intestine showed that the jejunal crypt depth was decreased in the AAL group (p < 0.05) while elevating the jejunal villus height/crypt depth (p < 0.05). 16S rRNA sequencing results showed the Artemisia annua increased the diversity of cecum microbiota, increasing the relative abundance of Bacteroides, Fecalibacterium, and Paraprevotella. In conclusion, the addition of 1% Artemisia annua to the diet could improve the growth performance, antioxidant and immune ability of geese, as well as improve the development of the jejunum intestinal tract of geese, and change the structure of the cecum microbiota, which had a positive effect on the growth and development of geese. Artemisia annua can be further developed as a feed additive.

Artemisia annua Extract Attenuate Doxorubicin-Induced Hepatic Injury via PI-3K/Akt/Nrf-2-Mediated Signaling Pathway in Rats.

Doxorubicin (DOX), which is used to treat cancer, has harmful effects that limit its therapeutic application. Finding preventative agents to thwart DOX-caused injuries is thus imperative. Artemisia annua has numerous biomedical uses. This study aims to investigate the attenuative effect of Artemisia annua leaf extract (AALE) treatment on DOX-induced hepatic toxicity in male rats. A phytochemical screening of AALE was evaluated. Forty male rats were used; G1 was a negative control group, G2 was injected with AALE (150 mg/kg) intraperitoneally (i.p) daily for a month, 4 mg/kg of DOX was given i.p to G3 once a week for a month, and G4 was injected with DOX as G3 and with AALE as G2. Body weight changes and biochemical, molecular, and histopathological investigations were assessed. The results showed that AALE contains promising phytochemical constituents that contribute to several potential biomedical applications. AALE mitigated the hepatotoxicity induced by DOX in rats as evidenced by restoring the alterations in the biochemical parameters, antioxidant gene expression, and hepatic histopathological alterations in rats. Importantly, the impact of AALE against the hepatic deterioration resulting from DOX treatment is through activation of the PI-3K/Akt/Nrf-2 signaling, which in turn induces the antioxidant agents.

Metabolomic analysis and antibacterial and antioxidant activities of three species of Artemisia plants in Tibet.

BACKGROUND: Artemisia is important medicinal plants in China and are widely used in medicine, agriculture, and food. Pharmacologically active components of the plants remain to be investigated. METHODS: This study sought to identify and compare the chemical constituents of three species of Artemisia in Tibet using a widely-targeted metabolomics approach and their antibacterial and antioxidant capacities were determined. RESULT: A total of 1109 metabolites within 10 categories were detected from the three species of Artemisia, including lipids, amino acids, nucleotides, flavonoids, terpenes, coumarins, organic acids, and phenolic acids. 732 different metabolites have been identified between Artemisia sieversiana and Artemisia annua, 751 different metabolites were identified between Artemisia wellbyi and A. sieversiana, and 768 differential metabolites were differentially detected from A. wellbyi and A. annua. Differentially identified compounds included flavonoids, phenolic acids, artemisinins and coumarin. A. annua contained the highest relative content of artemisinin among three Artemisia. The antimicrobial experiments showed that the three Artemisia species had strong antibiotic activities against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Proteus mirabilis and Pseudomonas aeruginosa. The biochemical analysis showed that the three species of Artemisia have strong antioxidant capacity. CONCLUSIONS: This is the first reported attempt to comparatively determine the types of the metabolites of the three widely distributed Artemisia species in Tibet. The information should help medicinal research and facilitate comprehensive development and utilization of Artemisia species in Tibet.

RNA sequencing in Artemisia annua L explored the genetic and metabolic responses to hardly soluble aluminum phosphate treatment.

Artemisia annua L. is a medicinal plant valued for its ability to produce artemisinin, a molecule used to treat malaria. Plant nutrients, especially phosphorus (P), can potentially influence plant biomass and secondary metabolite production. Our work aimed to explore the genetic and metabolic response of A. annua to hardly soluble aluminum phosphate (AlPO4, AlP), using soluble monopotassium phosphate (KH2PO4, KP) as a control. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze artemisinin. RNA sequencing, gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to analyze the differentially expressed genes (DEGs) under poor P conditions. Results showed a significant reduction in plant growth parameters, such as plant height, stem diameter, number of leaves, leaf areas, and total biomass of A. annua. Conversely, LC-MS analysis revealed a significant increase in artemisinin concentration under the AlP compared to the KP. Transcriptome analysis revealed 762 differentially expressed genes (DEGs) between the AlP and the KP. GH3, SAUR, CRE1, and PYL, all involved in plant hormone signal transduction, showed differential expression. Furthermore, despite the downregulation of HMGR in the artemisinin biosynthesis pathway, the majority of genes (ACAT, FPS, CYP71AV1, and ALDH1) were upregulated, resulting in increased artemisinin accumulation in the AlP. In addition, 12 transcription factors, including GATA and MYB, were upregulated in response to AlP, confirming their importance in regulating artemisinin biosynthesis. Overall, our findings could contribute to a better understanding the parallel transcriptional regulation of plant hormone transduction and artemisinin biosynthesis in A. annua L. in response to hardly soluble phosphorus fertilizer.

Anti-Inflammatory Activities of Monoterpene and Sesquiterpene Glycosides from the Aqueous Extract of Artemisia annua L.

Artemisia annua L. is a Chinese medicinal herb, but the origin of its pharmacological properties, including its anti-inflammatory activity, remain unknown. In this study, five new monoterpene glycosides (1-5) and two new sesquiterpene glycosides (6 and 7) were isolated from the aqueous extract of the aerial parts of A. annua. The structures of these glycosides were determined using high-resolution electrospray ionization mass spectrometry, nuclear magnetic resonance spectroscopy, electronic circular dichroism calculations, and chemical hydrolysis methods. The anti-inflammatory activities of the isolated compounds were evaluated by down-regulating interleukin-6 (IL-6) in lipopolysaccharide-stimulated RAW 264.7 macrophages. Notably, all the new compounds significantly inhibited the expression of IL-6 in a dose-dependent manner.

Multivariate Analysis of Essential Oil Composition of Artemisia annua L. Collected from Different Locations in Korea.

Artemisia annua L. is distributed throughout the world and it is an important medicinal plant in Korea to treat various human diseases. Recently, A. annua has also been considered to be an effective ethnobotanical drug against COVID-19. A. annua contains an appreciable amount of essential oil with different biological properties. However, the composition of essential oils in aromatic plants can be varied depending on several factors, including geographic, genetic, ecological, etc. Hence, the present study aimed to investigate the chemical diversity of essential oils of Korean A. annua collected from different locations in Korea by multivariate analysis. For this purpose, the seeds of A. annua were collected from 112 different locations in Korea and were grown under the same environmental conditions. Except for nine individuals which decayed during the cultivation, essential oils were isolated from the aerial parts of 103 A. annua individuals (AEOs) using the steam distillation extraction method, and their chemical compositions were determined by GC-MS analysis. Furthermore, a multivariate analysis was performed to distinguish the difference between 103 individuals of A. annua based on their essential oil compositions. The yield of A. annua essential oils ranged from 0.04 to 1.09% (v/w). Based on the GC-MS data, A. annua individuals were grouped into six chemotypes such as artemisia ketone, camphor, ß-cubebene, eucalyptol, α-pinene, and ß-selinene. The multivariate analysis results revealed that Korean A. annua could be largely grouped into three clusters such as artemisia ketone, eucalyptol, and ß-selinene. Among 35 components selected for principal component analysis (PCA), PC1, PC2, and PC3 accounted for 82.55%, 8.74%, and 3.62%, respectively. Although all individuals of A. annua were cultivated under the same environmental conditions, there is an intraspecific chemical diversity that exists within Korean native species.

Metabolic engineering of the anthocyanin biosynthetic pathway in Artemisia annua and relation to the expression of the artemisinin biosynthetic pathway.

MAIN CONCLUSION: Four types of cells were engineered from Artemisia annua to produce approximately 17 anthocyanins, four of which were elucidated structurally. All of them expressed the artemisinin pathway. Artemisia annua is the only medicinal crop to produce artemisinin for the treatment of malignant malaria. Unfortunately, hundreds of thousands of people still lose their life every year due to the lack of sufficient artemisinin. Artemisinin is considered to result from the spontaneous autoxidation of dihydroartemisinic acid in the presence of reactive oxygen species (ROS) in an oxidative condition of glandular trichomes (GTs); however, whether increasing antioxidative compounds can inhibit artemisinin biosynthesis in plant cells is unknown. Anthocyanins are potent antioxidants that can remove ROS in plant cells. To date, no anthocyanins have been structurally elucidated from A. annua. In this study, we had two goals: (1) to engineer anthocyanins in A. annua cells and (2) to understand the artemisinin biosynthesis in anthocyanin-producing cells. Arabidopsis Production of Anthocyanin Pigment 1 was used to engineer four types of transgenic anthocyanin-producing A. annua (TAPA1-4) cells. Three wild-type cell types were developed as controls. TAPA1 cells produced the highest contents of total anthocyanins. LC-MS analysis detected 17 anthocyanin or anthocyanidin compounds. Crystallization, LC/MS/MS, and NMR analyses identified cyanidin, pelargonidin, one cyanin, and one pelargonin. An integrative analysis characterized that four types of TAPA cells expressed the artemisinin pathway and TAPA1 cells produced the highest artemisinin and artemisinic acid. The contents of arteannuin B were similar in seven cell types. These data showed that the engineering of anthocyanins does not eliminate the biosynthesis of artemisinin in cells. These data allow us to propose a new hypothesis that enzymes catalyze the formation of artemisinin from dihydroartemisinic acid in non-GT cells. These findings show a new platform to increase artemisinin production via non-GT cells of A. annua.

Anticancer activity of an Artemisia annua L. hydroalcoholic extract on canine osteosarcoma cell lines.

Since ancient times, Artemisia annua (A. annua) has been used as a medicinal plant in Traditional Chinese Medicine. In addition, recent studies have investigated the cytotoxic effects of A. annua extracts towards cancer cells. The leading aim of the present research is to evaluate the cytotoxic effects of an hydroalcoholic extract of A. annua on two canine osteosarcoma (OSA) cell lines, OSCA-8 and OSCA-40, focusing on the possible involvement of ferroptosis. The quantitative determination of artemisinin concentration in the extract, culture medium and OSA cells was carried out through the use of an instrumental analytical method based on liquid chromatography coupled with spectrophotometric detection and tandem mass spectrometry (LC-DAD-MS/MS). OSCA-8 and OSCA-40 were exposed to different dilutions of the extract for the EC50 calculation then the uptake of artemisinin by the cells, the effects on the cell cycle, the intracellular iron level, the cellular morphology and the lipid oxidation state were evaluated. A concentration of artemisinin of 63.8 ± 3.4 µg/mL was detected in the extract. A dose-dependent cytotoxic effect was evidenced. In OSCA-40 alterations of the cell cycle and a significantly higher intracellular iron content were observed. In both cell lines the treatment with the extract was associated with lipid peroxidation and with the appearance of a "ballooning" phenotype suggesting the activation of ferroptosis. In conclusion the A. annua idroalcoholic extract utilized in this study showed anticancer activity on canine OSA cell lines that could be useful in treating drug resistant canine OSAs.

Structural Characterization and In-Vitro Antioxidant and Immunomodulatory Activities of Polysaccharide Fractions Isolated from Artemisia annua L.

Arimisia annua L. is an important anticancer herb used in traditional Chinese medicine. The molecular basis underpinning the anticancer activity is complex and not fully understood, but the herbal polysaccharides, broadly recognised as having immunomodulatory, antioxidant and anticancer activities, are potential key active agents. To examine the functions of polysaccharides from A. annua, their immunomodulatory and antioxidant potentials were evaluated, as well as their structural characterization. The water-soluble polysaccharides (AAPs) were fractionated using size-exclusion chromatography to obtain three dominant fractions, AAP-1, AAP-2 and AAP-3, having molecular masses centered around 1684, 455 and 5.8kDa, respectively. The antioxidant potentials of the isolated polysaccharides were evaluated by measuring radical scavenging activities against DPPH● (2,2-diphenyl-1-picrylhydrazyl radical), ABTS●+ (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid radical ion), and the OH● (hydroxyl radical). AAP-1 displayed high antioxidant activities against these radicals, which were 68%, 73% and 78%, respectively. AAP-2 displayed lower scavenging activities than the other two fractions. Immunostimulatory activities of AAPs were measured using mouse macrophages. The three polysaccharide fractions displayed significant antioxidant activities and stimulated the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). AAP-1 showed significant immunostimulatory activity (16-fold increase in the production of IL-6 compared to the control and 13-fold increase in the production of TNF-α) with low toxicity (>60% cell viability at 125 µg/mL concentration). Preliminary structural characterization of the AAPs was carried out using gas chromatography (GC) and FTIR techniques. The results indicate that AAP-1 and AAP-2 are pyranose-containing polysaccharides with ß-linkages, and AAP-3 is a ß-fructofuranoside. The results suggest that these polysaccharides are potential candidates for immunotherapy and cancer treatment.

The sweet wormwood essential oil and its two major constituents are promising for a safe control measure against fall webworm.

The fall webworm, Hyphantria cunea (Drury), is a harmful polyphagous global defoliator. The major chemical components of Artemisia annua essential oil (EO) was found to contain (±)-camphor (16.42%), 1,8-cineole (6.22%), α-pinene (6%), caryophyllene (5.19%), and α-selinene (5.17%). The highest toxicity was recorded for EO of A. annua (LD50 = 305.05 µg/larva), followed by (±)-camphor (LD50 = 465.03 µg/larva) and 1,8-cineole (LD50 = 573.49 µg/larva). The binary mixtures of compounds expressed a weaker activity compared to individuals. The (±)-camphor was found to be antagonistic to 1,8-cineole. The biochemical compounds of treated larvae were also determined. The activity level of alanin and aspartate aminotransferase decreased sharply while acid and alkaline phosphatase increased. Activity of lactate dehydrogenase was significantly higher than the control group at 24 h, but decreased significantly after 48 h in all treatments. The activity of esterases were decreased in the treated larvae. The glutathione S-transferase significantly increased in all time intervals. Overall the current results suggest that the sweet wormwood (A. annua) EO and its components could be a safe and environmentally friendly approach in possible control of fall webworm (H. cunea).

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.

Identification of Growth Factors, Cytokines and Mediators Regulated by Artemisia annua L. Polyphenols (pKAL) in HCT116 Colorectal Cancer Cells: TGF-ß1 and NGF-ß Attenuate pKAL-Induced Anticancer Effects via NF-κB p65 Upregulation.

The anticancer effects of natural phytochemicals are relevant to the modulation of cytokine signaling pathways in various cancer cells with stem-like properties as well as immune cells. The aim of this study was to elucidate a novel anticancer mechanism of Artemisia annua L. polyphenols (pKAL) involved in the regulation of growth factors, cytokines and mediators in stem-like HCT116 colorectal cancer cells. Through RayBiotech human L-1000 antibody array and bioinformatics analysis, we show here that pKAL-induced anticancer effects are associated with downregulation of growth factor and cytokine signaling proteins including TGFA, FGF16, PDGFC, CCL28, CXCR3, IRF6 and SMAD1. Notably, we found that TGF-ß signaling proteins such as GDF10, ENG and TGFBR2 and well-known survival proteins such as NGF-ß, VEGFD and insulin were significantly upregulated by pKAL. Moreover, the results of hematoxylin staining, cell viability assay and Western blot analysis demonstrated that TGF-ß1 and NGF-ß attenuated pKAL-induced anticancer effects by inhibiting pKAL-induced downregulation of caspase-8, NF-κB p65 and cyclin D1. These results suggest that certain survival mediators may be activated by pKAL through the TGF-ß1 and NGF-ß signaling pathways during pKAL-induced cell death and thus, strategies to inhibit the survival signaling are inevitably required for more effective anticancer effects of pKAL.

Cytotoxicity of an Innovative Pressurised Cyclic Solid-Liquid (PCSL) Extract from Artemisia annua.

Therapeutic treatments with Artemisia annua have a long-established tradition in various diseases due to its antibacterial, antioxidant, antiviral, anti-malaria and anti-cancer effects. However, in relation to the latter, virtually all reports focused on toxic effects of A. annua extracts were obtained mostly through conventional maceration methods. In the present study, an innovative extraction procedure from A. annua, based on pressurised cyclic solid-liquid (PCSL) extraction, resulted in the production of a new phytocomplex with enhanced anti-cancer properties. This extraction procedure generated a pressure gradient due to compressions and following decompressions, allowing to directly perform the extraction without any maceration. The toxic effects of A. annua PCSL extract were tested on different cells, including three cancer cell lines. The results of this study clearly indicate that the exposure of human, murine and canine cancer cells to serial dilutions of PCSL extract resulted in higher toxicity and stronger propensity to induce apoptosis than that detected by subjecting the same cells to Artemisia extracts obtained through canonical extraction by maceration. Collected data suggest that PCSL extract of A. annua could be a promising and economic new therapeutic tool to treat human and animal tumours.

Extraction, Isolation and Characterization of Bioactive Compounds from Artemisia and Their Biological Significance: A Review.

Diverse medicinal plants such as those from the genus Artemisia have been employed globally for centuries by individuals belonging to different cultures. Universally, Artemisia species have been used to remedy various maladies that range from simple fevers to malaria. A survey conducted by the World Health Organization (WHO) demonstrated that 80% of the global population is highly reliant on herbal medicine for their primary healthcare. WHO recommends artemisinin-based combination therapies (ACT) for the treatment of global diseases such as malaria. Artemisinin is a bioactive compound derived from Artemisia annua leaves. It is a sesquiterpene endoperoxide with potent antimalarial properties. This review strives to instill natural products to chemists and others in diverse fields with a heterogeneous set of knowledge compiled from multifaceted researchers and organizations in literature. In particular, the various Artemisia species and effective extraction, isolation, and characterization methodologies are discussed in detail. An in-depth investigation into the literature reveals that divergent species of Artemisia exhibit a vast array of biological activities such as antimalarial, antitumor, and anti-inflammatory activities. There is substantial potential for bioactive compounds from Artemisia to provide significant relief from differing human ailments, but more meticulous research in this field is needed.

Phytochemical Analysis and Anti-Inflammatory Activity of Different Ethanolic Phyto-Extracts of Artemisia annua L.

Artemisia annua L. (AA) has shown for many centuries important therapeutic virtues associated with the presence of artemisinin (ART). The aim of this study was to identify and quantify ART and other secondary metabolites in ethanolic extracts of AA and evaluate the biological activity in the presence of an inflammatory stimulus. In this work, after the extraction of the aerial parts of AA with different concentrations of ethanol, ART was quantified by HPLC and HPLC-MS. In addition, anthocyanins, flavanols, flavanones, flavonols, lignans, low-molecular-weight phenolics, phenolic acids, stilbenes, and terpenes were identified and semi-quantitatively determined by UHPLC-QTOF-MS untargeted metabolomics. Finally, the viability of human neuroblastoma cells (SH-SY5Y) was evaluated in the presence of the different ethanolic extracts and in the presence of lipopolysaccharide (LPS). The results show that ART is more concentrated in AA samples extracted with 90% ethanol. Regarding the other metabolites, only the anthocyanins are more concentrated in the samples extracted with 90% ethanol. Finally, ART and all AA samples showed a protective action towards the pro-inflammatory stimulus of LPS. In particular, the anti-inflammatory effect of the leaf extract of AA with 90% ethanol was also confirmed at the molecular level since a reduction in TNF-α mRNA gene expression was observed in SH-SY5Y treated with LPS.

In vitro efficacy of artemisinin-based treatments against SARS-CoV-2.

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration-response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC50) in different cell types: 7-12 µg/mL), followed by artemether (53-98 µg/mL), A. annua extracts (83-260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.

Antibacterial Potency of Medicinal Plants including Artemisia annua and Oxalis corniculata against Multi-Drug Resistance E. coil.

Antibacterial activity of ethanolic and aqueous extracts of two medicinal plants including Oxalis corniculata (EtOc, AqOc) and Artemisia annua (EtAa, AqAa) as well as A. annua essential oil (EoAa) was investigated on multi-drug resistance (MDR) E. coli. Microdilution and agar well diffusion methods were used to determine the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) as well as the inhibition zone. The phytconstituents of these products were analyzed using Reverse-phase High- performance liquid chromatography (RP-HPLC) and gas chromatography-mass spectrometry (GC-mass). The order of bacteriostatic and bacteriocide rate of the products can be shown as follows: EoAa>AqOc>EtAa = AqAa>EtOc, but the bactericidal effect of A. annua extracts is higher than of O. corniculata based on the MIC/MBC ratio and the order is as follows: EoAa>EtAa = AqAa>EtOc>AqOc. The most potent product, i.e. EoAa with a 56.7% inhibition of all isolates, has the potential to substitute 13 used antibiotics including oxacillin, amoxicillin, ampicillin, amoxicillin-clavulanic acid, tetracycline, streptomycin, ciprofloxacin, ceftriaxone, cefazolin, cefuroxime, cefotaxime, ceftazidime and cefixime (P <0.05). Different terpenoids were detected and measured in EoAa and catechin flavonoids in extracts of both plants, quercetin in extracts of O. corniculata but it was only possible to detect chlorogenic acid polyphenol in AqAa. Due to the antibacterial activities of the studied products, more effective than some antibiotics and their edible consumption, these products can be suggested as an alternative to some antibiotics and food preservatives to fight against MDR E. coli.

Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. has been used for millennia in Southeast Asia to treat "fever". Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria. AIM OF THE STUDY: SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2. MATERIALS AND METHODS: Using Vero E6 and Calu-3 cells, we measured anti SARS-CoV-2 activity against fully infectious virus of dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as the concentrations that inhibited viral replication by 50%; CC50s were also calculated and defined as the concentrations that kill 50% of cells. RESULTS: Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 µM, 0.01-0.14 µg, and 23.4-57.4 µg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet its hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 µM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 µM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values. CONCLUSIONS: A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections.