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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.
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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.
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Nanoparticles are known bio elicitors in plant biotechnology. Different concentrations of ZnO, CuO and CoO nanoparticles were used for the enhanced accumulation of secondary metabolites and antioxidant activities in the callus derived from root, shoot and leaf of Artemisia annua L. Biomass of callus was somehow affected on high concentrations of Nps. Phenolic content was observed maximum (60µg) in shoot callus at 0.1mg/l of CuONps. Total antioxidant activity was observed maximum (33µg) in root callus at 0.1mg/l of ZnOnps. Total reducing power maximum (33µg) was observed in root callus at concentration of 0.05 mg/l of CoONps. Maximum radical scavenging activity was observed in shoot callus at 0.05mg/l of ZnONps. Rutin gallic acid and caffic acid were also determined in most of the samples by HPLC. The study concludes that different Nps have positive effect on the induction of secondary metabolites in A.annua plant.
Nanopartículas são bio-elicitores conhecidos em biotecnologia de plantas. Diferentes concentrações de nanopartículas de ZnO, CuO e CoO foram usadas para o acúmulo aumentado de metabólitos secundários e atividades antioxidantes no calo derivado da raiz, parte aérea e folha de Artemisiaannua L. A biomassa do calo foi de alguma forma afetada em altas concentrações de Nps. O conteúdo fenólico foi observado no máximo (60 µg) no calo da parte aérea a 0,1 mg / l de CuONps. A atividade antioxidante total foi observada no máximo (33µg) no calo radicular a 0,1mg / l de ZnOnps. O poder de redução total máximo (33µg) foi observado no calo radicular na concentração de 0,05 mg / l de CoONps. Atividade máxima de eliminação de radicais foi observada no calo da parte aérea a 0,05mg / l de ZnONps. O ácido rutina-gálico e o ácido caffic também foram determinados na maioria das amostras por HPLC. O estudo conclui que diferentes Nps têm efeito positivo na indução de metabólitos secundários na planta de A.annua.
Subject(s)
Artemisia annua , Nanoparticles , Phenols , Plant Leaves , AntioxidantsABSTRACT
Objective: To study the constituents from the dried aboveground part of Artemisia annua. Methods: The chemical constituents were isolated by various chromatographic techniques and their structures were elucidated by spectroscopic analyses and comparison of NMR data with those reported in literatures. Results: Six compounds were obtained and characterized as artelignan (1), scopoletin (2), scoparone (3), chrysosplenol B (4), jaceidin (5) and mikanin (6). Conclusion: Compound 1 is identified as a new compound named artelignan.
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Objective: Artemisia annua is the chief source of artemisinin, a potent antimalarial agent, in which other bioactive phytochemicals are also present. Due to low levels of bioactive compounds including artemisinin and flavonoids, it is necessary to increase the level of the secondary metabolites by regulating the expression of rol genes in the plant. Methods: A hybrid variety of A. annua (Hyb1209r, Shennong)developed by the Centre for Novel Agricultural Products, University of York, UK, was selected to produce transgenics of rolB and rolC genes. Genetic transformation was carried out via Agrobacterium tumefaciens GV3101 harboring rolB and rolC genes of Agrobacterium rhizogenes cloned separately. HPLC was used for the qualitative and quantitative analysis of flavonoids and artemisinin. Furthermore, thin layer chromatography (TLC)was also used to analyze artemisinin content. Results: Comparative analysis via HPLC revealed considerable enhancement in the phytochemical content of transgenic A. annua plants as compared to the wild type plant. Transgenics of rolB gene showed an average increase of 321% in rutin, 97.2% in caffeic acid, and 218.4% in myricetin, respectively. In the case of rolC gene transgenics, an average increase of 197.5% in rutin, 76.3% in caffeic acid, and 209.3% in myricetin was observed. Transgenics of rolB and rolC genes showed a 14.3%–28.6% and 2.8%–12.7% increase in artemisinin content respectively by HPLC analysis. TLC analysis showed that an average 142.2% and 110.2% enhancement in artemisinin for rolB and rolC transgenics respectively, compared with the wild type. An enhanced production of total flavonoids (average 30.2% and 25.5% increase in rolB and rolC transgenics, respectively)and total phenolics (average 34.3% and 25.8% increase in rolB and rolC transgenics, respectively)was observed as a result of transformation. Transformed A. annua plants showed improved free radical scavenging activity (average 46.5% and 29.1% increase in rolB and rolC transgenics, respectively)and total reducing power (average 32.7% and 26.4% increase in rolB and rolC transgenics, respectively)compared with untransformed plant. Conclusion: rolB and rolC genes were effective for developing A. annua plants with an enhanced level of phytochemicals.
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As a commonly used Chinese materia medica, Artemisia annua mainly contains sesquiterpenoids, diterpenes, phenylpropionic acids, coumarins, flavonoids, volatile oil, and other chemical compositions. Its pharmacological activities are anti-malaria, anti-tumor, anti-microbial, anti-parasitic, antipyretic, anti-inflammatory, immunoregulation and so on. The significantly anti-malarial activity has led to its earlier use in the treatment of malaria. In this paper, the chemical constituents and pharmacological activities of A. annua in recent years are reviewed in order to provide a reference for the further development and rational utilization of this plant resource.
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Objective: To study the chemical constituents with high polarity from the whole herb of Artemisia annua. Methods: The constituents were isolated and purified by column chromatography on Sephadex LH-20, silica gel, ODS, and preparative TLC. The structures were elucidated by the analysis of the spectroscopic data. Results: Fourteen compounds were obtained from the methanol soluble fraction of the 70% ethanol extract of A. annua by maceration at room temperature and identified as artemisiannuside A (1), scopolin (2), 2,4-dihydroxy-6-methoxyacetophenone-4-O-β-D-glucopyranoside (3), methyl caffeate (4), salicylic acid (5), ferulic acid (6), vanillic acid (7), 2,4-dihydroxy-6-methoxyacetophenone (8), homoeriodictyol (9), jaceidin (10), penduletin (11), axillarin (12), eriodictyol (13), and ®-15,16-didehydrocoriolic acid (14), respectively. Conclusion: Compound 1 is a new acylated coumarin glycoside. Compound 14 is isolated from the Artemisia genus for the first time and compound 9 is firstly isolated from A. annua.
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Artemisinin is widely used for the treatment of malaria. Artemisia annua fielded low artemisinin, which was far behind the requirements. The study on the metabolic regulation of A. annua is an effective way to improve the yield of artemisinin. Metabolic regulation of artemisinin is an effective approach to improve the yield of artemisinin. In this paper, the variety of factors that affect artemisinin content was summarized, including biosynthesis in artemisinin and its branch pathway of key enzyme genes, transcription factors, phytohormones, environmental stress, induction factors, trichomes, etc. By reviewing the research progress of these factors in metabolic regulation of artemisinin, new strategies in conventional secondary metabolic engineering were generalized based on artemisinin and its branch biosynthesis pathway and fresh metabolic engineering based on trichomes. These strategies enriched the pathway in metabolic regulation of artemisinin and put forward a new idea to cultivate transgenic strain of A. annua with good quality and high yield.
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Abstract Nanoparticles are known bio elicitors in plant biotechnology. Different concentrations of ZnO, CuO and CoO nanoparticles were used for the enhanced accumulation of secondary metabolites and antioxidant activities in the callus derived from root, shoot and leaf of Artemisia annua L. Biomass of callus was somehow affected on high concentrations of Nps. Phenolic content was observed maximum (60µg) in shoot callus at 0.1mg/l of CuONps. Total antioxidant activity was observed maximum (33µg) in root callus at 0.1mg/l of ZnOnps. Total reducing power maximum (33µg) was observed in root callus at concentration of 0.05 mg/l of CoONps. Maximum radical scavenging activity was observed in shoot callus at 0.05mg/l of ZnONps. Rutin gallic acid and caffic acid were also determined in most of the samples by HPLC. The study concludes that different Nps have positive effect on the induction of secondary metabolites in A.annua plant.
Resumo Nanopartículas são bio-elicitores conhecidos em biotecnologia de plantas. Diferentes concentrações de nanopartículas de ZnO, CuO e CoO foram usadas para o acúmulo aumentado de metabólitos secundários e atividades antioxidantes no calo derivado da raiz, parte aérea e folha de Artemisiaannua L. A biomassa do calo foi de alguma forma afetada em altas concentrações de Nps. O conteúdo fenólico foi observado no máximo (60 µg) no calo da parte aérea a 0,1 mg / l de CuONps. A atividade antioxidante total foi observada no máximo (33µg) no calo radicular a 0,1mg / l de ZnOnps. O poder de redução total máximo (33µg) foi observado no calo radicular na concentração de 0,05 mg / l de CoONps. Atividade máxima de eliminação de radicais foi observada no calo da parte aérea a 0,05mg / l de ZnONps. O ácido rutina-gálico e o ácido caffic também foram determinados na maioria das amostras por HPLC. O estudo conclui que diferentes Nps têm efeito positivo na indução de metabólitos secundários na planta de A.annua.
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Artemisinin is the first choice for malaria treatment. The plastidial MEP pathway provides 5-carbon precursors (IPP and its isomer DMAPP) for the biosynthesis of isoprenoid (including artemisinin). Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) is the last enzyme involved in the MEP pathway, which catalyzes HMBPP to form IPP and DMAPP. In this study, we isolated the full-length cDNA of HDR from Artemisia annua L. (AaHDR2) and performed functional analysis. According to gene expression analysis of AaHDR2 (GenBank:KX058541) and AaHDR1 reported ever (GenBank:ADC84348.1) by qPCR, we found that AaHDR1 and AaHDR2 had much higher expression level in trichomes than that in roots, stems, leaves and flowers. AaHDR2 had much higher expression level in flowers than that in leaves. Further, the plant hormones such as MeJA and ABA respectively up-regulated the expression level of AaHDR1 and AaHDR2 significantly, but GA3 up-regulated the expression level of AaHDR2 only. The gene expression analysis of AaHDR1 and AaHDR2 showed that AaHDR2 had a greater contribution than AaHDR1 to isoprenoid biosynthesis (including artemisinin). We used AaHDR2 for the following experiments. Bioinformatic analysis indicated that AaHDR2 belonged to the HDR family and the functional complementation assay showed that AaHDR2 did have the enzymatic function of HDR, using E. coli mutant MG1655araHDR as host cell. The subcellular localization assay showed that AaHDR2 fused with GFP at its N-terminal specifically targeted in chloroplasts. Finally, AaHDR2 was overexpressed in Arabidopsis thaliana. The AaHDR2-overexpressing plants produced the isoprenoids including chlorophyll a, chlorophyll b and carotenoids at significantly higher levels than the wild-type Arabidopsis plants. In summary, AaHDR2 might be a candidate gene for genetic improvement of the isoprenoid biosynthesis.
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The plastidial methylerythritol phosphate (MEP) pathway provides 5-carbon precursors to the biosynthesis of isoprenoid (including artemisinin). 2-C-Methyl-D-erythritol-4-phosphate cytidylyltransferase (MCT) is the third enzyme of the MEP pathway, which catalyzes 2-C-methyl-D-erythritol-4-phosphate to form 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol. The full-length MCT cDNA sequence (AaMCT) was cloned and characterized for the first time from Artemisia annua L. Analysis of tissue expression pattern revealed that AaMCT was highly expressed in glandular secretory trichome and poorly expressed in leaf, flower, root and stem. AaMCT was found to be a methyl jasmonate (MeJA)-induced genes, the expression of AaMCT was significantly increased after MeJA treatment. Subcellular localization indicated that the GFP protein fused with AaMCT was targeted specifically in chloroplasts. The transgenic plants of Arabidopsis thaliana with AaMCT overexpression exhibited a significantly increase in the content of chlorophyll a, chlorophyll b and carotenoids, demonstrating that AaMCT kinase plays an influential role in isoprenoid biosynthesis.
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Objective: To analyze the expression character, screen the core sequence of 3-hydroxy-3-methyl glutaryl coenzyme A (HMGR) promoter from Artemisia annua, and provide a theoretical basis for the efficient biosynthesis of artemisinin. Methods: In this study, β-glucoside acid enzyme (GUS) gene was used as the report gene and constructed five different length expression vectors, including the whole length of HMGR promoter, based on the cloned HMGR promoter sequences, and successfully obtained transgenic plants by the method of agrobacterium mediation transform tobacco, then the tobacco was stained by GUS. Results: The GUS staining showed that the blue color could detect in the roots, stems, leaves, flowers, and fruit pods. And the leaves in the lower part could easily get the blue color than the leaves in the top part, this may due to that the accumulation of HMGR metabolite product is more and the permeability in leaves is better in the mature leaves. The results also showed that the color showed shallow in heat and drought stresses, and with no significant difference in cold stress, the tobacco leaves with paragraph of P-HMGR-1, P-HMGR-2, and P-HMGR-3 showed blue color but did not find blue in leaves with paragraph of P-HMGR-4, P-HMGR-5. Conclusion: HMGR promoter has a stable expression during the whole growth period of tobacco. HMGR promoter was not sensitive with cold environment, but sensitive with heat and draught resistance. The key area of HMGR promoter was in P-HMGR-3. And there are some regulatory elements but no necessary part of HMGR promoter between the non overlap area of P-HMGR-3 and P-HMGR-2.
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BACKGROUND/OBJECTIVES: Artemisinin, a natural product isolated from Gaeddongssuk (artemisia annua L.) and its main active derivative, dihydroartemisinin (DHA), have long been used as antimalarial drugs. Recent studies reported that artemisinin is efficacious for curing diseases, including cancers, and for improving the immune system. Many researchers have shown the therapeutic effects of artemisinin on tumors such as breast cancer, liver cancer and kidney cancer, but there is still insufficient data regarding glioblastoma (GBM). Glioblastoma accounts for 12-15% of brain cancer, and the median survival is less than a year, despite medical treatments such as surgery, radiation therapy, and chemotherapy. In this study, we investigated the anti-cancer effects of DHA and transferrin against glioblastoma (glioblastoma multiforme, GBM). MATERIALS/METHODS: This study was performed through in vitro experiments using C6 cells. The toxicity dependence of DHA and transferrin (TF) on time and concentration was analyzed by MTT assay and cell cycle assay. Observations of cellular morphology were recorded with an optical microscope and color digital camera. The anti-cancer mechanism of DHA and TF against GBM were studied by flow cytometry with Annexin V and caspase 3/7. RESULTS: MTT assay revealed that TF enhanced the cytotoxicity of DHA against C6 cells. An Annexin V immune-precipitation assay showed that the percentages of apoptosis of cells treated with TF, DHA alone, DHA in combination with TF, and the control group were 7.15 ± 4.15%, 34.3 ± 5.15%, 66.42 ± 5.98%, and 1.2 ± 0.15%, respectively. The results of the Annexin V assay were consistent with those of the MTT assay. DHA induced apoptosis in C6 cells through DNA damage, and TF enhanced the effects of DHA. CONCLUSION: The results of this study demonstrated that DHA, the derivative of the active ingredient in Gaeddongssuk, is effective against GBM, apparently via inhibition of cancer cell proliferation by a pharmacological effect. The role of transferrin as an allosteric activator in the GBM therapeutic efficacy of DHA was also confirmed.
Subject(s)
Annexin A5 , Antimalarials , Apoptosis , Brain Neoplasms , Breast Neoplasms , Cell Cycle , Cell Proliferation , DNA Damage , Drug Therapy , Flow Cytometry , Glioblastoma , Immune System , In Vitro Techniques , Kidney Neoplasms , Liver Neoplasms , Therapeutic Uses , TransferrinABSTRACT
Objective: To obtain the indispensable key enzyme involved in the MEP pathway, the 2C-methyl-D-erythritol-2, 4-cyclodi-phosphate synthase gene (MCS) was cloned from Artemisia annua, and bioinformatic analysis, prokaryotic expression, and tissue-specific expression were conducted. Methods: According to AaMCS EST sequence, the full length of cDNA and genomic sequences were obtained by the design of specific primers, rapid-amplification of cDNA ends (RACE) and genome amplification. The coding region of MCS gene was cloned into the expression vector pET-21a (+) by gene recombination technology, the recombinant plasmid pET-21a (+)-MCS was transformed into E. coli BL21 (DE3), and the expression of recombinant protein was induced by IPTG. Semi-quantitative RT-PCR was used to detect the expression of AaMCS transcripts. Results: The AaMCS cDNA was found to be 994 bp containing an open reading frame (ORF) of 681 bp that translated into a putative peptide of 226 amino acid, The full length of MCS was 2540 bp consisting of three exons and two introns. A recombinant pET-21a (+)-MCS was constructed by genetically fusing the MCS to pET-21a (+) vector system, and was successfully expressed in E. coli BL21. The tissue expression patterns indicated that the expression level of AaMCS transcripts in flowers was higher than that in the roots and stems. Conclusion: The MCS gene is cloned from A. annua, and the stable prokaryotic expression system of pET-21a (+)-MCS is constructed. This work is helpful for investigating the activities and other physiological functions of MCS protein.
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Objective: To study the regularity of dry matter accumulation and its absorption and distribution of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) in Artemisia annua, and to provide a scientific fertilizer application for the standardized cultivation. Methods: The sampling was carried out in the different growing periods of A. annua under field condition, and the amount of dry matter and the contents of N, P, K, Ca, and Mg in all parts of the plant were determined, respectively. Results: The dry matter mainly distributed in leaves within 40 d after transplanting, and this period could be defined as seedling period. The period within 40-100 d could be defined as vegetative growth period and the dry matter mainly distributed in leaves and branches. The period within 100-180 d could be defined as reproductive growth period. And the period of 100-140 d could be defined as earlier reproductive growth period, and the dry matter mainly distributed in branches and flower buds during this time. The period of 140-180 d could be defined as later reproductive growth period, the dry matter mainly distributed in branches and seeds during this time. The accumulation of nutrition was the lowest in seedling period; but the uptake of nutrition increased in vegetative growth period, and mainly distributed in leaves and branches. Nutrition mainly distributed in leaves earlier reproductive growth period, but transferred to flower buds gradually. Nutrition mainly distributed in seeds in the later reproductive growth period. The dry matter accumulation lineally increased with the assimilation and accumulation of N, K, Mg, Ca, and P. The uptake proportion of them was 4.21:4.17:0.74:0.63:0.24 in the whole growth period. Conclusion: Low nutrition should be applied during seedling stage to controlling the loss of nutrition. During the period of 40-140 d after transplanting, A. annua grows quickly, and mineral nutrition must be supplied especially N and K. The maximum uptake period of P, Ca, and Mg is in 100-120 d, so Ca, Mg, and P fertilizers should be applied earlier or by foliage spray to promote the formation of flower buds and seeds.
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Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 μL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions.
Subject(s)
Antimalarials/pharmacology , Artemisia annua/chemistry , Plant Extracts/pharmacology , Plasmodium falciparum/drug effects , Artemisinins/pharmacology , Brazil , Chloroquine/pharmacology , Drug Synergism , Parasitic Sensitivity Tests/methods , Quinine/pharmacologyABSTRACT
Objective To obtain the indispensable key enzyme-hydroxide methyl enylamino 4-cyclodiphosphate synthase (HDS) gene involved in the MEP pathway cloned from Artemisia annua and conduct bioinformatic and functional complementation analysis. Methods To perform multiple sequence alignment for the nucleotide acid sequence of the other reported seed plants' HDS gene, to select conservative areas for designing degenerate primers, and to gain the aim gene from A. annua through homologous expanding and cDNA bottom speedily expanding technique. To perform sequence alignment using BLAST, to identify open reading frame (ORF) using ORF Finder, and to construct phylogenetic tree using neighbor joining (NJ) ways in MEGA3.0. Results The obtained HDS cDNA sequence was 2 324 bp containing a 1 854 bp ORF and encoding a 617-amino acid protein. Bioinformatic analysis showed that AaHDS was homologous with HDS derived from other seed plant species. Functional complementation analysis indicated that AaHDS could make up the short HDS function of mutant Escherichia coli MG1655 araHDS. It could make the mutant get back to upgrowth, which showed AaHDS had typical HDS gene function. Conclusion The cloning HDS gene from A. annua for the first time provides a good basis for further study on the metabolization project of artemisinin.
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OBJECTIVE: To investigate the DNA protection of polysaccharide and flavonoids in 14 kinds of Chinese herbs of Artemisia annua L. from different growing places such as Hunan, Chongqing, Beijing etc. METHODS: The fluorescence integration intensity of DNA, EB and extracts containing polysaccharide and flavonoids from 14 kinds of Artemisia annua L. were determined with EB as a sensitive fluorescence probe. Constant D was used to directly denote the degree of interaction between the drug molecules with DNA. According to the size of D, the protective action of polysaccharide and flavonoids on DNA was discussed. RESULTS: The order of interaction degree of polysaccharide in extracts with DNA was as follows; Hunan Huaihua > Beijing > Shanxi Yushe > Hunan (Xinzhou cultivation) > Shanxi Xinzhou cultivation in room > Chongqing (Xinzhou cultivation) > Hebei Fuping > Shanxi Dingxiang Nanzhuang > Chongqing > Shanxi Xinzhou Luye > Shanxi Yuxian > Shanxi Grey artemisia (Yangqu) ; The order of interaction of flavonoids in extracts with DNA through determination was as follows; Shanxi Xinzhou Luye > Hunan(Xinzhou cultivation) > Shanxi Xinzhou cultivation in room > Chongqing(Xinzhou cultivation) > Wild Artemisia(Shanxi Xinzhou) > Grey artemisia (Shanxi Yangqu) > Shanxi Dingxiang Nanzhuang > Chongqing > Hebei Fuping > Beijing > Shanxi Yushe > Iron rod Artemisia(Shanxi Xinzhou) > Shanxi Yuxian > Hunan Huaihua. The interaction of artemisia polysaccharide and flavonoids with DNA showed a definite dose-effect relationship. CONCLUSION: The extracts of Artemisia annua L. could interact with DNA, but the degree of interaction was different. The bigger the D was, the stronger the interaction was. Copyright 2012 by the Chinese Pharmaceutical Association.
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Objective: Trying to find the ways to enhance the expression of cyp71av1 gene encoding cytochrome P450 mono-oxygenase which is a key enzyme in artemisinin biosynthesis pathway accelerating the artemisinin synthesis, the promoter of cyp71av1 was isolated and characterized. Methods: 5′ untranslated regions of cyp71av1 were isolated from Artemisia annua with thermal asymmetric interlaced PCR. For functional characterization, the isolated fragments were fused with β-glucuronidase GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5′ untranslated regions of cyp71av1 in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay. Results: Two DNA fragments upstream of cyp71av1 coding sequence, a long fragment and a truncated fragment, were isolated from A. annua and introduced into N. tabacum respectively. Histochemical staining showed that two isolated fragments confered stable GUS expression in transgenic plants, and no significant difference was found between the two fragments on the GUS activity. The quantitative results also showed that the GUS activity in transgenic tobacco plants treated by dehydration, low-temperature (4 °C), and ultraviolet irradiation were 1.4 to 2.7 folds higher than that in the controls. Conclusion: It suggests that the isolated fragments has promoter activity and may be responsive to adverse environmental stresses.
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Objective: The genetic diversity of Artemisia annua germplasmic resources in natural populations of China was analyzed by SRAP marker. Methods: Taking 60 germplasm of A. annua version in main distribution areas of natural population in China as testing materials, the relative parameters were calculated by Popgene version 1.31 and Treeconw software. The systematic diagram of genetic relationship was made up by Treeconw software and clustered by UPGMA method. Results: The 232 DNA bands were amplified with 24 pairs of SRAP primer combinations, and the percentage of polymorphic bands (PPB) was 81.47%. At species level: Nei's gene diversity (H) was 0.190 5 using SRAP marker. Shannon's information index (I) was 0.300 0 by SRAPs; Among the four areas with higher level of artemisinin: PPB were 50.00%-61.21%, the average was 55.82%. He were 0.155 7-0.179 3 by SRAP marker. I were 0.237 9-0.276 6 by SRAP maker; Genetic distance were 0.643 2-0.872 7, the average was 0.754 7. Conclusion: SRAP marker is an efficient method in revealing the abundant genetic diversity of wild A. annua in China, which provides the material basis for promising germplasm of further breeding.