Heme activates artemisinin more efficiently than hemin, inorganic iron, or hemoglobin

Artemisinin derivatives appear to mediate their anti-malarial through an initial redox-mediated reaction. Heme, inorganic iron, and hemoglobin have all been implicated as the key molecules that activate artemisinins. The reactions of artemisinin with different redox forms of heme, ferrous iron, and deoxygenated and oxygenated hemoglobin were analyzed under similar in vitro conditions. Heme reacted with artemisinin much more efficiently than the other iron-containing molecules, supporting the role of redox active heme as the primary activator of artemisinin.     

Enhancement of artemisinin content in tetraploid Artemisia annua plants by modulating the expression of genes in artemisinin biosynthetic pathway

Tetraploid Artemisia annua plants were successfully inducted by using colchicine, and their ploidy was confirmed by flow cytometry. Higher stomatal length but lower frequency in tetraploids were revealed and could be considered as indicators of polyploidy. The average level of artemisinin in tetraploids was increased from 39% to 56% than that of the diploids during vegetation period, as detected by high-performance liquid chromatography-evaporative light scattering detector. Gene expressions of 10 key enzymes related to artemisinin biosynthetic pathway in different ploidy level were analyzed by semiquantitative polymerase chain reaction and significant upregulation of FPS, HMGR, and artemisinin metabolite-specific Aldh1 genes were revealed in tetraploids. Slight increased expression of ADS was also detected. Our results suggest that higher artemisinin content in tetraploid A. annua may result from the upregulated expression of some key enzyme genes related to artemisinin biosynthetic pathway.     

Ferryl-oxo heme intermediate in the antimalarial mode of action of artemisinin

Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopies have been employed to investigate the reductive cleavage of the O-O bond of the endoperoxide moiety of the antimalarial drug artemisinin and its analog trioxane alcohol by hemin dimer. We have recorded FTIR spectra in the nu(O-O) and nu(as)(Fe-O-Fe) regions of artemisinin and of the hemin dimer that show the cleavage of the endoperoxide and that of the hemin dimer, respectively. We observed similar results in the trioxane alcohol/hemin dimer reaction. The RR spectrum of the artemisinin/hemin dimer reaction displays a vibrational mode at 850 cm(-1) that shifts to 818 cm(-1) when the experiment is repeated with (18)O-O(18) endoperoxide enriched trioxane alcohol. The frequency of this vibration and the magnitude of the (18)O-O(18) isotopic shift led us to assign the 850 cm(-1) mode to the Fe(IV) = O stretching vibration of a ferryl-xoxo heme intermediate that occurs in the artemisinin/hemin dimer and trioxane alcohol/hemin reactions. These results provide the first direct characterization of the antimalarial mode of action of artemisinin and its trioxane analog, and suggest that artemisinin appears to react with heme molecules that have been incorporated into hemozoin and subsequently the heme performs cytochrome P450-type chemistry.     

Proposed reductive metabolism of artemisinin by glutathione transferases in vitro

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.     

Bioconversion of artemisinin to its nonperoxidic derivative deoxyartemisinin through suspension cultures of Withania somnifera Dunal

Biotransformation of artemisinin was investigated with two different cell lines of suspension cultures of Withania somnifera. Both cell lines exhibited potential to transform artemisinin into its nonperoxidic analogue, deoxyartemisinin, by eliminating the peroxo bridge of artemisinin. The enzyme involved in the reaction is assumed to be artemisinin peroxidase, and its activity in extracts of W. somnifera leaves was detected. Thus, the non-native cell-free extract of W. somnifera and suspension culture-mediated bioconversion can be a promising tool for further manipulation of pharmaceutical compounds.     

Modulation of artemisinin biosynthesis by elicitors,inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.     

Effect of Development Stage on the Artemisinin Content and the Sequence Characterized Amplified Region （SCAR） Marker of High-Artemisinin Yielding Strains of Artemisia annua L.

The effects of development states on the artemisinin content of clone S1 of Artemisia anuua L. grown in a greenhouse were investigated in the present study. The artemisinin content increased gradually during the phase of vegetative growth and reached its highest level at 8-9 mg/g dry weight （DW） when the S1 was 6 months old on a long day （LD） photoperiod. Treatment with 9-18 d of short day （SD） photoperiod resulted in the artemisinin content reaching and being maintained at a higher level （2.059-2.289 mg/g DW）, twofold that of control plants and plants of S1 presented at the pro-flower budding and flower-budding stages. The artemisinin content varied in different parts of the plant. The artemisinin content of leaves was higher than that of florets and branches. The artemisinin content in middle leaves was higher than that of bottom leaves, and then top leaves. Different densities of capitate glands （the storage organ of artemisinin） located on the surface of leaves, florets, and branches explained the variations in artemisinin content in these parts of the plant. The correlation coefficient between artemisinin content and density of capitate glands on the surface of different organs was 0.987. The genetic marker for artemisinin content was screened using random amplified polymorphic DNA （RAPD） and sequence characterized amplified region （SCAR） techniques. The random primer OPAl5 （5＇-TTCCGAACCC-3＇） could amplify a specific band of approximately 1 000 bp that was present in all high-artemisinin yielding strains, but absent in all low-yielding strains in three independent replications. This specific band was cloned and its sequence was analyzed. This RAPD marker was converted into a SCAR marker to obtain a more stable marker.     

Responses of Artemisia annua L. to lead and salt-induced oxidative stress

The Artemisia annua L. plants were subjected separately to NaCl (0–160 mM) and lead acetate (0–500 μM) at the age of 90 days (S1 treatment) and 120 days (S2 treatment). The treated plants were studied on 100, 130 and 160 days after sowing (DAS) in S1, and on 130 and 160 DAS in S2 treatments for lipid peroxidation rate, photosynthetic rate (Pn), chlorophyll content, artemisinin concentration and artemisinin yield in leaf samples and for total biomass accumulation. The treatments enhanced lipid peroxidation at all stages of plant growth and increased the concentration and yield of artemisinin at 100 and 130 DAS in S1 and S2, respectively, while other parameters declined at all growth stages. The magnitude of changes was greater in lead-treated than in salt-treated plants. Both treatments induced oxidative stress which might have damaged the photosynthetic apparatus resulting in a loss of chlorophyll content and a decline in photosynthetic rate, biomass accumulation and artemisinin production. The increase in artemisinin content, observed during the early phase of plant growth, might be due to a sudden conversion of its precursors (e.g. artemisinic acid/dihydroartemisinic acid) to artemisinin by activated oxygen species under oxidative stress.     

Alkylation of human hemoglobin A0 by the antimalarial drug artemisinin

In vitro, the heme cofactor of human iron(II) hemoglobin was efficiently and quickly alkylated at meso positions by the peroxide-based antimalarial drug artemisinin, leading to heme-artemisinin-derived covalent adducts. This reaction occurred in the absence of any added protease or in the presence of an excess of an extra non-heme protein, or even when artemisinin was added to hemolysed human blood. This activation of artemisinin by the heme moiety of non-digested hemoglobin clearly indicates the high affinity of this drug for heme, and its efficient alkylating ability under very mild conditions.     

Synthesis of artemisinin dimers using the Ugi reaction and their in vitro efficacy on breast cancer cells

The Ugi four-component reaction was used to prepare a series of artemisinin monomers and dimers. We found that the endoperoxide group in artemisinin remains intact during the reaction. The new artemisinin dimers showed potent anti-cancer activity against two human breast cancer cell lines, MDA-MB-231 and BT-474. One of the Ugi artemisinin dimers showed an IC₅₀ value of 12 nM when tested on BT474 cells, more than 600 times more potent than artesunate. Furthermore, the same Ugi artemisinin dimer showed a low toxicity when tested on MCF10A, a nontumorigenic cell line, resulting in a selectivity index of more than 8000.     

Simultaneous analysis of artemisinin and flavonoids of several extracts of Artemisia annua L. obtained from a commercial sample and a selected cultivar

Artemisia annua L. (Qinghao) is a promising and potent antimalarial herbal drug. This activity has been ascribed to its component artemisinin, a sesquiterpene lactone that is very effective against drug-resistant Plasmodium species with a low toxicity. Our studies indicate that several flavonoids of A. annua can promote and enhance the reaction of artemisinin with hemin. These data are in good agreement with previous investigations on the in vitro potentiation of antimalarial activity of artemisinin by such flavonoids. As a consequence, in view of a possible use of the phytocomplex rather than pure artemisinin, an HPLC/DAD/MS method is proposed for the simultaneous detection and quantification of both flavonoids and artemisinin. Different extracts, obtained from two different herbal drugs, a commercial sample and a selected cultivar, were analyzed in order to determine which solvents provide the best yields of both artemisinin and flavonoids. Qualitative and quantitative results obtained using an HPLC method are described, which will be useful for developing highly effective herbal drug preparations.     

Alkylating ability of artemisinin after Cu(I)-induced activation

The reductive activation of artemisinin by copper(I)-dipyrrin or copper(I)-(2-Clip-Phen) complexes generates an artemisinin derived alkylating species leading to covalent artemisinin–copper complex adducts. The reactivity of the peroxide function of artemisinin toward Cu(I) complexes is similar to that of Fe(II) analogues, even though the reaction is more sluggish and product distribution slightly different.     

Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L.

A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Δ11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1α was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.     

黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养,第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸,青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径,当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理,可使代谢向合成青蒿素的方向移动,青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ,黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞;经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ,黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞,比靠激素诱导提高了８倍多,与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中,缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此,在青蒿素合成的过程中通过体外调节,     

Differentially Expressed Genes during Contrasting Growth Stages of Artemisia annua for Artemisinin Content

Artemisia annua is the source of antimalarial phytomolecule, artemisinin. It is mainly produced and stored in the glandular secretory trichomes present in the leaves of the plant. Since, the artemisinin biosynthesis steps are yet to be worked out, in this investigation a microarray chip was strategized for the first time to shortlist the differentially expressing genes at a stage of plant producing highest artemisinin compared to the stage with no artemisinin. As the target of this study was to analyze differential gene expression associated with contrasting artemisinin content in planta and a genotype having zero/negligible artemisinin content was unavailable, it was decided to compare different stages of the same genotype with contrasting artemisinin content (seedling - negligible artemisinin, mature leaf - high artemisinin). The SCAR-marked artemisinin-rich (∼1.2%) Indian variety ‘CIM-Arogya’ was used in the present study to determine optimal plant stage and leaf ontogenic level for artemisinin content. A representative EST dataset from leaf trichome at the stage of maximal artemisinin biosynthesis was established. The high utility small scale custom microarray chip of A. annua containing all the significant artemisinin biosynthesis-related genes, the established EST dataset, gene sequences isolated in-house and strategically selected candidates from the A. annua Unigene database (NCBI) was employed to compare the gene expression profiles of two stages. The expression data was validated through semiquantitative and quantitative RT-PCR followed by putative annotations through bioinformatics-based approaches. Many candidates having probable role in artemisinin metabolism were identified and described with scope for further functional characterization.     

Automated calculation of docking of artemisinin to heme

We report automated molecular docking of artemisinin to heme. The effects of atomic charges, and ligand and heme structures on the docking results were investigated. Several charge schemes for both artemisinin and heme, artemisinin structures taken from various optimization methods and X-ray data, and five heme models, were employed for this purpose. The docking showed that artemisinin approaches heme by pointing O1 at the endoperoxide linkage toward the iron center, a mechanism that is controlled by steric hindrance. This result differs from that reported by Shukla et al. which suggested that heme binds with artemisinin at the O2 position. The docking results also depended on the structures of both artemisinin and heme. Moreover, the atomic charges of heme have a significant effect on the docking configurations.     

Synthesis of a novel series of artemisinin dimers with potent anticancer activity involving Sonogashira cross-coupling reaction

A series of C-10 acetal artemisinin dimers were synthesized using Sonogashira cross-coupling reaction. All these novel semisynthetic artemisinin dimers exhibited excellent growth inhibitory activity against Lung A-549 human cancer cell line.