Modeling the decomposition mechanism of artemisinin

A theoretical study on artemisinin decomposition mechanisms is reported. The calculations have been done at the HF/3-21G and B3LYP/6-31G(d,p) theoretical levels, by using 6,7,8-trioxybicyclo[3.2.2]nonane as the molecular model for artemisinin, and a hydrogen atom, modeling the single electron transfer from heme or Fe(II) in the highly acidic parasite's food vacuole, as inductor of the initial peroxide bond cleavage. All relevant stationary points have been characterized, and the appearance of the final products can be explained in a satisfactory way. Several intermediates and radicals have been found as relatively stable species, thus giving support to the current hypothesis that some of these species can be responsible for the antimalarial action of artemisinin and its derivatives.     

Sensitive and selective electrochemical detection of artemisinin based on its reaction with p-aminophenylboronic acid

The electrochemical detection of artemisinin generally requires high oxidation potential or the use of complex electrode modification. We find that artemisinin can react with p-aminophenylboronic acid to produce easily electrochemically detectable aminophenol for the first time. By making use of the new reaction, we report an alternative method to detect artemisinin through the determination of p-aminophenol. The calibration curve for the determination of artemisinin is linear in the range of 2 μmol L⁻¹ to 200 μmol L⁻¹ with the detection limit of 0.8 μmol L⁻¹, which is more sensitive than other reported electrochemical methods. The relative standard deviation is 4.83% for the determination of 10 μM artemisinin. Because the oxidation potential of p-aminophenol is around 0 V, the present method is high selective. When 40 μM, 90 μM and 140 μM of artemisinin were spiked to compound naphthoquine phosphate tablet samples, the recoveries are 107.6%, 105.4% and 101.7%, respectively. This detection strategy is attractive for the detection of artemisinin and its derivatives. The finding that artemisinin can react with aromatic boronic acid has the potential to be exploited for the development of other sensors, such as fluorescence artemisinin sensors.     

The role of C-centered radicals on the mechanism of action of artemisinin

Artemisinin is a sesquiterpene lactone with an endoperoxide function that is essential for its antimalarial activity. Endoperoxides are supposed to act on heme leading to the reduction of the peroxide bond and production of radicals, which can be responsible for killing the parasite. The geometries of artemisinin, radical anions and neutral species generated by rearrangement after reduction of the peroxide bond were fully optimized with the AM1 and PM3 semi-empirical methods, in order to characterize the intermediates formed during the process. Among the radicals calculated along the pathway for reductive decomposition of artemisinin, the secondary radical centered on carbon C₄ has the highest stability when compared to other radicals that can be achieved either by hydrogen migration to the original O-centered radical or by homolytic break of C–C bond. This suggests that the C₄-centered radical may be the species responsible for killing the parasite, as has been indicated previously in experimental studies.     

The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua L. (Qinghao)

The Chinese medicinal plant Artemisia annua L. (Qinghao) is the only known source of the sesquiterpene artemisinin (Qinghaosu), which is used in the treatment of malaria. Artemisinin is a highly oxygenated sesquiterpene, containing a unique 1,2,4-trioxane ring structure, which is responsible for the antimalarial activity of this natural product. The phytochemistry of A. annua is dominated by both sesquiterpenoids and flavonoids, as is the case for many other plants in the Asteraceae family. However, A. annua is distinguished from the other members of the family both by the very large number of natural products which have been characterised to date (almost six hundred in total, including around fifty amorphane and cadinane sesquiterpenes), and by the highly oxygenated nature of many of the terpenoidal secondary metabolites. In addition, this species also contains an unusually large number of terpene allylic hydroperoxides and endoperoxides. This observation forms the basis of a proposal that the biogenesis of many of the highly oxygenated terpene metabolites from A. annua - including artemisinin itself - may proceed by spontaneous oxidation reactions of terpene precursors, which involve these highly reactive allyllic hydroperoxides as intermediates. Although several studies of the biosynthesis of artemisinin have been reported in the literature from the 1980s and early 1990s, the collective results from these studies were rather confusing because they implied that an unfeasibly large number of different sesquiterpenes could all function as direct precursors to artemisinin (and some of the experiments also appeared to contradict one another). As a result, the complete biosynthetic pathway to artemisinin could not be stated conclusively at the time. Fortunately, studies which have been published in the last decade are now providing a clearer picture of the biosynthetic pathways in A. annua. By synthesising some of the sesquiterpene natural products which have been proposed as biogenetic precursors to artemisinin in such a way that they incorporate a stable isotopic label, and then feeding these precursors to intact A. annua plants, it has now been possible to demonstrate that dihydroartemisinic acid is a late-stage precursor to artemisinin and that the closely related secondary metabolite, artemisinic acid, is not (this approach differs from all the previous studies, which used radio-isotopically labelled precursors that were fed to a plant homogenate or a cell-free preparation). Quite remarkably, feeding experiments with labeled dihydroartemisinic acid and artemisinic acid have resulted in incorporation of label into roughly half of all the amorphane and cadinane sesquiterpenes which were already known from phytochemical studies of A. annua. These findings strongly support the hypothesis that many of the highly oxygenated sesquiterpenoids from this species arise by oxidation reactions involving allylic hydroperoxides, which seem to be such a defining feature of the chemistry of A. annua. In the particular case of artemisinin, these in vivo results are also supported by in vitro studies, demonstrating explicitly that the biosynthesis of artemisinin proceeds via the tertiary allylic hydroperoxide, which is derived from oxidation of dihydroartemisinic acid. There is some evidence that the autoxidation of dihydroartemisinic acid to this tertiary allylic hydroperoxide is a non-enzymatic process within the plant, requiring only the presence of light; and, furthermore, that the series of spontaneous rearrangement reactions which then convert this allylic hydroperoxide to the 1,2,4-trioxane ring of artemisinin are also non-enzymatic in nature.     

青蒿素研究进展

青蒿素是目前治疗疟疾的特效药。本文对自青蒿素发现以来的最新研究进展进行了比较详尽的综述。内容包括: 青蒿素的发现及历史, 青蒿素的来源, 青蒿素的全合成,青蒿素的生物合成, 青蒿素衍生物以及植物组织培养生产青蒿素。     

Fluorescent Coumarin–Artemisinin Conjugates as Mitochondria‐Targeting Theranostic Probes for Enhanced Anticancer Activities

Mitochondria‐targeting theranostic probes that enable the simultaneously reporting of and triggering of mitochondrial dysfunctions in cancer cells are highly attractive for cancer diagnosis and therapy. Three fluorescent mitochondria‐targeting theranostic probes have been developed by linking a mitochondrial dye, coumarin‐3‐carboximide, with a widely used traditional Chinese medicine, artemisinin, to kill cancer cells. Fluorescence images showed that the designed coumarin–artemisinin conjugates localized mainly in mitochondria, leading to enhanced anticancer activities over artemisinin. High cytotoxicity against cancer cells correlated with the strong ability to accumulate in mitochondria, which could efficiently increase the intracellular reactive oxygen species level and induce cell apoptosis. This study highlights the potential of using mitochondria‐targeting fluorophores to selectively trigger and directly visualize subcellular drug delivery in living cells.     

Synthesis and cytotoxicity studies of artemisinin derivatives containing lipophilic alkyl carbon chains

[reaction: see text] Cytotoxic artemisinin derivatives have been synthesized by a modular approach of "artemisinin + linker + lipophilic alkyl carbon chain". A strong correlation between the length of the carbon chains and the cytotoxicities against human hepatocellular carcinoma (HepG2) was revealed. Notably, compared with artemisinin (IC(50) = 97 microM), up to 200-fold more potent cytotoxicity (IC(50) = 0.46 microM) could be achieved by attachment of a C(14)H(29) carbon chain to artemisinin via an amide linker.     

Simple and rapid micro‐scale quantification of artemisinin in living Artemisia annua L. by improved gas chromatography with electron‐capture detection

Malaria threatens 300–500 million people and kills more than one million people annually. Artemisinin has been widely used as part of the artemisinin‐based combination therapies against malaria. However, its supply is seriously short due to very small amounts of production of artemisinin in Artemisia annua. Molecular biologic researches aimed at increasing the artemisinin yield in plant have received more and more attention and therefore corresponding quantification methods for artemisinin analysis are urgently needed. A variety of methods for determination of artemisinin have been developed but they cannot be applied when only very little plant material is available or the material should be kept live, which often occurs in molecular biologic researches. The present work developed a simple, fast and low toxic micro‐scale analysis procedure for determination of artemisinin in a single leaf or flower of living Artemisia annua using improved gas chromatography with electron‐capture detection. The recovery of >95% was achieved by vortex of a piece of fresh leaf in 1 mL ethyl acetate for 2 min at room temperature. This method provides a powerful tool for biosynthesis study of artemisnin, high‐throughput screening high‐yield clone in an early stage, or real‐time quality control of Artemisia annua crop. Copyright © 2009 John Wiley & Sons, Ltd.     

A hydrogen peroxide based access to qinghaosu (artemisinin)

Attachment of H(2)O(2) onto the highly hindered quaternary C-12a in an advanced qinghaosu (artemisinin) precursor has been achieved through a facile perhydrolysis of a spiro epoxy ring with the aid of a previously unknown molybdenum species without involving any special equipment or complicated operations. The resultant β-hydroxyhydroperoxide can be further elaborated into qinghaosu, illustrating an entry fundamentally different from the existing ones to this outstanding natural product of great importance in malaria chemotherapy.     

青蒿素及其衍生物电化学性质的研究Ⅱ: 青蒿素在 氯化血红素存在下的还原

用电化学方法研究了青蒿素与氯化血红素之间的相互作用。青蒿素在玻璃碳电极上于-1.08V处发生一个2电子转移的不可逆还原。但是,即使在低至4.0×10^-^8mol/L氯化血红素存在下,青蒿素仍可被催化还原,阴极过电位降低了600mV。配合物EDTA-Fe(Ⅲ)具有类似氯化血红素的催化性质,它降低了QHS阴极过电位590mV。在这个体系中,青蒿素在碳电极上的还原是一个借助于氯化血红素催化的还原过程,氯化血红素的存在降低了青蒿素还原活化能,促进了青蒿素的分解。文中讨论了该反应的还原机理。     

Synthesis and antimalarial activities of structurally simplified 1,2,4-trioxanes related to artemisinin

Eleven derivatives of the clinically useful, antimalarial, 1,2,4-trioxane artemisinin have been synthesized in only several steps from commercial cyclohexanones. Of these simple, tricyclic 1,2,4-trioxanes, 10 showed considerable in vitro antimalarial activity, with one being as potent as artemisinin. Some structure-activity relationship generalizations are made from this series of artemisinin analogs. Triethylsilyl hydrotrioxide (Et₃SiOOOH), prepared in situ from ozone and triethylsilane, is shown to be a mild, fastacting, and effective dioxetane-forming reagent with vinyl ethers and with a vinyl thioether on relatively small (50–100 mg) scale.     

Development of the protocol for purification of artemisinin based on combination of commercial and computationally designed adsorbents

A polymeric adsorbent for extraction of the antimalarial drug artemisinin from Artemisia annua L. was computationally designed. This polymer demonstrated a high capacity for artemisinin (120 mg g^?1), quantitative recovery (87%) and was found to be an effective material for purification of artemisinin from complex plant matrix. The artemisinin quantification was conducted using an optimised HPLC‐MS protocol, which was characterised by high precision and linearity in the concentration range between 0.05 and 2 μg mL^?1. Optimisation of the purification protocol also involved screening of commercial adsorbents for the removal of waxes and other interfering natural compounds, which inhibit the crystallisation of artemisinin. As a result of a two step‐purification protocol crystals of artemisinin were obtained, and artemisinin purity was evaluated as 75%. By performing the second stage of purification twice, the purity of artemisinin can be further improved to 99%. The developed protocol produced high‐purity artemisinin using only a few purification steps that makes it suitable for large scale industrial manufacturing process.     

Synthesis of N~(11)-anchoring biotinylated artemisinin derivatives and their preliminary biological assessment

Unique endoperoxide moiety of artemisinin and its derivatives has been considered the functionality exhibiting highly potent antimalarial and anticancer activities.To investigate the mechanisms of their biological actions,development of suitable molecular probes including biotinylated derivatives is of extreme significance.The synthesis and preliminary biological assessment of four new biotinylated artemisinin derivatives have been reported in this work.     

Synthesis of N 11-anchoring biotinylated artemisinin derivatives and their preliminary biological assessment

Unique endoperoxide moiety of artemisinin and its derivatives has been considered the functionality exhibiting highly potent antimalarial and anticancer activities. To investigate the mechanisms of their biological actions, development of suitable molecular probes including biotinylated derivatives is of extreme significance. The synthesis and preliminary biological assessment of four new biotinylated artemisinin derivatives have been reported in this work.     

Understanding the interactions between artemisinin and cyclodextrins: spectroscopic studies and molecular modeling

Artemisinin extracted from Artemisia annua L. proved to be currently, with its derivatives, the most effective drugs against simple and severe malaria, and is also effective on the chloroquine-resistant forms. The advantageous effect of some cyclodextrins (CDs) on artemisinin solubilization was demonstrated by different authors. The present work aims to confirm the effect of several CDs on artemisinin solubilization and to analyse the complexes formed between these CDs and artemisinin in order to understand their solubilization capacities. In this context, solubility studies, liquid-state NMR spectroscopy (¹H NMR studies and ROESY experiments) as well as theoretical studies (molecular modeling) have been performed. Randomly methylated-βCD, Crysmeb^? and hydroxypropylated-γCD were also found to improve the aqueous solubilization of artemisinin as well as βCD, γCD and hydroxypropylated-βCD whose effects were already demonstrated. The best solubilization ability was found with Crysmeb^?. The spectroscopic studies showed a lot of interactions between artemisinin and all the CDs studied, but mainly outside the cavity. Molecular modeling confirmed that artemisinin and CDs formed non-inclusion complexes.     

大孔吸附树脂提取青蒿素的研究

探讨了大孔吸附树脂提取青蒿素的方法。以青蒿素的吸附量,青蒿素含量,青蒿素收率和提取率为考察指标,确定大孔吸附树脂提取青蒿素的工艺条件。研究结果表明,ADS-17树脂对青蒿素的吸附量大,解吸容易,可用于提取黄花蒿中青蒿素的工业化生产,其工艺条件为:青蒿素最大吸附量为112.30mg/g,吸附流速为2BV/h,洗脱剂为90%乙醇,解吸流速为2BV/h,青蒿素含量大于99%,收率高达0.3%,提取率高达75%以上。     

Reversed-Phase High-Performance Liquid Chromatographic Determination of Artemisitene in Artemisinin

Abstract

A new, simple and selective reversed-phase HPLC assay is developed for the determination of the clinically undesirable artemisitene in the antimalarial agent artemisinin (qinghaosu). It involves the use of an internal standard (santonin) and the determination time is less than 5 minutes. Detection was accomplished using a UV detector set at 216 nm and limits were as low as 15ng for a 10μl injection. Being simple and selective this method is particularly useful for the routine analysis of artemisinin to check its purity. In addition, the method can be used for preparative scale purification of these compounds. It has been applied for the evaluation of crystalline samples of artemisinin without prior preparation.     

Direct analysis of artemisinin from Artemisia annua L. using high-performance liquid chromatography with evaporative light scattering detector, and gas chromatography with flame ionization detector

Since the isolation of artemisinin 32 years ago, it has been analyzed by different chromatographic techniques. This work compared the analysis of artemisinin from crude plant samples by GC with flame ionization detection (GC-FID) and HPLC with evaporative light scattering detector (HPLC-ELSD). Data is also presented indicating that GC is suitable for the quantification of two of artemisinin precursors (arteannuin B and artemisinic acid) if a mass spectrometer is available. GC-FID and HPLC-ELSD were chosen because of their low cost compared to other detection methods, their ease of operation compared to HPLC with electrochemical detection, and because neither require artemisinin derivatization. Both GC-FID and HPLC-ELSD provided sensitive (ng level) and reproducible results for the analysis of artemisinin from field plants, with a correlation coefficient of r(2)=0.86 between the two methods. Both methods could be easily adapted to the analysis of pharmaceutical-grade artemisinin.     

Identification of impurities in artemisinin, their behavior in high performance liquid chromatography and implications for the quality of derived anti-malarial drugs

Previous work [1] on the HPLC analysis of artemisinin tentatively identified the two impurities present above trace levels. This identification was based on LC-MS results and NMR of impurities isolated from artemisinin. In this work the impurities have been synthesized allowing verification of their identity by LC-MS. It is found that the previously suggested elution order is incorrect. A determination of relative response factors strongly impacts suggested limits on impurity levels and explains the erroneous peak assignment. The fates of the identified impurities are explored in the transformation of artemisinin to its derivative active pharmaceutical ingredients. A survey of a wide variety of artemisinin samples isolated from different geographical regions, different growing seasons, different plant backgrounds and using different extraction and purification approaches showed that artemisinin has sufficient purity for its intended use as a raw material for anti-malarial drug products.     

Decomposition mechanism of an artemisinin-type compound via hemin-electrocatalysis

The interaction between a typical derivative of artemisinin and hemin was investigated by electrochemical and spectroelectrochemical methods. This derivative can be reduced via hemin-catalysis at the glassy carbon electrode, the cathodic overpotential is decreased by ca. 650 mV. A HPLC method for separating the products of the catalytic reaction was established. They were identified either in H(2)O-CH(3)CN solution or in tetrahydrofuran, respectively. The structures of these products show that the hemin-catalyzed decomposition of an artemisinin-type compound on the glassy carbon or reticulated vitreous carbon electrode can be achieved by both electrochemical reduction and rearrangement. The conclusion that the reaction of artemisinin with hemin is a critical step in the antimalarial mechanism of artemisinin can be drawn.