Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographical origin: proof for the existence of chemotypes.

The time course of the levels of artemisinin, its biosynthetic precursors and the biosynthetically related sesquiterpenes was monitored during a vegetation period of Artemisia annua plants of different geographical origin. Considerable differences in contents of artemisinin and its direct precursors artemisinic acid and dihydroartemisinic acid were found between these A. annua's. For the first time the A. annua plants of different geographical origin were found to belong to different chemotypes. A chemotype with a high artemisinin level was found to have also a high dihydroartemisinic acid level but a relatively low artemisinic acid level. Reversibly, a chemotype with low levels of artemisinin and dihydroartemisinic acid contained a high artemisinic acid level. Artemisinic acid is considered to be the direct precursor of dihydroartemisinic acid in the biosynthetic pathway of artemisinin. The observed accumulation of artemisinic acid in one of the A. annua chemotypes may indicate the presence of a rate-limiting step in the biosynthetic pathway of artemisinin. The enzymatic reduction of artemisinic acid into dihydroartemisinic acid is probably a "bottle neck" in the biosynthetic pathway of artemisinin in varieties with high artemisinic acid and consequentially low artemisinin levels. After a night-frost period, the level of artemisinin was increased, in the Vietnamese A. annua plants, while the dihydroartemisinic acid level was decreased. This phenomenon is in accordance with our hypothesis that stress triggers the conversion of dihydroartemisinic acid to artemisinin. It is suggested that the presence of high levels of dihydroartemisinic acid may be an adaptation to stress conditions (e.g., night-frost), during which relatively high levels of 1O2 are formed. Dihydroartemisinic acid gives the plant protection by reacting with these reactive oxygen species yielding artemisinin as stable end-product.

Artemisinin-derived sesquiterpene lactones as potential antitumour compounds: cytotoxic action against bone marrow and tumour cells.

We determined the in vitro cytotoxic activity of the sesquiterpene lactone endoperoxide artemisinin (1) and some chemically prepared derivatives, which have been found to display cytotoxicity to cloned murine Ehrlich ascites tumour (EAT) cells and human HeLa cells and against murine bone marrow using a clonogenic assay for committed progenitor cells of the granulocyte-monocyte lineage (CFU-GM assay). Comparing artemisinin (1) to deoxyartemisinin (2), the endoperoxide group appeared to play a role in cytotoxicity to CFU-GM cells. Dimers of dihydroartemisinin and dihydrodeoxyartemisinin revealed that the stereochemistry of the ether linkage of the dimers was a more important determinant for this cytotoxic activity. The nonsymmetrical dimer of dihydroartemisinin (3) and the corresponding endoperoxide-lacking dimer of dihydrodeoxyartemisinin (5) were equally cytotoxic to CFU-GM cells. Despite the differences between both systems, it may be stated that most compounds displayed higher cytotoxicity to CFU-GM cells than to EAT cells. Dimers of dihydroartemisinin (3, 4) were selected as potential antitumour compounds and subjected to the National Cancer Institute drug-screening programme consisting of about sixty human cancer cell lines derived from nine different tissues. Both compounds displayed the same specific cytotoxicity pattern. Throughout the screen dimer 3 was more active than 4.

Stereochemistry-dependent cytotoxicity of some artemisinin derivatives.

We determined the cytotoxicity of some artemisinin derivatives against EN2 tumor cells using the MTT assay. Artemisinin (1) was clearly more cytotoxic than deoxyartemisinin (2), which lacks the endoperoxide bridge. Ether-linked dimers of dihydroartemisinin with defined stereochemistry were found to differ in the extent of cytotoxic effect on EN2 cells. The nonsymmetrical dimer (3) was more cytotoxic than the symmetrical dimer (4). The nonsymmetrical dimer of dihydrodeoxyartemisinin (5) lacking the endoperoxide bridges was also effective in the MTT assay, although less cytotoxic than 3 and 4. Similarly, the symmetrical dimer (6) was less effective than 5. Epoxides of artemisitene also showed that stereochemistry was an important factor for cytotoxicity. The results suggested that the endoperoxide bridge was not crucial for cytotoxicity to the tumor cells, but contributed to the cytotoxic effect apparently exerted by the ether linkage of the dimers. Flow cytometry data indicated that the dimers 3 and 4 caused an accumulation of the cells in the G1-phase of the cell cycle. In contrast, artemisinin (1) caused a slight increase of S-phase cells.

Structure-cytotoxicity relationships of some helenanolide-type sesquiterpene lactones.

This study deals with the cytotoxicity of helenanolide-type (10 alpha-methylpseudoguaianolide) sesquiterpene lactones. We determined the influence of substitution patterns on the toxicity of 21 helenanolides to a cloned Ehrlich ascites tumor cell line, EN2. Within a series of helenalin esters, the acetate (2) and isobutyrate (3) were more toxic than helenalin itself (1). Esters with larger acyl groups (tiglate 4 and isovalerate 5) exhibited a decreased toxicity compared with the parent alcohol (1). Similar relationships were observed between the 6,8-diastereomer of helenalin, mexicanin I (6) and its acetate (7) and isovalerate (8). In contrast, cytotoxicity within a series of 11 alpha, 13-dihydrohelenalin esters (9-12) was shown to be directly related to the size and lipophilicity of the ester side chain, dihydrohelenalin (9) being the least toxic compound in this group. Investigation of several 2,3-dihydrohelenalin derivatives (13-21) with 2 alpha-hydroxy-4-oxo- and 2 alpha,4 alpha-dihydroxy- or -O-acyl-substituted cyclopentane rings (arnifolins and chamissonolides, respectively), for which no pharmacological data have been reported so far, revealed further interesting influences of the substitution pattern on cytotoxicity. The results may be interpreted in terms of lipophilicity and steric effects on the accessibility of the reactive sites considered responsible for biological activity.

Stability of artemisinin in aqueous environments: impact on its cytotoxic action to Ehrlich ascites tumour cells.

We have recently shown artemisinin to be cytotoxic against Ehrlich ascites tumour cells. The aim of this study was to investigate the stability of this compound in the aqueous environment of the in-vitro Ehrlich ascites tumour cell system (RPMI 1640 cell culture medium supplemented with 10% foetal bovine serum (RPMI/FBS) with reference to its cytotoxic action. Literature data show that artemisinin can react with Fe2+ yielding reactive intermediates leaving artemisinin G as a major end-product. The current study showed that only excess addition of Fe2+ to artemisinin in distilled water, phosphate-buffered saline (PBS) and RPMI/FBS and incubation for 24 h led to degradation of artemisinin and yielded artemisinin G. If Fe2+ was not added results from HPLC analysis were indicative of complete recovery of artemisinin from distilled water and RPMI/FBS, with or without cells, at 37 degrees C for at least 24 h. In addition, incubation of artemisinin in RPMI/FBS with or without cells at 37 degrees C for 24 h before cytotoxicity assay did not change its cytotoxic action. On the basis of these results, we suggest that cytotoxicity to tumour cells was caused by unchanged artemisinin. This is not so for the antimalarial activity of artemisinin and derivatives, for which the presence of a pool of (haem) Fe2+ is a prerequisite resulting in free radicals or electrophilic intermediates or both.

Cytotoxicity and mode of action of aeroplysinin-1 and a related dienonefrom the sponge Aplysina aerophoba.

Aeroplysinin-1 (1) and the structurally related dienone 2 were cytotoxic to Ehrlich ascites tumor (EAT) cells and HeLa tumor cells in the microculture tetrazolium (MTT) and clonogenic assays. Both compounds are bromotyrosine derivatives, isolated from the marine spong Aplysina aerophoba. As the effective concentrations in the MTT assay were lower than in the clonogenic assay, 1 and 2 are able to cause growth inhibition as well as actual cell death in these cell lines. With an IC50 value of 8.2 microM (MTT assay, 2-h incubation, EAT cells), 1 was the more toxic compound. When the cells were depleted of glutathione by pretreatment with buthionine sulfoximine, they were significantly more sensitive toward 1 and 2 in the MTT assay. A dose-enhancement factor as high as 11.8 was found in EAT cells after 2-h incubation with 2. Using electron paramagnetic resonance we were able to measure free radical formation of 1 and 2, yielding the semiquinone structures 3 and 4, respectively, in a culture medium with tumor cells. It is concluded that free radicals are, at least in part, responsible for the cytotoxicity of 1 and 2. This conclusion is in line with expectations derived from the chemical structures of both compounds.

Progress in the research of artemisinin-related antimalarials: an update.

Artemisinin, a sesquiterpene lactone endoperoxide isolated from Artemisia annua L., and a number of its semisynthetic derivatives have shown to possess antimalarial properties. They are all effective against Plasmodium parasites that are resistant to the newest and commonly used antimalarial drugs. This article gives a survey of the literature dealing with artemisinin-related antimalarial issues that have appeared from the end of 1989 up to the beginning of 1994. A broad range of medical and pharmaceutical disciplines is covered, including phytochemical aspects like the selection of high-producing plants, analytical procedures, and plant biotechnology. Furthermore, the organic synthesis of artemisinin derivatives is discussed, as well as their mechanism of action and antimalarial activity, metabolism and pharmacokinetics, clinical studies, side-effects and toxicology, and biological activities other than antimalarial activity.

Detection of protein deposition on contact lens type polymeric hydrogels by Coomassie blue R staining.

Using an aqueous solvent of high methanol content, we have been able to extend the use of Coomassie blue R protein staining to contact lens-type acrylate hydrogels of 35-80% water content. Protein deposition on a wide range of hydrogels was compared after exposure to protein and there was good agreement between in vitro and in vivo studies to assess deposit resistance. Staining was sensitive down to a 2 micrograms lysozyme/cm2 zone extending from one polymer surface to the other, and linear with protein content up to 40 micrograms/cm2. The staining method permits unusual deposit morphologies to be easily visualized and is best used for qualitative or semiquantitative evaluation of protein deposition during the development of new polymeric materials. We propose a new classification system for protein deposition based on the degree of Coomassie blue R staining.