[Psychotropic drugs dispensed on the island of Bonaire]. / Psychofarmacaverstrekkingen op Bonaire.

BACKGROUND: Up till now little is known about psychiatric disorders in relation to the use of psychotropics drugs on the Dutch Antilles, with the exception of Curaçao. AIM: To map the quantity and type of psychotropics prescribed for Bonairians in 2009. METHOD: We performed a retrospective data analysis of the antipsychotics dispensed by the pharmacies on Bonaire in 2009. Our analysis focused on the benzodiazepines and related compounds, antipsychotics, lithium, antidepressants and ADHD- and narcolepsy-medication. With regard to antipsychotics and antidepressants, we also investigated 'the age distribution of the persons to whom the psychotropics were dispensed'. In addition, we mapped the frequency with which the drugs were dispensed: once only, infrequently, regularly. RESULTS: At least one psychotropic drug was delivered to 18.37% of (N=2365) Bonairians in 2009. Benzodiazepines and related compounds in particular were the most commonly dispensed drugs. CONCLUSION: One in five Bonairians received at least one prescription for psychotropic drugs in 2009.

A fast and simple GC MS method for lignan profiling in Anthriscus sylvestris and biosynthetically related Plant species.

A new GC-MS method for monitoring lignans was developed to study the variation in plants and elucidate the biosynthetic steps. A simple and fast extraction procedure for lyophilised plant material was developed, giving a lignan-rich extract. A GC-MS method was set up using an apolar WCOT fused silica column using a high temperature programme (150 degrees C to 320 degrees C at 15 degrees C min(-1)). This new GC-MS method gave a clear lignan profile of plant material. It was possible to show the large variation in the concentrations of deoxypodophyllotoxin (DOP), yatein and anhydropodorhizol (AHP) in Anthriscus sylvestris (L.) Hoffm. plants growing on different locations using cinchonidin as an internal standard. In contrast with existing GC methods for lignan analysis no derivatisation is needed. It is also possible to use this method for the detection of different classes of lignans in biosynthetically related plant species.

Deoxypodophyllotoxin 6-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum flavum involved in the biosynthesis of cytotoxic lignans.

Cell-suspension cultures of Linum flavum L. (Linaceae) synthesize and accumulate aryltetrahydronaphthalene lignans with 6-methoxypodophyllotoxin as the main component. The experimental data indicate that the biosynthesis of 6-methoxypodophyllotoxin occurs via deoxypodophyllotoxin, beta-peltatin, and beta-peltatin-A methyl ether. The enzyme catalyzing the introduction of the hydroxyl group in position 6 is deoxypodophyllotoxin 6-hydroxylase (DOP6H). The enzyme was shown to be a cytochrome P450-dependent monooxygenase by blue-light reversion of carbon monoxide inhibition and inhibition by cytochrome c. DOP6H is a membrane-bound microsomal enzyme with a pH optimum of 7.6 and a temperature optimum of 26 degrees C. Deoxypodophyllotoxin is specifically accepted with an apparent Km of 20 microM and a saturation concentration of 200 microM; 4'-demethyldeoxypodophyllotoxin is the only other tested substrate accepted for hydroxylation. DOP6H predominantly accepts NADPH as electron donor; NADH can only sustain low hydroxylation activities. A synergistic effect of NADPH and NADH is not observed. The enzyme is saturated around 250 microM NADPH; the apparent Km for this substrate is 36 microM.

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.

Isolation and identification of dihydroartemisinic acid hydroperoxide from artemisia annua: A novel biosynthetic precursor of artemisinin

Dihydroartemisinic acid hydroperoxide (2) was isolated for the first time as a natural product from the plant Artemisia annua in a 29% yield. Its structure was identified by (1)H and (13)C NMR spectroscopy. Compound 2 is known as an intermediate of the photochemical oxidation of dihydroartemisinic acid (1) leading to artemisinin (3). The presence of 1 and 2 in the plant and the conditions under which 1 can be converted into 2, which can very easily oxidize to 3, provide evidence for a nonenzymatic, photochemical conversion of 1 into 3, in vivo, in the plant.

Isolation and identification of dihydroartemisinic acid from artemisia annua and its possible role in the biosynthesis of artemisinin

Dihydroartemisinic acid (2) was isolated as a natural product from Artemisia annua in a 66% yield, and its structure was confirmed by 1H and 13C NMR spectroscopy. Compound 2 could be chemically converted to artemisinin (4) under conditions that may also be present in the living plant. The results suggest that the conversion of 2 into 4 in the living plant might be a nonenzymatic conversion.

Cytotoxicity of natural ginseng glycosides and semisynthetic analogues.

The cytotoxicity of natural glycosides from Ginseng, semisynthetic analogues and related triterpenes of the dammarane series, isolated from the leaves of the Far-East species of the genus Betula was studied in order to elucidate structure-activity relationships. Some of the compounds studied were active against the human lung carcinoma GLC4 and adenocarcinoma COLO 320 cell lines. The natural glycosides displayed the lowest cytotoxicity. The triterpenes of the dammarane series used as starting aglycones for semisynthetic derivatives were moderately cytotoxic. The dammarane triterpenes possessing keto groups and their semisynthetic glucosides were the most active compounds tested. Cytotoxic effects of the dammarane glucosides were inversely proportional both to the number of sugars attached to the aglycones and to the number of hydroxy groups of the aglycones. The type of side chain and the configuration of the hydroxy group at C-3 in aglycones did not have a significant influence on the cytotoxicity.

Seasonal variations of Artemisinin and its biosynthetic precursors in tetraploid Artemisia annua plants compared with the diploid wild-type.

Using colchicine we induced tetraploidy in Artemisia annua L. plants. During a vegetation period we monitored the time course of the levels of artemisinin, its direct precursors, the biosynthetically related sesquiterpenes and the essential oil content in the diploid (wild-type) and tetraploid A. annua plants. The averaged artemisinin level in tetraploids was 38% higher than that of the wild-type as measured over the whole vegetation period. In contrast, the averaged essential oil content of the tetraploids over this period was 32% lower. This might suggest a reciprocal correlation between artemisinin (sesquiterpenes) and the essential oil content (monoterpenes). The averaged biomass of the leaves of the tetraploid plants was lower compared to the wild-type plants. Therefore, the artemisinin yield per m2 tetraploids was decreased by 25%. Although the tetraploid plants were smaller than the wild-type plants, certain individual organs like the leaves were considerably larger, and seeds obtained by cross pollination between tetraploid A. annua plants had a spectacular size. In principle, tetraploid A. annua can be a useful starting material for a breeding program in order to obtain larger and faster growing plants, which produce higher levels of artemisinin.

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 a series of natural and semi-synthetic simple coumarins as assessed in two human tumour cell lines.

The cytotoxicity of 22 natural and semi-synthetic simple coumarins was evaluated in GLC4, a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values > 100 microM, following a continuous (96 h) incubation, most coumarins exhibited only low cytotoxicity. Several compounds, however, displayed significant potencies. As far as the structure--cytotoxicity relationship is concerned, it is conspicuous that all the potentially active natural compounds possess at least two phenolic groups in either the 6,7- or 6,8-positions. In addition, the 5-formyl-6-hydroxy substituted semi-synthetic analogue was found to be potent, reflecting the importance of at least two polar functions for high cytotoxicity.

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.

Isolation of two cytotoxic diterpenes from the fern Pteris multifida.

From aerial parts of the fern Pteris multifida Poir. (Polypodiaceae) two diterpenes, entkaurane-2 beta, 16 alpha-diol and ent-kaur-16-ene-2 beta, 15 alpha-diol, were isolated by repeated column chromatography using silica gel and silica gel impregnated with silver nitrate. The structures were confirmed by spectroscopic methods. Both compounds showed a moderate cytotoxicity to Ehrlich ascites tumour cells.

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.

Synthesis and cytotoxicity of novel lignans.

In this study the syntheses of 11 novel lignans are described. Their cytotoxicities are studied in GLC4, a human small cell lung carcinoma cell line, using the microculture tetrazolium (MTT) assay. Ten of these compounds were substituted with a menthyloxy group on the 5-position of the lactone. These compounds can easily be prepared in (novel) 'one-pot', three- or four-step syntheses. In addition, methods for controlling the stereogenic centers are described. Furthermore, five naturally occurring podophyllotoxin-related compounds were tested. The cytotoxicities of all lignan compounds, and of three non-lignan intermediates originating from the syntheses, were compared with the clinically applied anticancer agents etoposide, teniposide, and cisplatin. Most compounds showed moderate to high activities against GLC4, and two of the compounds containing a menthyloxy group showed activities comparable to the reference cytotoxic agents.

Decreased helenalin-induced cytotoxicity by flavonoids from Arnica as studied in a human lung carcinoma cell line.

The effect of the flavones apigenin, luteolin, hispidulin and eupafolin, and of the flavonols kaempferol, quercetin, 6-methoxykaempferol and patuletin from Arnica spp. on the cytotoxicity of the sesquiterpene lactone helenalin was studied in the human lung carcinoma cell line GLC4 using the microculture tetrazolium (MTT) assay. The tumour cells were exposed to the test compounds for 2h. Helenalin concentrations around its control IC(50) value, 0.5 µM, were combined with flavonoid concentrations ranging from 0.01 to 20 µM. At non-toxic concentrations, up to 10µM, all flavonoids except kaempferol significantly reduced the helenalin-induced cytotoxicity. Hispidulin and patuletin displayed their modulating effect on helenalin-induced cytotoxicity in the broadest concentration range. The strongest effect was found with 5 and 10µM hispidulin, 0.05 µM quercetin, and 1 µM patuletin, increasing the IC(50) value of helenalin with circa 40%. No dose-dependency was found in the concentration range tested.

Cytotoxicity of flavonoids and sesquiterpene lactones from Arnica species against the GLC4 and the COLO 320 cell lines.

The cytotoxicity of 21 flavonoids and 5 sesquiterpene lactones, as present in Arnica species, was studied in GLC4, a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. Following continuous incubation, most flavonoids showed moderate to low cytotoxicity, as compared with the reference compound cisplatin (IC50 = 1.1 microM against GLC4 and 2.9 microM against COLO 320). Their IC50 values varied from 17 to > 200 microM. The most toxic compound was the flavone jaceosidin. Of the sesquiterpene lactones tested, helenalin, possessing both the reactive alpha-methylene-gamma-lactone moiety and a reactive alpha,beta-unsubstituted cyclopentenone ring, displayed the strongest cytotoxicity. For 2 h exposure, its IC50 value was 0.44 microM against GLC4 and 1.0 microM against COLO 320. COLO 320 was more sensitive than GLC4 for many flavonoids (especially for flavones), but more resistant to the cytotoxic effect of the sesquiterpene lactones bearing an exocylic methylene group fused to the lactone function.

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.

Artemisinin, Related Sesquiterpenes, and Essential Oil in Artemisia annua During a Vegetation Period in Vietnam.

The active principle of ARTEMISIA ANNUA L., artemisinin, is currently being developed to a registered antimalarial drug. For production purposes, plants with a high artemisinin content are required. We followed the development of the artemisinin content and of the biosynthetically related sesquiterpenes artemisinic acid, arteannuin B, and artemisitene in A. ANNUA plants, during a vegetation period in Vietnam, where this species is indigenous. In addition, the essential oil content and composition were studied. Samples of leaves, buds, flowers, or post-bloom flowers and fruits were taken at different stages: vegetative (5, 6, and 8 months old), at mass formation of buds (9 months), at full bloom (10 months), and post-bloom (10S months). The highest artemisinin content (0.86% dry wt) was present in the leaves of 5 months-old plants. At this stage also the highest leaf yield was found. Subsequently, the artemisinin content gradually dropped. At the age of 5 months the highest artemisinic acid and arteannuin B contents, 0.16 and 0.08% dry wt, respectively, were found as well. Artemisitene was present at all stages of development, ranging from 0.002 to 0.09% dry wt. With 1.9% v/w, the essential oil content was maximal just before flowering and was composed of 55% monoterpenes and 45% sesquiterpenes. At all other stages (0.4 - 1.0% v/w oil) this ratio was ca. 30%/70%. The main components of the oil were camphor and germacrene-D.