[Seperation and structure elucidation of alkaloids from Chinese drug buzhaye, Folium Microcos].

From the chloroform extracts of the dried Folium Microcos, four compounds were isolated by using repeated column chromatography on silica gel and recrystallization and their structures were elucidated by physicochemical properties and UV, MS and NMR, separately. They are N-methyl-6alpha-(deca-1', 3', 5'-trienyl)-3beta-methoxy-2beta-methylpiperidine, 6-(deca-1', 3', 5'-trienyl)-3-methoxy-2-methylpiperidine, N-methyl-6-(deca-1', 3', 5'-trienyl)-2, 3-dimethylpiperidine and N-methyl-6-(deca-1', 3', 5'-trienyl)-2-methylpiperidine, named as micropiperidine A, micropiperidine B, micropiperidine C and micropiperidine D, respectively. The latter three are new compounds.

Proton NMR lipid profile of Leishmania donovani promastigotes.

The proton nuclear magnetic resonance (NMR) lipid profile of Leishmania donovani was obtained in the one-dimensional and two-dimensional modes. Partial assignments of lipid classes and individual lipids were obtained purely from the proton NMR spectrum of the mixture. A more complete assignment and quantitative analysis was achieved by prior separation of the lipids by high pressure liquid chromatography (HPLC) followed by proton NMR analysis of the fractions. This work showed that proton NMR spectroscopy could facilitate lipid analysis and classification of various parasitic protozoa and serve as a basis for rapid studies of comparative lipid metabolism in parasites.

In vitro studies on the mode of action of quassinoids with activity against chloroquine-resistant Plasmodium falciparum.

Using the incorporation of [3H]isoleucine or [3H]hypoxanthine into acid-insoluble products as indices of protein- and nucleic acid-synthetic activity, respectively, it was shown that seven plant-derived quassinoids with differing chemical substitutions all inhibited protein synthesis more rapidly than nucleic acid synthesis in human erythrocytes infected with Plasmodium falciparum, in vitro. Five quassinoids (ailanthinone, bruceantin, bruceine B, glaucarubinone and holacanthone) were effective within 30 min at doses 10 times their 48 hr in vitro IC50 values. Chaparrin and glaucarubol differed in that they did not inhibit protein synthesis during the time course of these experiments when applied at 10 times their in vitro IC50 values. When these compounds were used at 209 and 114 times their respective IC50 values, their observed effects were identical to those of the other quassinoids studied. The time (t50) at which nucleic acid synthesis was reduced to 50% of control was directly proportional to the t50 for protein synthesis, suggesting that failure of nucleic acid synthesis is a consequence of inhibition of protein synthesis. It is concluded that in the malaria parasite, as in eukaryote models, quassinoids are rapid and potent inhibitors of protein synthesis, and that this is most likely due to effects upon the ribosome, rather than upon nucleic acid metabolism.

Comparison of the in vitro activities of quassinoids with activity against Plasmodium falciparum, anisomycin and some other inhibitors of eukaryotic protein synthesis.

Using the inhibition of incorporation of [3H]hypoxanthine as an index of viability of malaria parasites, it was shown that a chloroquine-sensitive strain of Plasmodium falciparum (T9-96) and a chloroquine-resistant strain (K1) did not differ in their sensitivities to the quassinoids ailanthinone, bruceantin and chaparrin. Similarly, there were no differences between the strains in their sensitivities to the protein synthesis inhibitors anisomycin, deacetylanisomycin, cephalotaxine, homoharringtonine, cycloheximide, puromycin and puromycin aminonucleoside. The IC50 values derived for ailanthinone and bruceantin, cycloheximide, homoharringtonine and puromycin were in the nanomolar range, whereas those for the anisomycins, cephalotaxine and the aminonucleoside of puromycin were micromolar or greater. Those drugs tested which contain an ester moiety (ailanthinone, bruceantin, anisomycin, homoharringtonine) were more active than the related drugs (chaparrin, deacetylanisomycin, cephalotaxine) that do not. Cross-resistance to inhibitors of protein synthesis appeared not to accompany resistance to chloroquine.

A matter of some sensitivity.

The development of sensitive chromatographic and spectroscopic techniques for the isolation and structure determination of natural products has greatly facilitated phytochemical investigations. Chemical investigations of herbarium material have resulted in the isolation of indole, quinoline and isoquinoline alkaloids from a wide number of plants. Examples of novel natural products from higher plants are given and include alkaloids, terpenoids, phenolics and quinones. Some plants investigated have not yielded the types of constituents which would have been predicted from them. Plant tissue cultures provide alternative sources of biologically active compounds and examples investigated include Cinchona, Ailanthus, Brucea and Artemisia for antiprotozoal compounds and Datura for tropane alkaloids. Biological tests are useful for bioassay-guided fractionation of plant extracts and examples of the isolation of a series of natural products with antiprotozoal and cytotoxic activities are given. Chemical and biological investigations into the traditional medicine Dragon's blood (Croton lechleri) from S. America and a Chinese prescription for the treatment of atopic eczema are described. The use of radio-ligand binding assays for the detection of a wide range of biological activities is discussed. Sensitivity of chemical and biological techniques has greatly improved prospects for finding new drug entities from plants and for investigating traditional medicines. Basic phytochemical investigations should continue to be encouraged especially in view of the rapid loss of plant species.

Phytochemistry and medicinal plants.

A truncated history of the contribution of plants to medicine is given with reference to some of the less well known ancestors of the Harborne family. Six of the top 20 prescriptions dispensed in 1996 were natural products and the clinical use of drugs such as artemisinin, etoposide and taxol has once more focussed attention on plants as sources of novel drug entities. High through-put robotic screens have been developed by industry and it is possible to carry out 50,000 tests per day in the search for compounds which have specificity of action against a key enzyme or a subset of receptors. Bioassay-guided fractionation of plant extracts linked to chromatographic separation techniques leads to the isolation of biologically active molecules whose chemical structures can readily be determined by modern spectroscopic methods. The role of academics in the search for new drugs is discussed by reference to some of our research into natural products with activity on the central nervous system, on pain receptors, the malaria parasite Plasmodium falciparum, the wound healing properties of the sap of species of Croton (Dragon's blood), and a traditional Chinese medicine used to treat eczema. Expertise in phytochemistry has been essential for this research and the strong lead shown by Professor Jeffrey Harborne is gratefully acknowledged.

Anthranilate synthase in microorganisms and plants.

The enzymology of anthranilate synthase (EC 5.4.99.6) in microorganisms and plants is reviewed. Aminoacid sequences of the enzyme subunits in different species are compared, and the mechanism of reaction is discussed.

Bioactive anthraquinone glycosides from Picramnia antidesma spp. fessonia.

A bioactivity guided fractionation, using KB cells and brine shrimp assays, of the methanolic extract from the leaves of Picramnia antidesma yielded two known anthraquinones, aloe-emodin and aloe-emodin anthrone, and three new aloe-emodin C-glycosides, named picramnioside A, picramnioside B and picramnioside C. Structures were established by spectroscopic methods (UV, IR, mass spectrometry, 1H and 13C and 2D NMR including COSY 45, HMQC, HMBC and ROESY). CD was used to establish the absolute configuration of the picramniosides.

Plant polyphenols: biologically active compounds or non-selective binders to protein?

Twenty phenolic compounds, representatives of proanthocyanidins and gallic acid/hexahydroxyldiphenic acid esters of glucose, have been assessed for their ability to inhibit binding of specific radioligands to 16 receptors. The receptors utilized were alpha 1-, alpha 2- and beta-adrenoceptors, adenosine 1, dopamine 1 and 2, muscarinic, Ca2+ channel, sulphonylureas, 5HT1, 5HT1A, 5HT2, histamine 1, benzodiazepine, opiate and Na+/K/ATPase. These phenolic compounds failed to inhibit ligands binding to 10 of the receptors under the test conditions. The most susceptible receptors to phenolic binding were beta-adrenergic, 5HT1 and opiate. Some of the compounds tested showed selectivity for a single or for two receptors. The inhibition of binding of radioligands to receptors by the phenolic compounds cannot be explained solely in terms of phenolic-protein binding. The results indicate that the removal of tannins from plant extracts prior to screening for receptor activities may result in missing biologically active compounds with specificity of action.

Anti-leishmanial activity of neolignans from Virola species and synthetic analogues.

Surinamensin, a neolignan isolated from Virola surinamensis, 3,4,5-trimethoxy-8-[2',6'-dimethoxy-4'-(E)-propenylphenoxy]-phenylpropane, a neolignan isolated from Virola pavonis, and 25 of its synthetic analogues or correlated substances with ether linkages and their corresponding C-8 sulphur and nitrogen analogues, were tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Some were active against L. donovani promastigotes at 30 microM but inactive against intracellular amastigotes. The natural neolignan from V. pavonis was active against promastigotes at 100 microM. The highest selective activity was found in those compounds with sulphur bridges. The beta-ketosulfide (3,4-dimethoxy)-8-(4'-methylthiophenoxy)-propiophenone produced 42% inhibition of L. donovani amastigotes in the liver of BALB/c mice at 100 mg/kg given once daily for five consecutive days (P>0.05).

Terpenoids from Guarea rhophalocarpa.

Four new terpenes including, two sandaracopimaradiene diterpenoids, ent-8(14),15-sandaracopimaradiene-2alpha,18-diol, and ent8(14),15-sandaracopimaradiene-2beta,18-diol, and two lanostane triterpenoids, 23-hydroxy-5alpha-lanosta 7,9(11),24-triene-3-one, and 5alpha-lanosta-7,9(11),24-triene-3alpha,23-diol, were isolated from the methanolic extract prepared from the leaves of G. rhopalocarpa together with the known steroid stigmasterol and the coumarin, scopoletin. The isolates showed weak antiprotozoal activity against Leishmania donovani promastigotes, and Trypanosoma brucei brucei blood stream trypomastigotes, and were devoid of interesting activity towards Plasmodium falciparum. The isolates did not show significant cytotoxic activity against KB cells.

Natural products as drugs.

Higher plants, many of which are threatened with extinction, are used as sources of pharmaceuticals and as ingredients of traditional medicines and are of value in new drug discovery. Artemisinin, taxol and camptothecin are examples of natural products which are undergoing clinical and commercial development. Several natural products isolated from plants used in traditional medicine have potent antiplasmodial action in vitro and represent potential sources of new antimalarial drugs. Plant biotechnology offers the possibility of improved production methods of cultivated medicinal plants as well as alternative approaches to the production of natural products for the preparation of pharmaceuticals.

Medicinal plants in tropical medicine. 1. Medicinal plants against protozoal diseases.

The active principles obtained from some of the traditional medicinal plants which are used worldwide for the treatment of protozoal diseases are reviewed. Among the active molecules considered from recent literature are bisbenzylisoquinoline, protoberberine and indole alkaloids, sesquiterpenes, quassinoids and limonoids. This review indicates that there are many antiprotozoal natural products already known which require further scientific investigation. There is a strong possibility that other antiprotozoal compounds with novel chemical structures and potentially novel modes of action will be discovered in plants.

Determination of anti-giardial activity in vitro by means of soluble formazan production.

A new microplate assay for the determination of activity in vitro against Giardia intestinalis has been developed in which viability is measured by soluble formazan production from a tetrazolium reagent. Metabolic reduction of the latter gives rise to a coloured product which may be determined directly by optical density measurement. Using this method the anti-giardial activities of more than 20 anti-microbial compounds have been assessed. The results obtained with a number of known anti-giardial agents, including metronidazole (50% inhibitory concentration [IC50] = 2.98 microM) and furazolidone (IC50 = 4.14 microM), compare well with previously published data; mepacrine, however, was found to produce a biphasic dose-response curve from which two IC50 values (0.816 and 6.83 microM) were obtained. This method provides a convenient means by which the search for new anti-giardial agents may be facilitated.

Potentiation of the antimalarial activity of qinghaosu by methoxylated flavones.

Interaction between the flavones casticin and artemetin and the antimalarial activity of chloroquine and qinghaosu (QHS) was examined using an in vitro growth assay based on [3H]hypoxanthine incorporation in synchronized cultures of a cloned line of Plasmodium falciparum. Casticin, and to a lesser extent artemetin, selectively enhanced the inhibition of growth by QHS, but had little effect on the activity of chloroquine. The findings suggest that flavones indigenous to Artemisia annua, from which QHS is isolated, might significantly alter the clinical potential of this novel antimalarial drug in the treatment of chloroquine-resistant malaria.

Cascarosides A and B.

Electron-impact and field desorption mass spectrometry, together with NMR and circular dichroism spectroscopy, were used to confirm that cascarosides A and B are C-10 isomers of 8-O-(beta-D-glucopyranosyl)barbaloin. Several batches of cascarosides A and B were prepared and oxidatively hydrolyzed to aloe-emodin. The results are discussed in relation to the assay for cascara given in the European Pharmacopoeia, 1971.

St John's wort (Hypericum perforatum L.): a review of its chemistry, pharmacology and clinical properties.

The chemical composition of St. John's wort has been well-studied. Documented pharmacological activities, including antidepressant, antiviral, and antibacterial effects, provide supporting evidence for several of the traditional uses stated for St John's wort. Many pharmacological activities appear to be attributable to hypericin and to the flavonoid constituents; hypericin is also reported to be responsible for the photosensitive reactions that have been documented for St. John's wort. With regard to the antidepressant effects of St John's wort, hyperforin, rather than hypericin as originally thought, has emerged as one of the major constituents responsible for antidepressant activity. Further research is required to determine which other constituents contribute to the antidepressant effect. Evidence from randomised controlled trials has confirmed the efficacy of St John's wort extracts over placebo in the treatment of mild-to-moderately severe depression. Other randomised controlled studies have provided some evidence that St John's wort extracts are as effective as some standard antidepressants in mild-to-moderate depression. There is still a need for further trials to assess the efficacy of St John's wort extracts, compared with that of standard antidepressants, particularly newer antidepressant agents, such as the selective serotonin reuptake inhibitors (recent comparative studies with fluoxetine and sertraline have been conducted). Also, there is a need for further studies in well-defined groups of patients, in different types of depression, and conducted over longer periods in order to determine long-term safety. St John's wort does appear to have a more favourable short-term safety profile than do standard antidepressants, a factor that is likely to be important in patients continuing to take medication. Concerns have been raised over interactions between St John's wort and certain prescribed medicines (including warfarin, ciclosporin, theophylline, digoxin, HIV protease inhibitors, anticonvulsants, selective serotonin reuptake inhibitors, triptans, oral contraceptives); advice is that patients taking these medicines should stop taking St John's wort, generally after seeking professional advice as dose adjustment of conventional treatment may be necessary.

Metabolic N-oxidation of atropine, hyoscine and the corresponding nor-alkaloids by guinea-pig liver microsomal preparations.

Incubation of guinea-pig liver microsomal preparations with atropine or hyoscine resulted in the formation of the corresponding nor-alkaloids and both isomers of atropine N-oxide from atropine and one isomer of hyoscine N-oxide from hyoscine. Separate incubations of guinea-pig liver microsomal preparations with nor-atropine and nor-hyoscine yielded the corresponding hydroxylamines. The N-oxide and hydroxylamine metabolites were identified by comparison of their t.l.c. behavior and m.x. with prepared compounds and also by their reduction to the corresponding tertiary or secondary amines.

Can ethnopharmacology contribute to the development of antimalarial agents?

The resistance of Plasmodium falciparum, the cause of tertian malaria, to synthetic antimalarials, together with the resistance of the vector mosquitoes to insecticides, has resulted in a resurgence in the use of quinine and a search for new antimalarial agents. In recent years, artemisinin, isolated from Artemisia annua which is used in Chinese traditional medicine for the treatment of malaria, has proved to be effective in the treatment of cerebral malaria due to chloroquine-resistant strains of P. falciparum. The development of in vitro tests utilising P. falciparum obtained from malaria patients means that it is possible to use bioassay guided fractionation of active extracts in order to isolate active principles. A number of laboratories throughout the world are currently investigating plants used in traditional medicine for their active constituents. Some of their results will be described and in particular two aspects of our investigations with species of Simaroubaceae and Menispermaceae will be discussed. There is every possibility that such approaches which use leads from Ethnopharmacology will result in the development of new antimalarial agents. It is vitally important to those populations relying on traditional medicines for the treatment of malaria that the safety and efficacy of such medicines be established, their active principles determined and that reproducible dosage forms be prepared and made available for use.

Ethnopharmacology and Western medicine.

The contribution of plants to western medicine is briefly considered using alkaloids as examples of one class of pharmacologically active natural product. Plants and plant products are present in 14 of the 15 therapeutic categories of pharmaceutical preparations which are currently recommended to medical practitioners in the U.K. and they form an important part of our health-care system in the western world. There is considerable scope for new drug discovery from traditional medicines which are used throughout the world and some recent developments are commented upon and conclusions are made.