Kurt Hostettmann

7. Bioactivity in plants: the link between phytochemistry and medicine

Abstract The development of medicinal plant research over the last 30 years is reviewed with reference to the search for new active principles. Difficulties inherent to activity guided isolation and the specific requirements of bioassays are discussed. An overview is given on currently used systems for various bioactivities, with emphasis on simple bioassays for phytochemical laboratories. The progress in medicinal plant research is illustrated by selected examples of plant derived compounds of importance as drugs or pharmacological tools.

Evaluation of quadrupole time-of-flight tandem mass spectrometry and ion-trap multiple-stage mass spectrometry for the differentiation of C-glycosidic flavonoid isomers.

LC-MS-MS is becoming a very important tool for the on-line identification of natural products in crude plant extracts. For an efficient use of this technique in the dereplication of natural products, a careful study of the parameters used to generate informative MS-MS spectra is needed. In this paper, the collision-induced dissociation (CID) MS-MS spectra of ubiquitous C-glycosidic flavonoids have been systematically studied using hybrid quadrupole time-of-flight and ion-trap (IT) mass analysers under various CID energy conditions. Efficient differentiation of flavonoid C-glycoside isomers was possible, based on the comparison of CID-MS-MS spectra of particular C-glycoside unit fragments. Striking differences between 6-C and 8-C flavonoid glycosides were especially observed in the product ion spectra of their 0.2X+ fragments ([M+H-120]+). Some guidelines for the on-line characterisation of C-glycosidic flavonoids by LC-MS-MS or LC-multiple-stage MS are given.

Antibacterial phloroglucinols and flavonoids from Hypericum brasiliense

Three known phloroglucinols (japonicine A, uliginosin A and isouliginosin B) and a new phloroglucinol (hyperbrasiol A) have been isolated from a petrol extract of the leaves and flowers of Hypericum brasiliense. Their structures were established by spectroscopic methods (UV, DCI-MS, 1H and 13CNMR, including SINEPT, HMBC, HSQC, DQFCOSY experiments). The substitution pattern of hyperbrasilol A was confirmed by X-ray crystallography. All four phloroglucinols were antibacterial against Bacillus subtilis in a TLC bioautographic assay. The flavonoids, kaempferol, luteolin, quercetin, quercitrin, isoquercitrin, hyperoside and guaijaverin, were isolated from a methanol extract of the same organs.

Liquid chromatography with ultraviolet absorbance–mass spectrometric detection and with nuclear magnetic resonance spectrometry: a powerful combination for the on-line structural investigation of plant metabolites

In order to discover new bioactive compounds from plant sources which could become new leads or new drugs, extracts should be submitted at the same time to chemical screening and to various biological or pharmacological targets. Metabolite profiling using hyphenated techniques such as LC/UV, LC/MS and more recently LC/NMR, quickly provides plenty of structural information, leading to a partial or a complete on-line de novo structure determination of the natural products of interest. As a complement to this approach, bioassays performed after LC/microfractionation of the extracts allow efficient localisation of the bioactive LC-peaks in the chromatograms. The combination of metabolite profiling and LC/bioassays provides the possibility of distinguishing between already known bioactive compounds (dereplication) and new molecules directly in crude plant extracts. Thus, the tedious isolation of compounds of low interest can be avoided and targeted isolation of new bioactive products or constituents presenting novel or unusual spectroscopic features can be undertaken. Several examples of rapid localisation of bioactive compounds, based on post-chromatographic bioautographic testing of LC/NMR microfractions and subsequent on-line identification will be illustrated. Application of hyphenated techniques for the efficient characterisation of labile constituents or constituents difficult to separate at the preparative scale will also be mentioned. The possibilities and limitations of LC/UV/NMR/MS and LC/bioassay as well as future development expected in this field will be discussed.

Review article number 6 : Plant molluscicides

Abstract A review on the application of plant molluscicides in the control of schistosomiasis is presented. Laboratory bioassays are discussed, together with criteria for activity. An attempt has been made to provide a comprehensive list of known molluscicidal natural products.

The potential of LC-NMR in phytochemical analysis

The coupling of high performance liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) is one of the most powerful methods for the separation and structural elucidation of unknown compounds in mixtures. The recent progress in pulse field gradients and solvent suppression, the improvement in probe technology, and the construction of high field magnets have given a new stimulus to this technique, which has emerged since the mid 1990s as a very efficient method for the on-line identification of organic molecules. LC-NMR thus represents a potentially interesting complementary technique to LC-UV-MS in phytochemical analysis for the detailed on-line structural analysis of natural products. Recent applications have fully demonstrated the usefulness of this technique. A brief review of the applications of LC-NMR in natural product chemistry is presented in this paper, and a summary of the basic principles and modes of operation of LC-NMR is provided. Selected examples of LC-NMR analyses of plant metabolites in crude extracts or in enriched fractions are outlined and used to illustrate the different strategies for employing the technique. The practical possibilities and limitations of LC-NMR in its application to the analysis of crude plant extracts are discussed by means of several examples. Analytical strategies involving LC multi-coupled (hyphenated) techniques for the chemical screening and dereplication of crude plant extracts are presented. An analysis of the future development of the technique with respect to its application in phytochemical analysis is also given.

Antifungal and antibacterial naphthoquinones from Newbouldia laevis roots.

From a dichloromethane extract of Newbouldia laevis roots, four new (6-hydroxydehydroiso-alpha-lapachone, 7-hydroxydehydroiso-alpha-lapachone, 5,7-dihydroxydehydroiso-alpha-lapachone and 3-hydroxy-5-methoxydehydroiso-alpha-lapachone) and six known naphthoquinones have been isolated. Their structures were established by spectroscopic methods (UV, EI mass spectrometry, 1H and 13C NMR) and that of 7-hydroxydehydroiso-alpha-lapachone was confirmed by X-ray crystallography. All naphthoquinones showed antifungal activity against Cladosporium cucumerinum and Candida albicans, and activity against the bacteria Bacillus subtilis and Escherichia coli.

An antifungal γ-pyrone and xanthones with monoamine oxidase inhibitory activity from Hypericum brasiliense

A new gamma-pyrone (hyperbrasilone), three known xanthones (1,5-dihydroxyxanthone, 5-hydroxy-1-methoxyxanthone and 6-deoxyjacareubin) and betulinic acid have been isolated from a dichloromethane extract of stems and roots of Hypericum brasiliense. Their structures were established by spectroscopic methods (UV, EI-MS, 1H and 13C NMR) and that of the gamma-pyrone was confirmed by X-ray crystallography. Hyperbrasilone and the xanthones were all antifungal against Cladosporium cucumerinum, while the three xanthones showed differing degrees of inhibition of monoamine oxidase A and B.

Antimicrobial flavonoids from Psiadia trinervia and their methylated and acetylated derivatives

Abstract From a dichloromethane extract and a hydrolysed methanolic extract from the leaves of Psiadia trinervia, 13 3-methylated flavonols have been isolated. Their structures were established by the usual spectroscopic methods (UV, EIMS, 1H and 13C NMR). Ayanin, casticin, chrysosplenol-D and 5,7,4′-trihydroxy-3,8-dimethoxyfiavone were responsible for the antifungal activity found in the preliminary screening. Chrysosplenol-D, isokaempferide, 5,7,4′- trihydroxy-3,3′-dimethoxyflavone and 5,7,4′-trihydroxy-3,8-dimethoxyflavone displayed antibacterial activity. Twenty-nine derivatives were prepared by permethylation and selective methylation of the free hydroxyl group at C-5. The antimicrobial activities of the isolates and derivatives were determined by bioautographic assays using C. cucumerinum and B. cereus as test organisms.

ANTIMICROBIAL STEROIDS FROM THE FUNGUS FOMITOPSIS PINICOLA

Phytochemical examination of the dichloromethane extract of the European fungus Fomitopsis pinicola led to the isolation of a new lanostanoid derivative identified from spectral and chemical evidences as 3 alpha-(4-carboxymethyl-3-hydroxy-3-methylbutanoyloxy)-lanosta++ +-8,24-dien-21-oic acid. In addition, seven known triterpenes, polyporenic acid C, 3 alpha-acetyloxylanosta-8,24-dien-21-oic acid, ergosta-7,22-dien-3 beta-ol,21-hydroxylanosta-8,24-dien-3-one, pinicolic acid A, trametenolic acid B and pachymic acid, were also isolated. Antimicrobial activity against Bacillus subtilis in a TLC bioassay was observed for five of the isolated steroids.