Search

Phytochemical investigation of dichloromethane (DCM) extract from the stems of Paragonia pyramidata var. pyramidata L. Rich. (Bur.) resulted in the isolation and characterization of two new triterpenoids 3β,19β-dihydroxylup-12, 20(29)-diene-28-oic acid (1) and 3β,19β-dihydroxylup-12-en-28-oic acid (2), three known triterpenoids lupeol (3), spinosic acid A (4) and oleanolic acid (5), together with four known steroids (20R)-22E-24-ethylcholesta-4,22-dien-3-one (6), (20R)-24-ethylcholest-4-en-3-one (7), stigmasterol (8) and β-sitosterol (9). HREIMS, GC-MS and NMR experiments including HSQC, HMBC, COSY and NOESY were used for the determination of the structures and NMR spectral assignments. This is the first report about the chemical constituents for this plant.

The methanol extract from the stem bark of Diospyros sanza-minika as well as five norbergenin derivatives isolated from this crude extract were evaluated for their in vitro activity against Plasmodium falciparum K1 and cytotoxicity on MRC-5 cells. 4-O-(3'-methylgalloyl)norbergenin was found to be the most potent compound (IC(50) 0.6 μg/mL; CC(50) 24.7 μg/mL), followed by 4-O-galloylnorbergenin (IC(50) 3.9 μg/mL; CC(50) > 64 μg/mL) and 11-O-p-hydroxy-benzoyl-norbergenin (IC(50) 4.9 μg/mL; CC(50) > 64 μg/mL). Norbergenin and 4-O-syringoylnorbergenin were inactive (IC(50) > 32 μg/mL; CC(50) > 64 μg/mL). The antimalarial activity of the pure constituents and of the methanol extract from the stem bark of Diospyros sanza-minika is reported for the first time. The results provide interesting baseline information for the potential use of the crude extract well as some of the isolated compounds in the search for novel antimalarial compounds.

Introduction – Rhodiola rosea is a broadly used medicinal plant with largely unexplored natural variability in secondary metabolite levels. Objective – The aim of this work was to develop a non-target procedure for 1H NMR spectroscopic fingerprinting of rhizome extracts for pattern recognition analysis and identification of secondary metabolites responsible for differences in sample composition. To achieve this, plants from three different geographic areas (Swiss Alps, Finland, and Altai region in Siberia) were investigated. Results – A sample preparation procedure was developed in order to remove polymeric polyphenols as the 1H NMR analysis of low-molecular-weight metabolites was hampered by the presence of tannins. Principal component analysis disclosed tight clustering of samples according to population. PCA models based on the aromatic region of the spectra showed that the first two components reflected changes in the content of salidroside and rosavin, respectively, the rosavin content being negatively correlated to that of rhodiocyanoside A and minor aromatics. Score plots and non-parametric variance tests demonstrated population-dependent changes according to harvest time. Data consistency was assessed using score plots and box-and-whisker graphs. In addition, a procedure for presenting loadings of PCA models based on bucketed data as high-resolution plots, which are reminiscent of real 1H NMR spectra and help to identify latent biomarkers, is presented. Conclusion – This study demonstrated the usefulness of the established procedure for multivariate non-target 1H NMR metabolic profiling of Rhodiola rosea. Copyright © 2010 John Wiley & Sons, Ltd.

Three glucosylated resveratrol analogues (piceid, piceatannol glucoside, resveratroloside) were successfully isolated from the crude MeOH extract of the invasive plant species Polygonum cuspidatum by semi-preparative high-speed countercurrent chromatography with a two-phase solvent system composed of cyclohexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v). Piceid (23 mg), resveratroloside (17 mg), piceatannol glucoside (15 mg) of purities over 80% were isolated from 500 mg crude MeOH extract in one step. Subsequent passage over a SPE column was used to quickly bring their purities to over 90%. The purities were determined by HPLC analysis and their structures were elucidated by proton nuclear magnetic resonance ((1)H-NMR), HMBC, ESI-MS and HR-MS.

From the leaves of Withania adpressa, a plant endemic to Sahara of Morocco and Algeria, the novel steroidal lactone (22R)-14 alpha;,15 alpha;,17ß,20ß-tetrahydroxy-1-oxowitha-2,5,24-trien-26,22-olide (= (15S,17S)-14,15,17,20-tetrahydroxy-22,26-epoxyergosta-2,5,24-triene-1,26-dione; 1), was isolated, along with three known compounds, withanolides F (2), J (3), and oleanolic acid. Their structures were mainly solved by in-depth 1D- and 2D-NMR (including ADEQUATE) experiments, as well as by HR-MS analyses and chemical evidence.

The dichloromethane extract of the aerial parts of Blumea gariepina (Asteraceae) was shown to be active against the phytopathogenic fungus Cladosporium cucumerinum and to inhibit acetylcholinesterase. In order rapidly to identify the active principles, the crude extract was analysed by on-flow HPLC-H-1-NMR. HPLC-micro-fractionation was performed and all peaks collected were submitted to assays against C. cucumerinum and acetylcholinesterase. By this means, the biological activities could be efficiently associated with selected HPLC peaks. Complementary on-line structural data for all peaks of interest in the crude extract were obtained from HPLC-MS and from HPLC-UV with post-column addition of UV shift reagents. This chemical screening strategy with integrated bioassays permitted the on-line identification of a number of constituents and gave useful information for an efficient isolation procedure. Copyright (C) 2005 John Wiley & Sons, Ltd.

In a search for potential acetylcholinesterase (AChE) inhibitors, the ethanol extract of the bulbs of Crinum X powellii (Amaryllidaceae) was found to demonstrate a marked inhibition of this enzyme. Using a bio-guided isolation strategy, linoleic acid ethyl ester has been identified as the compound responsible for this inhibition. Three other molecules - the alkaloid hippadine, the glycosylated benzyl alcohol derivative calleryanin and 4'-hydroxy-7-methoxyflavan - were also isolated and characterized for the first time from Crinum x powellii. The structures of these compounds were elucidated by spectrometric methods including EI, D/CI mass spectrometry, H-1, C-13 and 2D NMR experiments. Linoleic acid was also found to inhibit AChE.

Investigation of the methanol extract of the roots of Gnidia involucrata (Thymelaeaceae) led to the isolation and characterization of two new 3,8"-biflavonoid diastereoisomers, named GB-4 (6a) and GB-4a (6b). Their absolute configurations were determined in mixture by on-line LC/CD measurements, which also allowed the revision of absolute configurations of the billavanoids GB-1 and GB-2, and the configurational assignment of GB-3.

LC-UV-MS analysis of the methanol extract of Jamesbrittenia fodina O. M. Hilliard (Scrophulariaceae) revealed the presence of cinnamic ester derivatives. Two isomeric pairs of these constituents were detected, but could not be isolated. In order to identify these unstable compounds, LC-H-1-NMR spectra were obtained for each individual isomer and standard NMR measurements were performed in-mixture. The spectra clearly demonstrated that the fractions consisted of mixtures of cis and trans cinnamoyl catalpol glycoside esters.

A total of 148 crude ethanol extracts from 115 plant species were tested in vitro against Gram-negative strains (Escherichia coli, Pseudomonas aeruginosa) and the Gram-positive Staphylococcus aureus and Enterococcus faecalis. Moreover, they were submitted to antifungal assays against Candida albicans and Cladosporium cucumerinum, a human and a plant pathogenic microorganism, respectively, known to be good indicators of antifungal activity. No activity was detected against the Gram-negative bacteria, while 14.8% and 10.8% of the extracts showed Gram-positive bactericidal or bacteriostatic effects on S. aureus and E. faecalis, respectively. An antifungal activity was observed with 15 extracts (10.1%). Two species were particularly active against the fungi: Dioscorea minutiflora and Erythrina vogelii. The young tubers of D. minutiflora contain metabolites with a specific effect on fungi and were not active against the bacteria. On the other hand, E. vogelii was highly effective against the Gram-positive bacteria and the fungi.

The stem bark CH2Cl2 extract of Ravensara crassifolia showed antifungal activity against the phytopathogenic fungus Cladosporium cucumerinum in a bioautographic TLC assay. Activity-guided fractionation afforded two new alpha -pyrones : (6S)-5,6-dihydro-6-[(2R)-2-hydroxy-6-phenylhexyl]-2H-pyran-2-one (1) and (6R)-6-[(4R,6R)-4,6-dihydroxy-10-phenyldec-1-enyl]-5,6-dihydro-2H-pyran-2-one (2). Their structures and absolute configurations were established by NMR spectroscopy, chemical methods, and CD spectroscopy. The antifungal activity against C. cucumerinum was determined for both compounds.

A total of 78 different extracts from 20 medicinal plants belonging to 14 plant families from Mali were tested for their antifungal, larvicidal, molluscicidal, antioxidant and radical scavenging activities. Dichloromethane, methanol, water and ethanol extracts were used. TLC autobiography for antifungal activity was run with Cladosporium cucumerinum and Candida albicans. Extracts were also tested on the larvae of the mosquitoes Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Molluscicidal activities were established with the snails Biomphalaria glabrata, Biomphalaria pfeifferi and Bulinus truncatus. beta-Carotene and DPPH solutions sprayed on TLC plates were used for antioxidant and radical scavenging assays. Of the extracts investigated, 20% were antioxidant and radical scavengers, 19% fungicidal, 30% were larvicidal and 11% were molluscicidal. Three of the plant extracts, from Cussonia barteri (Araliaceae), Glinus oppositifolius (Aizoaceae) and Lannea velutina (Anacardiaceae) gave positive responses in all four tests.

Fifty-nine xanthones (= 9H-xanthen-9-ones) of natural or synthetic origin were investigated for their inhibitory activity toward monoamine-oxidase A (MAO-A) and MAO-B. The compounds demonstrated reversible. time-independent activities, with selectivity toward MAO-A. The most active inhibitor had an IC50 of 40 nM. Electron absorption spectroscopy revealed the formation of a 1:1 charge-transfer complex between lumiflavine and xanthones. 3D-QSAR Studies according to the CoMFA/GOLPE procedure provided information on the relationship between steric and electrostatic fields and MAO-A inhibition. The ALMOND procedure yielded additional topographical information on structural factors favoring activity

Two new 'quinone methide' diterpenes with a cassane skeleton were isolated from the root bark of Bobgunnia madagascariensis (DESV.) J.K. KIRKBR. & WIERSEMA (Leguminosae). The two compounds occurred as a diastereoisomer mixture 1/2, Their structure was established as (4R,6aS,10aS)- and (4S,6aS,10aS)-1,4,6a,7,8,9,10,10a-octahydro-4,11-dihydroxy-7,7,10a-trimethyl-2H-phenanthro[1,2-c]pyran-6,12-dione by spectroscopic methods and by comparison with the data obtained for another 'quinone methide' diterpene isolated previously from the same plant and identified by single-crystal X-ray analysis. H-1- and C-13-NMR signals were assigned by extensive in-mixture 2D correlation experiments, and H-1-NMR spectra of the separated diastereoisomers 1 and 2 were recorded by on-now LC/NMR. The interconversion of 1 and 2 was followed by repeated stop-flow LC/NMR experiments over a two-hour period, Compounds 1 and 2 showed moderate antifungal properties towards human pathogenic fungi, in particular the yeast Candida albicans.

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 held 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-MMR 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 photochemical analysis is also given. Copyright (C) 2001 John Wiley & Sons, Ltd.

Two minor saponins obtained from the methanolic extract of the leaves of Ilex paraguariensis have been characterised by 13C-NMR, 1H-NMR, API-MS and chemical hydrolysis as oleanolic acid-3-O-(beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl)-(28-->1)- beta-D-glucopyranosyl ester (guaiacin B) and oleanolic acid-3-O-(beta-D-glucopyranosyl-(1-->3)-(alpha-L-rhamnopyranosyl- (1-->2))-alpha-L-arabinopyranosyl)-(28-->1)-beta-D-glucopyranosyl ester (nudicaucin C). Both are isomeric forms of the known matesaponins 1 (MSP 1) and 2 (MSP 2) and differ only by the nature of the aglycone: they have oleanolic acid instead of ursolic acid, as found in the matesaponins. These minor saponins have not been fully separated from their major isomers MSP 1 and 2 and were characterised by in-mixture NMR analysis, LC-MS and LC-MSn experiments.

A liquid chromatographic/mass spectrometric (MS) method involving electrospray ionization together with up-front collision-induced dissociation, tandem MS or MSn fragmentation has been developed for rapid on-line characterization of triterpene glycosides. Sensitive detection was obtained by post-column addition of alkaline buffer, providing intense deprotonated molecular ions. The experimental method has provided information on the sugar sequence and aglycone of each saponin within a crude plant extract. The method has been applied for the rapid estimation of saponin content in methanolic and aqueous extracts of dried berries of Phytolacca dodecandra (Phytolaccaceae). Copyright © 1999 John Wiley & Sons, Ltd.

Five new compounds, the sesquiterpene lactones 1 and 2, the 2H-pyran-2-one 9, the flavone glycoside 10, and the organic diacid 11 were isolated from the CH2Cl2 extract (1 and 2) and from the BuOH part of the MeOH extract (9 – 11) of the aerial parts of Inulanthera nudaKallersjo (Asteraceae). The structures were established by spectroscopic methods, including UV, IR, EI-MS, D/CI-MS and TSP-MS, 1H- and 13C-NMR as well as two-dimensional NMR techniques. The sesquiterpene lactones were identified as 1α,4β,10β-trihydroxyguaia-2,11(13)-dieno-12,6α-lactone (1) and 4β,10β-dihydroxy-1α-methoxyguaia-2,11(13)-dieno-12,6α-lactone (2), while 9 was found to be 4-(β-D-glucopyranosyloxy)-6-methyl-2H-pyran-2-one, 10 was determined as luteolin 7-(2″-O-acetyl-β-D-glucuronoside) and 11 as (Z)-2-(4-hydroxybenzyl)butenedioic acid. The new compounds were accompanied by six more compounds (3 – 8) from the CH2Cl2 extract and ten compounds from the BuOH extract (including 12 – 16). Compounds 3 and 4 were identified as (E)- and (Z)-O-acetyldendranthemenol, while the structures of 5 – 8 were elucidated as the flavones salvigenin, 5-hydroxy-3′,4′,6,7-tetramethoxyflavone, eupatorin, and cirsilineol, respectively, from their spectroscopic data (EI- and D/CI-MS, UV, 1H- and 13C-NMR). Compounds 12 – 16 were determined as scopolin, maltol 3-glucoside, and the flavone glycosides luteolin 7-glucuronide, apigenin 7-glucuronide, and scutellarein 7-glucuronide based on spectral evidences (D/CI- and TSP-MS, UV, 1H- and 13C-NMR). The additional five compounds from the BuOH extract were identified on-line by HPLC-MS as chlorogenic acid, 3,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, and 3,4-dicaffeoylisoquinic acid. The identification was based on comparison of retention times and UV and mass spectra with authentic samples.

A new acylated dimeric secoiridoid glycoside, seemannoside A (2), has been isolated from the aerial parts of Lisianthius seemannii (GRISEB) O. KUNTZE (Gentianaceae). The structure was established by spectroscopic analysis (UV, MS, 1H- and 13C-NMR, and 2D-NMR experiments) and chemical reactions as (E-4′-O-p-coumaroyl)lisianthioside. The structure of the (Z)-isomer (seemannoside B, 3), also present in the plant, was confirmed by LC/UV/1H-NMR analysis. The active principle, 6, responsible for the antifungal activity of the apolar extract against Cladosporium cucumerinum, has been isolated. Its structure has been established by NMR spectroscopy and X-ray crystallographic analysis as a rare type of aglycone monoterpene dimer.

The CH2Cl2 extract of Monotes engleri Gilg. (Dipterocarpaceae) showed antifungal activity against the yeast Candida albicans in our bioautographic TLC assays. After a first fractionation of the crude extract, the bioactivity was located in one of the fractions. To perform an efficient targeted isolation of the active compounds, LC/UV/MS and LC/UV/NMR analyses of the crude extract and the active fraction were performed. LC/UV/, LC/MS, and LC/NMR data (1D and 2D) allowed the identification of 1 as (2S)-2,3-dihydro-5,7-dihydroxy–{3-hydroxy-4-[(3-methylbut-2-enyl) oxy]phenyl}-4H-1-benzopyran-4-one, a new prenylated flavanone, named monoteson A. Subsequent isolation of 1 has permitted the determination of its absolute configuration on the basis of CD measurements. Theree other prenylated flavanoes 2–4 were isolated from the same extract. Compound 3 was identified as 2- (3, 5-dihydroxyphenyl) -2,3-dihydro-5, 7-dihydroxy-6, 8-bis (3-methylbut-2-enyl)-4 H-1-benzopyran-4-one, another new natural product, named monotesone B. The structures of 2 and 4 were established as selinone and lonchocarpol A, respectively. The antifungal activity against Candida albicans was determined for all compounds.

Plants provide a rich source of novel biologically active compounds. Biological and chemical screenings are complementary approaches for the rapid detection and isolation of interesting new plant constituents. Biological screening followed by activity-guided fractionation has been used successfully in our laboratories for the discovery of antifungal, larvicidal and molluscicidal compounds. High performance liquid chromatography (HPLC) coupled to UV spectroscopy (LC/UV), mass spectrometry (LC/MS) and nuclear magnetic resonance (LC/ NMR) has proved to be highly efficient for the chemical screening of crude plant extracts. In particular LC/MS and LC/MS/MS used with different ionisation techniques such as thermospray (TSP), continuous flow-FAB (CF-FAB) and electrospray (ES) have proved to be very efficient for the early recognition of molluscicidal saponins in Swartzia madagascariensis and Phytolacca dodecandra. The combination of LC/UV/NMR/MS was of great value for the investigation of polyphenols and bitter principles in Gentianaceae species. Among other examples, LC/NMR analysis of the antifungal crude extract of the African plant Swertia calycina is presented.

Boophiline (1), a new sterol amide was isolated from the cattle tick Boophilus microplus (Ixodidae). The structure was assigned as N-[3-(sulfooxy)-25ξcholest-5-en-26-oyl]-L-isoleucine by detailed 2D NMR investigations in conjunction with FAB mass spectrometry and acidic hydrolyses. Complete assignment of the diastereotopic methylene protons of the ring system could be deduced from the NMR data. In agar dilution assays, 1 exhibited antifungal properties against Cladosporium cucumerinum and antibacterial activity against Bacillus subtilis and Escherichia coli.

Four new iridoid glucosides 1–4, named blumeosides A–D, were isolated from the methanolic stem-bark extract of Fagraea blumei G. DON. (Loganiaceae). They were accompanied by the benzyl-alcohol derivative di-O-methylcrenatin (5) and the flavone C-glucoside swertisin (6). The structures of 1–4 were established by spectroscopic methods, including FAB-MS, and 1H- and 13C-NMR, and by alkaline hydrolysis. Blumeosides A (1) and C (3) are 10-O-(2,5-dihydroxytercphthalo) adoxosidic acid and 10-O-(2-hydroxyterephthalo)adoxosidic acid, respectively. In blumeosides B (4) and D (2), both carboxylic groups of the terephthalic-acid moiety are esterified by adoxosidic-acid units, Blumeosides A–D (1–4) inhibited bleaching of crocin induced by alkoxyl radicals. Blumeosides A (1) and D (2) also demonstrated scavenging properties towards the 2,2-diphenyl-1-picryl-hvdrazvl (CDPPH) radical in TLC autographic and spectrophotometric assays.

Phytochemical investigation of Parinari capensis (Chrysobalanaceae) led to the isolation of three diterpene lactones 1–3. The structures of these compounds have been established by NMR spectroscopy and X-ray crystal-structure analysis. Addition of bromine onto the exocyclic methylidene group of 1 yielded the brominated derivative 1-Br. X-Ray analysis of 1-Br established unambiguously the absolute configuration, and 1 was then identified as the (4R, 9R)-10-hydroxy-13-methoxy-9-methyl-15-oxo-20-norkaur-16-en-18-oic acid γ-lactone. Diterpenes 2 and 3 were identified as the C(13)-demethoxylated and the C(13)-demethylated derivatives of diterpene 1, respectively. Antifungal activity against Cladosporium cucumerinum was determined for 1–3.

The chromonocoumarins frutinones A and B and lignans eudesmin, magnolin, yangambin and kobusin were isolated from lipophilic extract of P. gazensis. This species and two other related Polygala species were subsequently analysed by thermospray liquid chromatography–mass spectrometry and high pressure liquid chromatography coupled with photodiode-array detection. Frutinone A was also found in the roots and aerial parts of P. teretifolia and P. fruticosa, while the presence of frutinone B was limited principally to the roots. Lignans were present in the aerial parts of P. teretifolia. Frutinone A showed antifungal activity against Cladosporium cucumerinum and Candida albicans while eudesmin and kobusin were antifungal against Cladosporium cucumerinum only. © 1997 by John Wiley & Sons, Ltd.

A number of natural polyphenols (chlorogenic acid (9), cordigol (11), cordigone (12), danthrone (1), 1,5-dihydroxy-3-methoxyxanthone (2), eriosematin (7), flemichin D (8), frutinone A (6), mangiferin (4), quercetin (5), 1,3,6,7-tetrahydroxyxanthone (3) and verbascoside (10)) were investigated for their redox properties using cyclic voltammetry. The antioxidant properties of these compounds were also examined in two models, namely lipid peroxidation in rat synaptosomes and AAPH-mediated oxidation of serum albumin. Compounds with a catechol group (9, 4, 5, 3 and 10) were oxidized below 0.4 V and inhibited lipid peroxidation with IC50 values between 2 and 8 mu M. Compounds having one or more isolated phenolic groups and showing an oxidation potential between 0.45 and 0.8 V (11, 12 and 8) inhibited lipid peroxidation with IC50 between 7 and 9 mu M, except 2 (0.45 V), danthrone (0.96 V) and eriosematin which showed no or modest antioxidant activity. Some of the investigated compounds also protected albumin from oxidation, but no structure-activity relationship was apparent, suggesting that other factors beside redox potential influence this activity

Chemical screening of the secondary metabolites from Halenia corniculata L. (CORNAZ), by LC/UV,LC/TSP-MS (thermospray), and LC/ES-MS (electrospray) was used for the targeted isolation of corniculoside (1), a new biosidic ester secoiridoid. The structure was established as 7-β-[(E)-4′-O-(β-D-glucopyranosyl)caffeoyloxy]-sweroside by 1D- and 2D-NMR, LC/UV, LC/MS, and FAB-MS data, in combination with chemical reactions.

A lipophilic crude extract of Valeriana capense exhibited antifungal activity against the plant pathogenic fungus Cladosporium cucumerinum. Activity-guided fractionation of the extract afforded valtrate (1). Isovaltrate (2), didrovaltrate (3), chlorovaltrate (4), valtrate hydrine B4 (5), homovaltrate (6), dihomovaltrate (7), homodidrovaltrate (8), diavaltrate (9) and isovaleroxyhydroxydidrovaltrate (10) were subsequently isolated. The activities of compounds 1, 3 and 6–10 against different fungi were determined. Quantitation of 1 in roots and aerial parts of V. capense has been achieved. Investigation of the valepotriate content in crude extracts of some European Valerianaceae species was performed using thermospray liquid chromatography (TSP LC)-mass spectrometry (MS) and TSP LC–tandem MS. Lipophilic whole extracts of V. pyrenaica, V. celtica, V. globulariifolia, V. supina and the roots of Valerianella locusta have been analysed using valepotriates 1–10 as reference compounds. Several other valepotriates have been detected in the extracts and their structures tentatively assigned. Chlorovaltrate (4) was found not to be an artefact formed during the extraction procedure when extracts of the roots of Valeriana capense and V. officinalis obtained with chlorinated and non-chlorinated solvents were investigated by means of TSP LC–MS.

The novel flavonol laricitrin 3-O-(6″-acetyl)-glucoside has been characterized in needles of Norway spruce (Picea abies) together with quercetin 3-O-(6″-acetyl)-glucoside, the 3-O-glucoside and 3-O-rutinoside of syringetin, and 3′-O-caffeoyl-quinic acid, all detected for the first time from this species. Further, taxifolin, coniferin and Z-astringin are unreported together with 24 other phenolics of different classes. Juvenile needles contain significant levels of several flavonols, whereas stilbenes and simple phenolics are predominant in mature needles. Only gallocatechin, catechin, p-hydroxyacetophenone and taxifolin appear without sugar units. Characterization of the compounds was performed using high performance liquid chromatographic retention times, masses from thermospray ionisation, and ultraviolet spectral data.

The antiinflammatory activity of different extracts from 16 tropical plant species belonging to 15 different families has been determined. They were administered topically on TPA-induced mouse ear oedema and orally on carrageenan-induced mouse paw oedema. In the latter, extracts from Diospyros leucomelas, Erythrospermum monticolumn, Ficus polita, Heteromorpha trifoliata, Hypericum lalandii, Kirkia acuminata, Pterocarpus angolensis and Zanha africana significantly reduced the oedema 3 h after carrageenan injection. All the extracts were considerably active when assayed topically with only three exceptions.

Evaporative light scattering and thermospray mass spectrometry have been investigated as two alternative methods for the liquid chromatographic detection of ginkgolides and bilobalide. Both techniques were used to quantify these metabolites in leaf extracts and in some phytopharmaceuticals. Only a minimum of sample pre-purification was required. Results were compared with those obtained by gas chromatography (flame ionization detection), which is one of the most suitable methods for routine determinations of such compounds. The isolation of ginkgolides A, B, C, and J and bilobalide from leaves of Ginkgo biloba is also described.

The in vitro immunomodulatory activities of extracts from African medicinal plants have been studied. Certain extracts exerted enhancing effects in a concentration dependent manner on immune cells from three different strains of mice. Among these were an enhancement of phagocytic activity of peritoneal macrophages and tumoricidal potential of macrophages, and stimulation of the oxidative burst of macrophages and granulocytes. In contrast, the proliferative capacity of lymphocytes was only marginally affected. Enhanced macrophage and granulocyte activation was also detected using leucocytes of the LPS nonresponder strain C3H/HeJ. From the latter it can be concluded that the observed stimulatory effects of the plant extracts in vitro were not due to contamination with lipopolysaccharides.

Investigation of the metabolites from Gentiana, rhodentha FR. (Gentianaceae) by LC-UV and LC-MS resulted in the isolation and identification of a new type of secoiridoid glycoside, rhodenthoside A (1), together with the known secoiridoids sweroside (2) and swertiamarin (3), as well as the known iridoids kingiside (4) and 8-epikingiside (5). The structures were established by 1D- and 2D-NMR, EI-MS, D/CI-MS, FAB-MS, CF-FAB, LC-MS, TSP, LC-MS, and LD-MS data in combination with chemical reactions.

The dichloromethane extract of the bark of Ocotea usambarensis Engl. (Lauraceae) showed antifungal activity against Cladosporium cucumerinum. This activity was shown to be linked with its essential oil content together with the presence of the lignan (+)-piperitol. Gas chromatography-mass spectrometric (GC-MS) analysis of the oil allowed the identification of about seventy compounds. In order to determine the fungicidal active components of the oil, further separation was required. Instrumental preparative thin layer chromatography (TLC) was chosen because it was a very suitable method to be followed by direct bioautography. Analysis of the fractions by GC-MS combined with instrumental bidimensional TLC and direct bioautography allowed the separation and identification of the four main active compounds. Nerolidol, α-terpineol, α-copaen- 11-ol and a further unidentified sesquiterpene alcohol were shown to be fungicidal. Identification was made by comparison of the mass spectra with a computerised data-bank and the literature, together with the comparison of retention indices on two GC columns with literature values and with some commercially available samples.

Liquid chromatography-mass spectrometry (LC-MS) has become a successful analytical tool and is no longer regarded as a pure research instrument without any routine practical applications. The development of the thermospray interface (TSP) has been of great help in this respect, allowing the direct use of standard HPLC conditions (1 mL/min, aqueous solvents) for the LC-MS coupling. TSP LC-MS has been used in phytochemical analysis as a powerful detection technique for on-line identification and for quantification of plant constituents, even in trace amounts. Applications of this technique to different classes of plant metabolites such as polyphenols, terpenes, sugars, glucosinolates, alkaloids and glycosides are described. The potential of TSP LC-MS in the analysis of crude plant extracts is discussed.

1. Previous studies have shown that extracts of feverfew (Tanacetum parthenium) and parthenolide, a sesquiterpene alpha-methylenebutyrolactone obtained from it, inhibit smooth muscle contractility in a time-dependent, non-specific and irreversible manner. 2. The hypothesis that this toxic effect is due specifically to the presence in the sesquiterpene lactone of the potentially reactive alpha-methylene function was tested on rabbit isolated aortic ring preparations. This was done (a) by comparing the effects of two plant-derived sesquiterpene lactones purified from yellow star thistle (Centaurea solstitialis): cynaropicrin (an alpha-methylenebutyrolactone) and solstitialin 13-acetate (lacking the alpha-methylene function), and (b) by chemically inactivating the alpha-methylene functions in cynaropicrin and parthenolide by reaction with cysteine. 3. The results show that the characteristic smooth muscle inhibitory profile is demonstrated by the two alpha-methylenebutyrolactones (parthenolide and cynaropicrin), but not by the compound lacking this functional group (solstitialin 13-acetate), or by those previously active compounds in which it has been inactivated with cysteine. 4. Thus the alpha-methylene function is critical for this aspect of the toxic pharmacological profile of the sesquiterpene butyrolactones, which are natural products widely distributed in the Compositae family of flowering plants.

The CH2Cl2 extract of the leaves Annona purpurea L. (Annonaceae) showed strong brine shrimp toxicity and pronounced activity against larvae of the yellow-fever mosquito Aedes aegypti. Activity-directed fractionation of the extract by a combination of column chromatography on silica gel, filtration, and high-pressure liquid chromatography led to the isolation of acetogenins 1–6 as the main active principles. The structures of two novel acetogenins named purpureacin 1(5) and purpureacin 2(6) were elucidated by spectroscopic analysis (UV, EI- and DCI-MS, EI-MS of Me3Si derivatives,1H- and 13C-NMR). The configuration of 5 and 6 was not established due to the limited sample amount. Compounds 3–6 also showed antifungal activity against Candida albicans, and 5 was slightly active against Bacillus subtilis.

Ingestion of yellow star thistle (Centaurea solstitialis L.) by horses produces parkinsonism due to nigro-pallidal degeneration. The toxin responsible has not been identified so far. A CH 2 Cl 2 extract from the aerial parts of C. solstitialis exhibited significant neurotoxicity against primary neuronal cultures of foetal rat brain. Activity-guided fractionation yielded the known sesquiterpene lactones solstitialin A (1), 13-O-acetylsolstitialin A (3), cynaropicrin (4), and the hitherto unknown 3-O-acetylsolstitialin A (2). In the bioassay with rat foetal full cell culture, 3 and 4 were toxic in a concentration-dependent maner and may be responsible for the ability of the plant to cause neurodegenerative changes in the brain of horses

As the root bark CH2Cl2 extract of Bauhinia rufescens showed antifungal activity in a bioassay with the plant pathogenic fungus Cladosporium cucumerinum, a phytochemical investigation was undertaken on material collected in Niger. Activity-guided fractionation of this extract, using different preparative chromatographic methods, allowed the isolation of the four new antifungal tetracyclic compounds 1–4. Their structures were established by 1H- and 13C-NMR data, and single-crystal X-ray analyses were used to confirm the structures of 1 and 3. The isolated compounds seem to be biogenetically related to a stilbene.

From a MeOH extra of Psiadia trinervia, seven phenolic compounds were isolated by gel filtration and reversed-phase chromatography. Six of them are known compounds, namely 3,4-di-O-caffeoylquinic acid (2), 3,5-di-O-caffeoylquinic acid (3), caffeic acid, and three 3-methoxyflavonoids. Compound 1 is a 3,4-di-O-caffeoyl derviative of (1S,3R,4R,5R)-1,3,4, 5-tetrahydroxycyclohexanecarboxylic acid, a novel steroisomer of (−)-quinic acid. Following hydrolytic treatment of the MeOH extract, ethyl 3-O-caffeoylquinate (4), ethyl 3,4-di-O-caffeoylquinate (5), and ethyl 3,5-O-caffeoylquinate (6) were isolated. The latter three compounds are artifacts. The configuration of 1-3 was established by NMR and CD (exciton chirality method).

Three new saponins, alatoside A–C (4a, 5, and 6a, resp.), with a 18,19-secours-12-ene skeleton were isolated from the aerial parts of Sesamum alatum THONN. (Pedaliaceae) by preparative liquid chromatography. Their structures were elucidated by spectroscopic methods, including X-ray diffraction analysis, and by chemical degradation (acidic and enzymatic hydrolyses). In addition, verbascoside (1) and two cyclohexylethanol derivatives, rengyol (2a) and isorengyol (3a), were isolated and identified.

Investigation of the metabolites from Gentiana, rhodentha FR. (Gentianaceae) by LC-UV and LC-MS resulted in the isolation and identification of a new type of secoiridoid glycoside, rhodenthoside A (1), together with the known secoiridoids sweroside (2) and swertiamarin (3), as well as the known iridoids kingiside (4) and 8-epikingiside (5). The structures were established by 1D- and 2D-NMR, EI-MS, D/CI-MS, FAB-MS, CF-FAB, LC-MS, TSP, LC-MS, and LD-MS data in combination with chemical reactions.

Three known phloroglucinols (japonicine A, uliginosin A and isouliginosin B) and a new phloroglucinol (hyperbrasilol 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 13C NMR, 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.

Chemical screening of the secondary metabolites from Halenia corniculata L. (CORNAZ), by LC/UV,LC/TSP-MS (thermospray), and LC/ES-MS (electrospray) was used for the targeted isolation of corniculoside (1), a new biosidic ester secoiridoid. The structure was established as 7-β-[(E)-4′-O-(β-D-glucopyranosyl)caffeoyloxy]-sweroside by 1D- and 2D-NMR, LC/UV, LC/MS, and FAB-MS data, in combination with chemical reactions.

Four new prenylated chromones have been isolated from a dichloromethane extract of the roots of Eriosema tuberosum. Their structures were elucidated by spectroscopic methods. Eriosematin A exhibited antifungal activity against Cladosporium cucumerinum and Candida albicans in TLC bioautographic assays.

Four new iridoid glucosides 1–4, named blumeosides A–D, were isolated from the methanolic stem-bark extract of Fagraea blumei G. DON. (Loganiaceae). They were accompanied by the benzyl-alcohol derivative di-O-methylcrenatin (5) and the flavone C-glucoside swertisin (6). The structures of 1–4 were established by spectroscopic methods, including FAB-MS, and 1H- and 13C-NMR, and by alkaline hydrolysis. Blumeosides A (1) and C (3) are 10-O-(2,5-dihydroxytercphthalo) adoxosidic acid and 10-O-(2-hydroxyterephthalo)adoxosidic acid, respectively. In blumeosides B (4) and D (2), both carboxylic groups of the terephthalic-acid moiety are esterified by adoxosidic-acid units, Blumeosides A–D (1–4) inhibited bleaching of crocin induced by alkoxyl radicals. Blumeosides A (1) and D (2) also demonstrated scavenging properties towards the 2,2-diphenyl-1-picryl-hvdrazvl (CDPPH) radical in TLC autographic and spectrophotometric assays.