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LC-MS/MS data of the flavones from extracts of nonmodified and transgenic sugarcane (“Bowman-Birk” and “Kunitz”) led to the proposition of characteristic fragmentations that can be applied to identify the monosaccharides glucose, arabinose, and rhamnose in C-glycosylflavones. Collision-induced dissociation (CID) MS/MS generated diagnostic ions for different linked sugars in C-glycosylflavone–O-glycoside and C-glycosylflavone–O-acid derivatives, and the combination of MS data with postcolumn derivatization using UV shift reagents (which provide complementary information to determine the substitution patterns in the flavone skeleton) allowed for the identification of seven flavones not previously reported in sugarcane: 6-methoxyluteolin-8-C-arabinosyl-7-O-glucoside; diosmetin-8-C-arabinosyl-7-O-arabinoside; vitexin-7-O-rhamnoside; diosmetin-8-C-rhamnosyl-7-O-glucoside; tricetin-8–C-(6‴-O-sinapoylglucoside); 7,4′ di-O-methylapigenin-8-C-arabinosyl-rhamnoside; and luteolin-8-C-rhamnosyl-7-O-rhamnoside.

A phytochemical study of Asplenium ruta-muraria L. (Aspleniaceae) led to the isolation of a new caffeic acid glycoside, 2-O-caffeoyl-β-D-fructofuranosyl-(2 → 1)-α-D-glucopyranoside and an (α, β)-isomeric pair of 2E-caffeoyl-D-glucopyranoside, together with kaempferol-3-O-β-D-[6-E-caffeoyl-β-D-glucopyranosyl-(1 → 2)glucopyranoside]-7-O-β-D-glucopyranoside, 1-O-caffeoyl glycoside, sucrose, diploptene and β-sitosterol. Their structures were established by means of MS and capillary NMR techniques. Additionally, aromatase inhibitory activity of the extracts and phenolic compounds was evaluated.

In order to find some medicinal value for invasive neophytes, inhibition of acetylcholinesterase of Reynoutria sachalinensis F. Schmidt ex Maxim. (Polygonaceae), Polygonum cuspidatum Siebold. Zucc. (Polygonaceae), Heracleum mantegazzianum Sommier Levier (Apiaceae), Buddleja davidii Franch. (Buddlejaceae), and Ambrosia artemisiifolia L. (Asteraceae) was prescreened by a bioautographic TLC assay. Activity was found in the methanol extract of the leaves of the common ornamental bush B. davidii. Its fractionation by centrifugal partition chromatography led to the isolation of linarin as the most active compound. While extracts of Buddleja nitida Benth. (Buddlejaceae) from Panama were inactive, some constituents were identified on the basis of a comparison with B. davidii by HPLC-MS/MS. Structure-activity relationships of linarin and related flavonoids were investigated. For this purpose, fortunellin and tilianin were prepared by methylation reactions. It appears that the structural elements important for acetylcholinesterase inhibition are not only the 4' -OMe group, but also the 7-substituted sugar and the pattern of substitution on the B-ring.

For many social and environmental reasons, over the last few decades, there has been an increase in chronic and life-threatening diseases including mycoses, hyperuricemia-related disorders and some mental illnesses such as depression, anxiety and Parkinson's disease. In order to fight these diseases, compounds acting on various biological targets, including enzymes such as xanthine oxidase or monoamine oxidase, have to be screened. The enzyme xanthine oxidase catalyses the oxidation of hypoxanthine to xanthine and then to uric acid, which plays a crucial role in hyperuricemiarelated disorders such as gout and renal stones. One of the therapeutic approaches to treat these diseases is the use of xanthine oxidase inhibitors that block the production of uric acid. Monoamine oxidases (E.C.1.4.3.4) A and B catalyse the oxidative deamination of monoamines in the central nervous system and peripheral tissues. Inhibitors of MAO A are clinically useful to treat anxiety and depression since they are expected to increase both noradrenalin and serotonin levels in the brain. On the other hand, inhibition of MAO B appears to be an effective approach for the prevention and adjunct treatment of Parkinson's disease. In traditional Chinese medical practice, many medicinal herbs have been used to treat chronic diseases such as fungal infections, hyperuricemia-based disorders and mental illnesses. This usage is indicative for the presumable presence of antifungal phytochemicals and inhibitors of xanthine and monoamine oxidases. Plants do not represent the only source for interesting natural products; some endophytes ('special' microorganisms living inside the healthy host plant) are also known to produce secondary metabolites of promising pharmaceutical and/or agricultural potential. The above observations prompted us to search for natural antifungal compounds and inhibitors of xanthine and monoamine oxidases in different Chinese plants and endophyte cultures. The active constituents isolated were mainly mono-, sesqui-, di-, and triterpenes, sterols, coumarins, flavonoids, phenylethanoids, stilbenoids, alkaloids and alcohols.

Rapid detection of biologically active natural products plays a key role in the phytochemical investigation of crude plant extracts. In order to perform an efficient screening of the extracts, both biological assays and HPLC analysis with various detection methods are used. Techniques such as HPLC coupled to UV photodiode array detection (LC/DAD-UV) and to mass spectrometry (LC/MS or LC/MS/MS) provide numerous on-line structural data on the metabolites prior to isolation. The recent introduction of HPLC coupled to nuclear magnetic resonance (LC/NMR) represents a powerful complement to LC/UV/MS screening. Various plant species belonging to the Gentianaceae and Leguminosae have been analysed by LC/UV, LC/MS, LC/MS/MS and LC/NMR. These hyphenated techniques allow a rapid structural determination of known plant constituents with only a minute amount of plant material. Simple bioautographic assays such as those used for screening antifungal constituents can also be performed on-line directly by collecting HPLC peaks and measuring the activity against the fungi of interest. These bioassays permit a rapid localisation of the bioactive natural products. With such a combined approach, the time consuming isolation of common natural products is avoided and an efficient targeted isolation of compounds presenting interesting spectroscopical or biological features is performed. Several representative applications of the use of LC/MS and LC/NMR for the dereplication and identification of antifungal constituents are presented in the present paper.

A total of 204 crude extracts of Indonesian plants (77 species from 43 families) have been screened for their fungicidal (Cladosporium cucumerinum, Candida albicans) and free radical scavenging [2,2-diphenyl-1-picrylhydrazyl (DPPH) radical] activities. Of the species investigated, 20 were active against Cladosporium cucumerinum, nine against Candida albicans and 24 exhibited radical scavenging properties.

Three antifungal xanthones have been isolated from the dichloromethane root extract of Marila laxiflora (Guttiferae). Their structures were established by spectrometric (UV, EI mass spectrometry, 1 H and 13 C nmr) and chemical methods. In addition, other compounds were isolated from the methanol extract: a fourth xanthone, rhamnetin, betulinic acid, and a derivative of benzoic acid.

A total of 153 crude extracts of Panamanian plants (28 species from 21 families) have been screened for molluscicidal activity against Biomphalaria glabrata, one of the intermediate hosts of schistosomiasis. When tested at 400 ppm, 15 extracts were active. The most potent of these came from Cyperus luzulae (Cyperaceae) and Hymenaea courbaril (Leguminosae).

Four saponins were isolated from the methanol extract of the pericarps of Guaiacum officinale using a bioassay with the snail Biomphalaria glabrata. Their structures were established as 3-O-(s-D-glucopyranosyluronic acid)-oleanolic acid (1), 3-O-{[a-L-rhamnopyranosyl(1?3)-a-L-rhamnopyranosyl(1?2)]-a-L-arabinopyranosyl (3?1) [-s-D-glucopyranosyl]}-30-norolean-12,20(29)dien-28-oic acid (2, guaianin D), a-D-glucopyranosyl 3-O-[a-L-rhamnopyranosyl (1?3)-s-D-glucopyranosyl(1?3)-a-L-arabinopyranosyl]-30-norolean-12,20-(29) dienoate (3, guaianin B) and s-D-glucopyranosyl 3-O-[a-L-rhamnopyranosyl (1?3)-s-D-glucopyranosyl(1?3)-a-L-arabinopyranosyl]-oleanolate (4, guaianin C). The LC 100 /24h of monodesmosidic saponins 1 and 2 were 2.5 and 10 ppm, respectively, while the bidesmosidic saponins 3 and 4 were inactive at 10 ppm. Basic hydrolysis of 3 and 4 produced the corresponding monodesmosidic saponins 5 (guaianin) and 6 (guaianin H). These saponins were active at 2.5 and 5 ppm, respectively.

Nineteen plant species from fourteen families used in the traditional medicine of North American Indians have been tested for their fungicidal (Cladosporium cucumerinum, Candida albicans), bactericidal (Bacillus subtilis, Escherichia coli), larvicidal (Aedes aegypti) and molluscicidal (Biomphalaria glabrata) activities. Of the species investigated, 9 were active against Cladosporium cucumerinum, 9 against Candida albicans, 12 against Bacillus subtilis, 18 against Escherichia coli, 3 against Aedes aegypti, and 11 against Biomphalaria glabrata.

Four compounds were isolated from a dichloromethane extract of the aerial parts of Chenopodium procerum by different chromatographic techniques: the isoflavones irilin A and irilin B, the flavanone dihydrowogonin, and the sesquiterpene pygmol. The latter three inhibited the growth of the plant pathogenic fungus Cladosporium cucumeri-num. Four flavonol glycosides were obtained from the non-fungicidal methanol extract by Sephadex LH-20 gel filtration and centifugal partition chromatography. The structures of the compounds were eluacidated by spectroscopic methods (UV, lH-NMR, 13C-NMR, MS).

Bioactivity guided fractionation of a dichloromethane extract of Morinda lucida Benth. (Rubiaceae) roots has led to the isolation often anthraquinones. Four of these were active against Cladosporium cucumerinum and Candida albicans in bioautographic (TLC) assays. In-depth investigation of activity against three human pathogenic fungi showed that alizarin-1-methyl ether displayed the highest activity.

Three iridoid glucosides, two phenylpropanoid derivatives and one flavonoid have been isolated from the methanolic whole plant extract of Bartsia alpina L. (Scrophulariaceae). Their structures were established by spectrometric (UV, D/CI and FAB mass spectrometry, 1 H and 13 C NMR) and chemical methods. Some of these compounds have radical scavenging activity against DPPH or antioxidant activity against s-carotene on TLC bioassay.

A two-pump method for the control of stationary to mobile phase ratios in a cartridge centrifugal partition chromatography system is described. This cartridge CPC set-up and a rotating coil instrument are used in the separation of natural products from complex mixtures contained in crude plant extracts.

Droplet counter-current chromatography (DCCC) has found wide application for preparative separation of plant constituents and other natural products. It is particularly indicated for the isolation of polar compounds. The use of non-aqueous solvent systems allows the separation of weakly polar substances. Large-bore columns can be employed without decreasing the resolution when the flow-rate is increased. New fields of applications include essential oils, triterpenoids and steroids, plant growth regulators (gibberellins), antibiotics.

Purification of natural products and the fractionation of crude plant extracts are processes which traditionally involve adsorption and/or molecular exclusion chromatography. While countercurrent chromatography avoids the irreversible adsorption and decomposition frequently encountered in adsorption chromatography, the classical countercurrent distribution technique is both time- and solvent-consuming, consequently it is of limited value today. Rotation locular countercurrent chromatography (RLCC) is one of three new countercurrent techniques, the others being droplet contercurrent chromatography (DCCC)1 and planet coil countercurrent chromatography (PCCCC)2, which have revived the chromatographic application of liquid-liquid partition. We have employed a commercially available RLCC apparatus in the isolation of several natural products with a broad range of polarity demonstrating the versatility of the RLCC with regard to the functionality of the compounds to be isolated.