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Many of the protein kinase activators and inhibitors presently under investigation are natural products or are derivatives of natural products. Since over 500 kinases are encoded by the human genome, the task is to discover modulatory compounds which have high specificity for a single enzyme. Many different low molecular weight compounds have been studied for activity. These come from various sources, such as plants, marine organisms, microorganisms and cyanobacteria. The search for specific inhibitors is mainly performed in cell-based assays.

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.

Five isopimarane diterpenes (7beta-hydroxyisopimara-8,15-dien-14-one, 14alpha-hydroxyisopimara-7,15-dien-1-one, 1beta, 14alpha-dihydroxyisopimara-7,15-diene, 7beta-hydroxyisopimara-8(14),15-dien-1-one and 7beta-acetoxyisopimara-8(14),15-dien-1-one) have been isolated from the leaves of Hypoestes serpens (Acanthaceae). All compounds exhibited antifungal activity against both the plant pathogenic fungus Cladosporium cucumerinum and the yeast Candida albicans; two of them also displayed an acetylcholinesterase inhibition. The structures of the Compounds were determined by means of spectrometric methods, including 1D and 2D NMR experiments and MS analysis. (C) 2003 Published by Elsevier Ltd.

A new triterpenoid saponin was isolated from the roots of Silene cucubalus and its structure was determined on the basis of one- and two-dimensional NMR spectroscopy as gypsogenin 3-O-beta-xylopyranosyl-(1-->3)-[beta-galactopyranosyl-(1-->2)]-beta-glucuronopyranoside. A known saponin, quillaic acid 3-O-beta-xylopyranosyl-(1-->3)-[beta-galactopyranosyl-(1-->2)]-beta-glucuronopyranoside, was also isolated.

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.

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.

Two new cyclobutane-type lignans, named moslolignans A and B, together with two known ones, andamanicin and magnosalin, were isolated from the whole plant of Mosla scabra. Their structures were established as 1 beta*,2 beta*,3 alpha*,4 alpha*-l,2-dimethyl-3-(3- methoxy-4,5-methylene-dioxyphenyl)-4-(2,4,5-trimethoxyphenyl)-cyclobutane and 1 beta*,2 beta*,3 alpha*,4 alpha"-1,2-dimethyl-3-(2,5-dimethoxy-3,4-methylenedioxyphenyl)-4-(2,4, 5-trimethoxyphenyl)-cyclobutane by spectroscopic methods. This is the first report of naturally-occurring cyclobutane-type lignans with asymmetrical substitutions. (C) 2000 Elsevier Science Ltd. All rights reserved.

In addition to the known cordiaquinones A and B, two novel meroterpenoid naphthoquinones, named cordiaquinones J and K, have been isolated from the roots of Cordia curassavica. Their structures were elucidated by spectrometric methods including EI, D/CI mass spectrometry, 1H, 13C and 2D-NMR experiments. The four naphthoquinones demonstrated antifungal activities against Cladosporium cucumerinum, Candida albicans and toxic properties against larvae of the yellow fever-transmitting mosquito Aedes aegypti.

Seven compounds have been isolated from methanol extracts of whole Saxifraga stellaris (Saxifragaceae) plants. They were identified as the new compound, 6- O -galloyl fructose, together with 3- O -[2- O -(β- d -xylopyranosyl)-β- d -galactopyranosyl]-kaempferol, 3- O -[2- O -(β- d -xylopyranosyl)-β- d -galactopyranosyl]-quercetin, trifolin, hyperin, resveratrol-3- O -glucoside, triandrin, by chemical and spectroscopic methods. Their free radical scavenging properties are also described.

A new flavonoid, dihydrorhamnetin 3- O - β -d-arabinopyranoside, was isolated from Pyrola elliptica , together with quercetin 3- O - β -d-glucopyranoside, quercetin 3- O - α -l-arabinofuranoside, quercetin 3- O - β -d-galactopyranoside, rhamnetin 3- O - β -d-galactopyranoside, taxifolin 3- O - β -d-arabinopyranoside, taxifolin 3- O - β -d-xylopyranoside, homoarbutin, isohomoarbutin and monotropein.

Analysis of the dichloromethane extracts of Kielmeyera coriacea leaves and stems by high performance liquid chromatography coupled with a photodiode array detector (LC-UV) and thermospray liquid chromatography-mass spectrometry (TSP/LC-MS) revealed the presence of several xanthones. Phytochemical investigation of these extracts resulted in the isolation and identification of ten xanthones, one biphenyl and two triterpenes. Their structures were established by chemical and spectroscopic methods (UV, EI-MS, D/CI-MS, 1 H NMR, 13 C NMR, HMQC and HMBC). One xanthone and two triterpenes are new compounds. Four xanthones and the biphenyl exhibited antifungal activity against the plant pathogenic fungus Cladosporium cucumerinum , while two prenylated xanthones inhibited the growth of Candida albicans .

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.

Phytochemical investigation of the methanolic extract of Gentiana linearis roots led to the isolation of two novel iridoids (7-O-coumaroyl-loganic acid; 7-O-(4″-O-glucosyl)coumaroyl-loganic acid) and a new secoiridoid (6″″-O-glucosyltrifloroside) together with trifloroside, gentiopicroside, isovitexin and isosaponarin. The structures have been elucidated by MS and NMR spectral data, enzymic and chemical degradation. The chemotaxonomic significance of the isolated secoiridoids and iridoids is briefly discussed.

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 new flavonol glycoside has been isolated from the leaves of Vernonia galamensis ssp. nairobiensis . Its structure has been established as isorhamnetin 3-O-β- d -apio- d -furanosyl (1→2)-β- d -galactopyranoside on the basis of spectral data (UV, 1 H and 13 C NMR, D/CIMS) and by acid hydrolysis. Characterization of the peracetate derivative confirmed the configuration of the apiosyl moiety. In addition, the known flavonol glycosides; quercetin 3-galactoside, quercetin 3-apiosyl(1→2)galactoside and quercetin 3-rhamnosyl(1→6)galactoside have been isolated and identified.

LC-UV-mass spectrometry and bioassay co-directed fractionation of an aqueous acetone extract of the roots of Gentiana macrophylla gave three new chromene derivatives and two novel and six known secoiridoids, along with kurarinone, kushenol I, beta-sitosterol, stigmasterol, daucosterol, beta-sitosterol-3-O-gentiobioside, alpha-amyrin, oleanolic acid, isovitexin, gentiobiose and methyl 2-hydroxy-3-(1-beta-D-glucopyranosyl)oxybenzoate. The structures of the new products were established from spectral and chemical evidence as 2-methoxyanofinic acid and macrophyllosides A-D. The six known secoiridoids were gentiopicroside, sweroside, 6'-O-beta-D-glucosylgentiopicroside, 6'-O-beta-D-glucosylsweroside, trifloroside and rindoside. The new acid (2-methoxyanofinic acid), its methyl ester, kurarinone and kushenol I were shown to be active against the plant pathogenic fungus Cladosporium cucumerinum. The methyl ester and kurarinone inhibited also the growth of the human pathogenic yeast Candida albicans. Structure-activity relationships were studied. Thus, addition of a methoxyl group to the benzene nucleus of anofinic acid (2,2-dimethyl-2H-1-benzopyran-6-carboxylic acid) increased the antifungal activity remarkably whereas glycosylation at the carboxylic moiety was found to remove the activity. Esterification of the new acid induced its activity against C. albicans, but decreased its growth inhibition properties against C. cucumerinum. Hydroxylation of kurarinone at the 3 beta-position removed its activity against C. albicans and decreased the inhibition of C. cucumerinum. In addition, the chemotaxonomic significance of the identified constituents is discussed.

Three new phloroglucinols (hyperbrasilols B and C, and isohyperbrasilol B) have been isolated from a petrol extract of the leaves and flowers of Hypericum brasiliense. Their structures were established by spectroscopic methods including two-dimensional-NMR heteronuclear correlations. EI and D/Cl-mass spectral, NMR, IR and UV data are reported. All three phloroglucinols were antibacterial against Bacillus subtilis in a TLC bioautographic assay.

An improved method is presented for the isolation of phenolic compounds from needles of Norway spruce ( Picea abies , Pinaceae). High-speed counter-current chromatography (HSCCC) has been employed as a key step in the semipreparative-scale separation and purification of 26 phenolics from juvenile and mature needles. The isolation of the three 3- O -(6″Ac)glucosides syringetin, isorhamnetin and kaempferol is reported together with other flavonols, dihydroflavonols, catechins, stilbenes, and other phenolics.

The novel flavonol, syringetin 3- O -(6″-acetyl)- β -glucopyranoside, has been isolated from needles of Norway spruce ( Picea abies ) together with the 3- O -(6″-acetyl)- β -glucopyranosides of isorhamnetin and kaempferol, the 3- O -(6″- α -rhamnopyranosyl)- β -glucopyranosides of laricitrin, isorhamnetin, myricetin, quercetin and kaempferol and the 3- O - β -glucopyranosides of laricitrin, isorhamnetin, myricetin, quercetin and kaempferol. Most of the flavonols have been isolated for the first time from Norway spruce. Kaempferol 3- O -(6″-acetyl)- β -glucopyranoside has previously been isolated from Senecio aureus , but without determination of the binding site of the acetyl group. Structure determination of the flavonols was achieved from TLC, 1 H NMR and UV shift reagent data, and, in most cases, 13 C NMR and MS.

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.

Counter-current chromatography is an all-liquid technique that is finding increasing use in separation problems, especially in the field of natural products. A broad selection of applications, involving both polar and non-polar compounds, is described here. Examples of the choice and utilization of aqueous and non-aqueous solvent systems are given. The emphasis is placed on new and rapid centrifugal partition techniques but droplet countercurrent chromatography is also treated, together with some applications of rotation locular counter-current chromatography.

Stem and leaves infusion of Chuquiraga spinosa (RP) Don. (Asteraceae) is used in the Peruvian traditional medicine for its anti-inflammatory properties and for the treatment of vaginal infections.This study evaluated the antioxidant, anti-inflammatory and antifungal activities of C. spinosa for the first time.Extracts of methanol, 50% methanol and water were obtained from C. spinosa aerial parts. Antioxidant activity of the extracts was evaluated (DPPH˙, ABTS˙(+) and superoxide radical-scavenging activity). The correlation between these results and total polyphenolic content was determined by Pearson's Correlation Coefficient. Anti-inflammatory activity of 50% methanol extract was evaluated with the rat model of carrageenan-induced acute inflammation and mouse model of TPA-induced acute inflammation. The antifungal activity of the extracts against Cladosporium cucumerinum and Candida albicans was studied by direct bioautography, and antifungal activity against phytopathogenic fungi was performed by culture in potato dextrose agar plates.All the extracts showed high antioxidant activity, and there was correlation between the activity and total polyphenolic compounds. As 50% methanol extract was administered orally, the paw edema in rats was reduced significantly (52.5%). This extract, by topical administration, produced a reduction of 88.07% of the edema TPA-induced in ear of mice. The aqueous and 50% methanol extracts were active against C. albicans (minimum inhibitory concentration of 2.5 and 6.25 µg, respectively). The aqueous extract showed antifungal activity against C. cucumerinum (MIC: 2.5 µg).Preliminary phytochemical screening and the analysis of the three extracts by high-performance liquid chromatography diode-array detection showed the majority compounds are flavonoids and phenolic acid derivatives. These compounds may be responsible of the radical-scavenging activity of these extracts as well as responsible of anti-inflammatory effect in vivo of 50% methanol extract. Several authors have demonstrated the antioxidant and anti-inflammatory properties of some flavonoids and phenolic acids. The antifungal activity of the extracts obtained from aerial parts of C. spinosa has been investigated here for the first time. Other studies are necessary to determine the mechanism of action and to identify the bioactive compounds of this plant.

Thirteen compounds have been isolated from the methanol extract of Saxifraga cuneifolia L. (Saxifragaceae) whole plant. These were identified as five new compounds, (E)-5-O-beta-D-glucopyranosyl-stilbene-3,3', 4,5'-tetraol, (E)-4-O-beta-D-glucopyranosyl-5-methoxy- stilbene-3,3',5'-triol, 4-[ 4'-O-(6''-O-galloyl-beta-D-glucopyranosyl)- 3'-hydroxyphenyl] butan-2-one, 3-O( 6''-O-galloyl-beta-D-glucopyranosyl)-epigallocatechin and 3-O-beta-D-glucopyranosyl-epigallocatechin, together with the known compounds 4-[4'-O-beta-D-glucopyranosyl- 3'-hydroxyphenyl]-butan-2-one, (R) rhododendrin, 1-O-galloyl-beta-D-glucose, catechin, 3-O-galloyl-epigallocatechin, gallocatechin, clitorin and rutin, by chemical and spectroscopic methods. Their free radical scavenging properties are also described.

From the latex of Euphorbia milii var. hislopii collected in Brazil, eight milliamines were isolated using a bioassay-guided protocol based on molluscicidal activity. Three were known milliamines A, D and E, the other five being new compounds of this class. Milliamine L, a dianthraniloyl peptide ester of ingenol, was found to be molluscicidal at 4 nM (2.5 μg 1 −1 ).

The dichloromethane extract from the roots of Polygala nyikensis afforded four simple xanthones, two of which are new compounds. Their structures were established by chemical and spectroscopic methods (UV, EIMS, 1 H and 13 C NMR, NOE difference spectroscopy). 1,7-Dihydroxy-4-methoxyxanthone and 1,7-dihydroxy-3,5,6-trimethoxyxanthone exhibit antifungal activity against the plant pathogenic fungus Cladosporium cucumerinum .

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.

Three new meroterpenoid naphthoquinones, the known cordiaquinone B and a new naphthoxirene have been isolated from the roots of Cordia linnaei. Their structures were established by spectrometric methods including EI, D/CI and FAB mass spectrometry, 1H, 13C and 2D NMR experiments. The naphthoquinones showed activity against Cladosporium cucumerinum, Candida albicans and the larvae of the yellow fever-transmitting mosquito Aedes aegypti, while the naphthoxirene derivative was found to be inactive in the same bioassays.

In addition to the known cordiaquinones A and B, two novel meroterpenoid naphthoquinones, named cordiaquinones J and K, have been isolated from the roots of Cordia curassavica. Their structures were elucidated by spectrometric methods including EI, D/CI mass spectrometry, 1H, 13C and 2D-NMR experiments. The four naphthoquinones demonstrated antifungal activities against Cladosporium cucumerinum, Candida albicans and toxic properties against larvae of the yellow fever-transmitting mosquito Aedes aegypti.

Mass spectrometry is essential for the structure determination of naturally occurring glycosides. Molecular weight and sugar sequence information can be obtained. Development of “soft” ionization techniques has allowed a direct mass spectrometric investigation of underivatized glycosides. Numerous different and complementary methods of ionization have been used for their study. For a given compound, the choice of a particular technique is crucial in order to obtain the structural information needed. A brief survey of these ionization techniques is given, together with examples of their use in major classes of plant glycosides.

International audience; Polygonum sachalinensis is a widespread invasive plant in Europe. Chemical profiles of its different organs were studied by HPLC-UV-ESI/MS. Seven major constituents quercetin-3-O-β-D-galactopyranoside, quercetin-3-O-arabinopyranoside, lapathoside D, N-trans-feruloyltyramine, lapathoside C, hydropiperoside, and vanicoside B were isolated and identified. The free radical-scavenging, α/β-glucosidase, and acetylcholinesterase inhibitory activities of crude MeOH extracts and isolated compounds were studied. The structure\textendashactivity relationships were discussed. The chemical profiles revealed flavonoids and phenylpropanoids are the major compounds of all the organs of this plant. Quercetin-3-O-arabinopyranoside, lapathoside D, N-trans-feruloyltyramine, lapathoside C and hydropiperoside were isolated from this species for the first time. In the α-glucosidase bioassay, quercetin-3-O-β-D-galactopyranoside, lapathoside D and N-trans-feruloyltyramine demonstrated stronger activities than the positive reference acarbose. The trend in scavenging power showed no relation to enzyme inhibition in the test models.

Introduction - The segregation between the genera Gentians and Gentianella among the Gentianaceae family is poorly defined. In order to clarify the classification of these genera, some researchers have tried to incorporate data about the chemical constitution, but this has not yet been achieved in a comprehensive way. Objective - To develop a fast and reproducible analytical method for the observation of characteristic fingerprints of secondary metabolites of each genus. Methodology - Seven species were investigated, three Gentianella and four Gentiana selected for their close taxonomic links within each genus. Ten xanthones previously isolated from one of these species were used as chemotaxonomic markers. A UPLC/ESI-TOF-MS method was developed to analyse the methanolic extracts. Results - The UPLC/TOF-MS provided clear metabolic fingerprints and elemental composition of the compounds. The profiles of the three Gentianella species were strikingly similar. On the contrary, metabolic profiles of Gentians species were very different from the Gentianella chromatograms and also from each other. Several compounds were unique to each genus and therefore could be used as biomarkers. Conclusion - UPLC/TOF-MS can be applied as a chemotaxonomic tool for the rapid screening of Gentianaceae species and for the distinction between closely related taxa from the Gentianaceae family.

Introduction - Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of beta-glucosidase inhibitors but not for alpha-glucosidase. Nonetheless, alpha-glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti-viral infections. Objective - To develop a TLC bioautographic method to detect alpha- and beta-glucosidase inhibitors in plant extracts. Methodology - The enzymes alpha- and beta-D-glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of alpha-D-glucosidase, and 37 degrees C for 20 min in the case of beta-D-glucosidase. For detection of the active enzyme, solutions of 2-naphthyl-alpha-D-glucopyranoside or 2-naphthyl-beta-D-glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for alpha-D-glucosidase) or 1 :4 (for beta-D-glucosidase) and sprayed onto the plate to give a purple background colouration after 2-5 min. Results - Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited alpha-D-glucosidase and beta-D-glucosidase down to 0.1 mu g. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 mu g for alpha-glucosidase and 50 mu g for beta-glucosidase. Conclusion - The screening test was able to detect inhibition of alpha- and beta-glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright (C) 2009 John Wiley & Sons, Ltd.

Centrifugal partition chromatography has been applied to the separation of flavonol glycosides from Epilobium parviflorum (Onagraceae). Using these glycosides, a method is described for their determination in different species of Epilobium by high-performance liquid chromatography on an octadecylsilyl column.

An investigation of Eleutherococcus senticosus roots, using centrifugal partition chromatography as a key intermediate separation step, has led to the straightforward isolation of eight constituents. Four of these, sinapaldehyde glucoside, coniferaldehyde glucoside, coniferin and 1,5-di-O-caffeoylquinic acid, have been described for the first time in this plant. The quantitative analysis of different E. senticosus samples has been performed by high performance liquid chromatography using eleutherosides B and E as external standards.

Chromatography is the lynchpin of phytochemistry and is the key to obtaining pure compounds for structure elucidation, for pharmacological testing or for development into therapeuticals. It also plays a fundamental role as an analytical technique for quality control and standardisation of phytotherapeuticals. Although liquid chromatography is barely 100 years old, an extraordinary variety of instrumental and ancillary equipment is available, notably in the domain of high-performance liquid chromatography. It is impossible to touch all areas of chromatography in such a review but certain areas are worthy of mention: HPLC, HPTLC, UPLC and countercurrent chromatography. Another important addition has been the development of hyphenated techniques involving HPLC: LC/UV, LC/MS, LC/MS(n) and LC/NMR. These are indispensable nowadays for the early detection and identification of compounds in crude plant extracts.

Recent innovations in liquid/liquid chromatographic techniques have led to a resurgence of interest in their use for the separation of natural products. Commercially available instruments which rely on centrifugal acceleration to speed up separation times are based either on a battery of cartridges or on a rotating multilayer coil and can be employed either as analytical tools or for preparative purposes. While solvent consumption is low, milligram to gram quantities are capable of being separated. Aqueous and non‐aqueous two‐phase solvent systems are compatible with these techniques and numerous applications involving simple and complex mixtures of natural products, both polar and non‐polar, have been demonstrated.

Centrifugal counter-current chromatography (CCCC) was examined as a means of measuring lipophilicity values. Using an aqueous buffer as the stationary phase and 1-octanol as the eluent, seventeen solutes of widely different structure and of log Doct [log (distribution coefficient) at pH 7.41 spanning 2.5 units were examined. A very good correlation was found with literature log P [log (partition coefficient) of neutral species] values measured by the classical shake-flask method. The efficiency and precision of the CCCC method makes it a promising technique for measuring lipophilicity.

Chromatography is the lynchpin of phytochemistry and is the key to obtaining pure compounds for structure elucidation, for pharmacological testing or for development into therapeuticals. It also plays a fundamental role as an analytical technique for quality control and standardisation of phytotherapeuticals. Although liquid chromatography is barely 100 years old, an extraordinary variety of instrumental and ancillary equipment is available, notably in the domain of high-performance liquid chromatography. It is impossible to touch all areas of chromatography in such a review but certain areas are worthy of mention: HPLC, HPTLC, UPLC and countercurrent chromatography. Another important addition has been the development of hyphenated techniques involving HPLC: LC/UV, LC/MS, LC/MS(n) and LC/NMR. These are indispensable nowadays for the early detection and identification of compounds in crude plant extracts.

Plants have a rich history as medicines for human use. Recently introduced plant-derived drugs include paclitaxel from Taxus (Taxaceae) species for the treatment of cancers, artemisinin for malaria therapy and the alkaloid galanthamine for the management of Alzheimer's disease. A judicious combination of chemical and biological screening techniques can provide a steady supply of lead compounds for the improved treatment of human ailments. By this means, candidate compounds can be rapidly selected for further development, as is the case for xanthones from gentians and coumarins from Peucedanum ostruthium (Apiaceae), which may be of relevance in Alzheimer's disease. Encouraging results are also being obtained for hypericin from Hypericum perforatum (Hypericaceae) in the photodynamic therapy of cancer. An aspect that must not be forgotten is that the usual biological screening procedures do not pick up prodrugs which have to be metabolized by the human body into active analogues. A good example is salicin from Salix species (Salicaceae) which is metabolized in the body to give the anti-inflammatory and analgesic compound salicylic acid. Investigations which include simulation of metabolism are thus of primordial importance.

A phytochemical study of the methanol extract of Juniperus communis berries was undertaken. The crude extract was analysed by HPLC-UV and the isolation of the minor compounds was performed by centrifugal partition chromatography. By this means, five diterpenes were isolated, one of which was a new labdane diterpene 15,16-epoxy-12-hydroxy-8(17),13(16),14-labdatrien-19-oic acid. The structures of the isolated compounds were elucidated by spectroscopic methods, including UV, NMR, MS and HR-MS.

Counter-current chromatography is a very versatile separation technique which does not require a solid stationary phase. It relies simply on the partition of a sample between the two phases of an immiscible solvent system. Some of the more recent applications of the method to the separation of plant-derived natural products are described here. Crude plant extracts and semi-pure fractions can be chromatographed, with sample loads ranging from milligrams to grams. Aqueous and non-aqueous solvent systems are used and the separation of compounds with a wide range of polarities is possible. The technique is complementary to other chromatographic methods and is compatible with gradient systems. The possibilities for solvent selection are almost limitless but some guidelines for the choice of successful systems are presented. (c) 2005 Elsevier B.V. All rights reserved.

A rapid qualitative HPLC-UV-MS method was developed for the detection of huperzine A in Lycopodiaceac species. HPLC coupled with mass spectrometry experiments allowed the identification of the alkaloid and enabled targeted analysis of complex matrices such as herbal extracts. Huperzine A was detected by single ion monitoring of the pseudomolecular ion [M + H](+) at m/z 243.2. The alkaloid was also detected on TLC in a bioautographic enzyme assay with acetylcholinesterase to show the activity of the compound. Four Lycopodiaceae species collected in Switzerland were tested by these methods and Huperzia selago (L.) Bernh. ex Schrank et Martius was the only species found to contain huperzine A. Copyright (c) 2006 John Wiley & Sons, Ltd.

The complexation of various flavonoids with cyclodextrins and cyclodextrin derivatives was studied using thin layer chromatography on cellulose. The results indicate that the flavonoids examined do not complex readily with α-cyclodextrin or with succinylated α-cyclodextrin. However, most do complex with β-cyclodextrin, succinylated β-cyclodextrin, succinylated γ-cyclodextrin, α-cyclodextrin polymer, β-cyclodextrin polymer and γ-cyclodextrin polymer. With γ-cyclodextrin elongated trails are obtained, suggesting that the kinetics are somewhat slower. © 1997 John Wiley & Sons, Ltd.

HPLC-UV-MS analysis of the methanol extract of Jamesbrittenia fodina (Wild) O. M. Hilliard (Scrophulariaceae) revealed the presence of different iridoid cinnamic esters; however, isolation of these constituents was prevented by instability problems. HPLC-UV-MS and HPLC-NMR analysis of the mixtures obtained after a tentative isolation indicated that, in the first instance, instability was due to a light-induced cisltrans isomerisation of the cinnamic moieties. Further investigation of related compounds showed an additional instability problem linked to other chemical transformations. A detailed HPLC-NMR-MS study of these fractions demonstrated that the modifications occurred on the rhamnose moiety of these iridoids. It could be concluded that the second type of instability was attributable to transesterification of the cinnamic moiety on the rhamnose unit. The recording of stop-flow HPLC-NMR spectra for specific HPLC peaks permitted the direct monitoring of these transformations. Based on these on-line data, six new unstable aucubin derivatives were efficiently characterised.

Different plant extracts have been screened by thin-layer chromatography (TLC) bioautography in an effort to discover new acetylcholinesterase (AChE) inhibitors. The CH2Cl2 extract of Peucedanum ostruthium. (L.) Koch roots exhibited significant inhibition of AChE -l activity. Active constituents were isolated by bioguided fractionation using almost exclusively centrifugal partition chromatography. Four coumarins (ostruthin, imperatorin, ostruthol, and oxypeucedanin hydrate) and a chromone derivative (peucenin) were found to inhibit AChE activity in this bioassay.

The bark of catuaba (Erythroxylum vacciniifolium Martins, Erythroxylaceae), a tree native to the northern part of Brazil, was investigated for its alkaloid content. With the aim of obtaining preliminary structure information on-line, the alkaloid extract was analysed by high-performance liquid chromatography coupled to diode array UV detection, to mass spectrometry and to nuclear magnetic resonance. Interpretation of on-line spectroscopic data obtained from this extract led to structural elucidation of six new alkaloids and partial identification of 18 potentially original alkaloids bearing the same tropane skeleton esterified in positions 3 and 6 by 1-methyl-1H-pyrrol-2-carboxylic acid and/or 4-hydroxy-3,5-dimethoxybenzoic acid. (C) 2003 Elsevier B.V. All rights reserved.

The development and validation of a rapid qualitative and quantitative method based on an HPLC-UV-MS technique with atmospheric pressure chemical ionisation and electrospray ionisation for the analysis of yohimbine in a number of commercial aphrodisiac products is reported. HPLC with multiple-stage mass spectrometry experiments allowed the identification of the target compound and increased the selectivity of complex analyses such as those involved with multi-botanical preparations. The precision and the robustness of the method were improved by the use of two internal standards: codeine for UV detection and deuterium-labelled yohimbine for MS detection. Twenty commercial aphrodisiac preparations were analysed and the amount of yohimbine measured and expressed as the maximal dose per day suggested on product labels ranged from 1.32 to 23.16 mg. Copyright (C) 2003 John Wiley Sons, Ltd.