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The Latin American medicinal plant carqueja got its name from a morphologically similar plant from the Iberian peninsula. The popular name came in general use to indicate all species from the section Caulopterae from the genus Baccharis, which are often difficult to distinguish. Phytochemical studies indicate that the different species from this section contain similar compound classes, especially essential oils, flavonoids and diterpenes, but much variation is encountered in relation to the individual compounds within the species. There are also some indications for the existance of different chemotypes within species. Recovered information from manuscripts, elaborated in the Jesuit missions, and originally authored by the Jesuit friar Pedro de Montenegro around 1710, indicate that carqueja was used by the Guarani indians of Southern Latin America for wound treatment. This traditional use was largely lost and nowadays the plant is mainly indicated for intestinal problems, as a tonic or for treatment of diabetes. It might be possible that this is caused by a confusion with quina, around 1800, which led to the alternative popular name “Quina de Condamine”. The confusion about the identification of individual species persists up to today. In pharmacological studies performed with crude extracts, doubts arise about the species used and its phytochemical composition. Also in medicinal teas different species are used indiscriminantly leading to great differences in phytochemical profile. In this paper information about the phytochemistry, medicinal use, botanical specimens and nomenclature is extensively reviewed and recommendations for further studies and increased reproducibility of pharmacological studies are given.

Ethanol extract obtained from dried leaves of Acmella oleracea afforded after a liquid/liquid partition procedure a larvicidal hexane fraction (LC50 = 145.6 ppm) and a non larvicidal dichloromethane one. From the inactive fraction, three amides were identified, two new structures, named deca-6,9-dihydroxy-(2E,7E)-dienoic acid isobutylamide (1), deca-8,9-dihydroxy-(2E,6Z)-dienoic acid isobutylamide (2) and the known nona-2,3-dihydroxy-6,8-diynoic acid 2-phenylethylamide (3). Bioassay-guided chromatographic fractionation of the hexane partition led to the identification of an amide mixture, nona-(2Z)-en-6,8-diynoic acid 2-phenylethylamide (4) and deca-(2Z)-en-6,8-diynoic acid 2-phenylethlylamide (5). This mixture was active against Aedes aegypti larvae at LC50 = 7.6 ppm. Low toxicity of crude extracts and derived fractions on Artemia salina nauplies showed the possibility of using them to control the A. aegypti mosquito larvae. This is the first report on larvicidal activity of acetylenic 2-phenylethylamides and their identification in A. oleracea leaves.

The present state-of-the-art of NMR in plant metabolomics is reviewed. Attention is paid to the different practical aspects of the application of NMR. The sample preparation, the measurement of the spectrum, quantitative aspects and data analysis are discussed. Each stage has its specific problems, which are pointed out and recommendations are made.

Cyathenosin A, a spiropyranosyl derivative of protocatechuic acid was isolated from the stem pith of Cyathea phalerata Mart. Its structure was determined by MS, 1D and 2D NMR spectroscopic analyses and confirmed by single crystal X-ray analysis. Cyathenosin A is the first example of a naturally occurring compound containing a spirocyclic orthoester pyranosidic structure.

From the aerial parts of Hypericum polyanthemum Klotzsch ex Reichardt (Guttiferae), three chromenes, 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran; 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran were isolated. Their structures were determined by NMR spectroscopic analyses.

A suspension culture of Cinchona robusta which under normal culture conditions does not produce anthraquinones, produces a range of these anthraquinones after elicitation. Eight new anthraquinones were identified, robustaquinones A–H, in addition to two known anthraquinones, 1,3,8-trihydroxy-2-methoxyanthraquinone and copareolatin 6-methyl ether. Their oxygenation pattern, characterized by a 6,7-disubstitution in the A-ring, is rare and raises questions about their biogenetic origin. They might be derived from phenylpropanoids and not from o -succinylbenzoic acid.

gilsane@farmacia.ufrgs.br; 2) PPG-Botanica, ICBS, 91509-900 Porto Alegre, RS, Brazil, fax (051) 33167670; 3) Grupo Metabolomica, LCQUI/CCT, Universidade Estadual do Norte Fluminense, 28015-620 Campos dos Goytacazes, RJ, Brazil; 4) Faculte de Pharmacie, Universite de Picardie Jules Verne, 80037 Amiens, France. Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 572-574, November-December, 2007. Original article submitted August 1, 2006.

Glandular and nonglandular trichomes are spread over the vegetative and reproductive organs of Plectranthus madagascariensis. Two morphologically distinct types of glandular trichomes (capitate and peltate) are described. Capitate trichomes have an ovoid unicellular head and a short stalk cell or an elongated two- to three-celled stalk slightly enlarged at the base. Peltate trichomes, which show in vivo a characteristic orange-to-reddish color, are large, flattened-to-depressed in the center, and often appear as a ring of minute gems. They are composed of a large head with eight glandular cells arranged in a single layer. An unusual kind of capitate trichomes, reported for the first time in Lamiaceae, occurs typically restricted to the calyx. These trichomes possess a two- or three-celled stalk and a long, unicellular conical, glandular head. The several types of trichomes differ in the secretion process The essential oils of P. madagascariensis, isolated by hydrodistillation and by distillation-extractio...

In 9,10-anthraquinones the chemical shift of a peri-hydroxyl proton is affected by the substituents in the other benzenoid ring. These effects are additive. They are useful for the determination of substitution patterns and have been used to revise the structures of six previously reported anthraquinones containing methoxyl, hydroxyl, methylenedioxy and β-methyl substituents. Because the chemical shifts of the other protons are hardly affected by substitutions in the other ring, the characteristic chemical shifts for a wide variety of substitution patterns could be derived.

The possible limitation of the rate of biosynthesis of terpenoid indole alkaloids by low enzyme levels was investigated in two cell lines of Tabernaemontana divaricata with different terpenoid indole alkaloid biosynthetic capacities. The activities of tryptophan decarboxylase (TDC), strictosidine synthase (SSS), strictosidine glucosidase (SG), isopentenyl pyrophosphate isomerase (IPP isomerase) and geraniol 10-hydroxylase (G10H) of both cell lines were compared. The activities of TDC, SSS and IPP isomerase did not show a direct relationship with the biosynthetic capacity but SG and G10H might be limiting. In order to test whether the availability of the terpenoid precursor limits the biosynthesis of the terpenoid indole alkaloids, loganin was fed to the cultures. Loganin-feeding did not influence any of the measured enzyme activities but increased the terpenoid indole alkaloid accumulation of both cell lines to similar levels. A five-fold increase was observed for the accumulating line and a more than 100-fold increase for the low-accumulating one. Strictosidine accumulated mainly in the low-accumulating cell line which has high TDC and low SG activity; the amounts and types of the other terpenoid indole alkaloids which accumulated were similar in both lines. From this it can be concluded that the biosynthesis of terpenoid indole alkaloids in both cultures is limited by the availability of terpenoid precursors; this pathway is not saturated with substrates under normal culture conditions.

Ajmalicine degradation was investigated in a low alkaloid-accumulating Catharanthus roseus cell culture, and compared with the chemical degradation which occurred in medium without cells. Degradation was found to be faster in the presence of cells. By adding [Me- 2 H 3 ] ajmalicine to the cell culture the production and the degradation rates were estimated. Both were relatively high at the beginning of the growth phase while the degradation rate was also high in the stationary phase.

Two cell lines of Tabernaemontana divaricata derived from the same suspension culture were compared with respect to their biotransformation capacity. One is a high indole alkaloid-producing culture which accumulated mainly O-acetylvallesamine. The other cell line biosynthesizes terpenoid indole alkaloids in much lower amounts. Both cell lines were cultured in medium containing either conopharyngine, coronaridine, vobasine or tabersonine. Chemical breakdown was followed in fresh and used culture medium in the absence of cell culture. All the alkaloids investigated underwent chemical transformation. Most of the biotransformation products accumulated by the cultures have been reported to occur in intact plants. Since all the alkaloids added were transformed in the same way by both cultures, the two cell lines seem to have the same biotransformation potential.

Inoculation of Vicia sativa subsp. nigra (V. sativa) roots with Rhizobium leguminosarum biovar. viciae (R.l. viciae) bacteria substantially increases the ability of V. sativa to induce rhizobial nodulation (nod) genes. This increase is caused by the additional release of flavanones and chalcones which all induce the nod genes of R.l. viciae (K. Recourt et al., Plant Mol Biol 16: 841-852). In this paper, we describe the analyses of the flavonoids present in roots of V. sativa. Independent of inoculation with R.l. viciae, these roots contain four 3-O-glycosides of the flavonol kaempferol. These flavonoids appeared not capable of inducing the nod genes of R.l. viciae but instead are moderately active in inhibiting the activated state of those nod genes. Roots of 7-day-old V. sativa seedlings did not show any kaempferol-glycosidase activity consistent with the observation that kaempferol is not released upon inoculation with R.l. viciae. It is therefore most likely that inoculation with infective (nodulating) R.l. viciae bacteria results in de novo flavonoid biosynthesis and not in liberation of flavonoids from a pre-existing pool.

A method for the rapid phytochemical characterization of plant cell cultures, using proton nuclear magnetic resonance spectroscopy (1H NMR), is described. Aqueous extracts were prepared from a large number of plant cell suspension cultures. Following lyophilization, the extracts were redissolved in D2O containing an internal standard to enable quantification and 1H NMR spectra were measured. In the spectra, signals from sugars and amino acids were observed. The patterns obtained were characteristic of cell lines, with related lines typically showing similar characteristics, e.g., Tabernaemontana suspension cultures are characterized by their ability to accumulate large amounts of arginine, while Catharanthus cultures mainly accumulate glutamine. The amounts of amino acids accumulated were found to be determined mainly by the medium on which the cell cultures were growing. Murashige and Skoog medium supported nitrogen storage in amino acids, while Gamborg B5 medium did not. Reference spectra of some of the most common constituents are presented, together with data enabling a direct quantification of these compounds in aqueous extracts.

Summary The uptake and accumulation of the indole alkaloid cinchonamine and the quinoline alkaloid quinine in suspension cultured cells of Cinchona robusta and Catharanthus roseus were studied. Both alkaloids are endogenous to Cinchona and are not found in Catharanthus . Uptake in Cinchona appeared to be the resultant of two processes: rapid uptake with an equilibrium after 2 h, and slow and steady uptake with equilibrium after about 6 h. Occasionally, the cells of Cinchona lost their brownish-orange colour. During this physiological state of the cells only rapid uptake was observed. Steady uptake in these (white) cells could be induced by vanadate. The time course of rapid uptake in Cinchona was almost identical to the time course of uptake in Catharanthus , and had characteristics of free diffusion of non-protonated alkaloids in combination with ion-trapping of protonated alkaloids. Slow and steady uptake of quinine in Cinchona cells was determined and simultaneously the time course of the pH of the vacuoles was measured with in vivo 31 P-NMR. The slow and steady uptake appeared to be correlated with a steady decrease in vacuolar pH. The results demonstrate that a strong basic alkaloid, like quinine, can be accumulated by free diffusion of its neutral form in combination with ion-trapping of its charged form. Slow and steady uptake, which is often observed in alkaloid transport, is not caused by additional accumulation mechanisms or alkaloid carriers, but is the effect of slow acidification of the vacuoles.

Flavonoids released by roots of Vicia sativa subsp. nigra (V. sativa) activate nodulation genes of the homologous bacterium Rhizobium leguminosarum biovar viciae (R. l. viciae). Inoculation of V. sativa roots with infective R. l. viciae bacteria largely increases the nod gene-inducing ability of V. sativa root exudate (A.A.N. van Brussel et al., J Bact 172: 5394-5401). The present study showed that, in contrast to sterile roots and roots inoculated with R. l. viciae cured of its Sym plasmid, roots inoculated with R. l. viciae harboring its Sym plasmid released additional nod gene-inducing flavonoids. Using 1H-NMR, the structures of the major inducers released by inoculated roots, 6 flavanones and 2 chalcones, were elucidated. Roots extracts of (un)inoculated V. sativa contain 4 major non-inducing, most likely glycosylated, flavonoids. Therefore, the released flavonoids may either derive from the root flavonoids or inoculation with R. l. viciae activates de novo flavonoid biosynthesis.

A cell suspension culture from Tabernaemontana divaricata was fed with 15 N-labelled ammonium or nitrate. The incorporation of label in free amino acids, protein amino acids and indole alkaloids was determined. Ammonium was found to be used more extensively than nitrate in the biosynthesis of these compounds. For tryptamine considerably lower labelling percentages were found than for the indole alkaloid O -acetylvallesamine and the amino acids. This indicates a vacuolar pool of tryptamine, formed at the beginning of the culture-period and not available for further alkaloid biosynthesis.

A new iridoid glucoside, 2′-apiosylgardoside together with the known compounds theviridoside, ipolamiide and verbascoside were isolated from leaves of Verbenoxylum reitzii. The structure of the new compound was determined mainly by NMR spectroscopy.

Angelonia integerrima gave the known iridoid glucosides galiridoside, harpagide, ajugol, 6-hydroxyantirrhide, antirrhide, daunoside, aucubin and stegioside II, together with a new compound, named angeloside.

Eucnide bartonioides yielded morroniside as the main iridoid constituent. In addition, six minor iridoid glucosides were isolated namely the known glucosides; kingiside, sweroside, secologanol, 8-epi-loganin and loganin as well as a novel iridoid glucoside, which by spectroscopic methods was assigned to be 5-hydroxyloganin. The compound 8-epi-loganin has so far been reported only from Scrophulariaceae and related families and it is rather unexpected in Loasaceae. © 1997 Elsevier Science Ltd. All rights reserved

Baccharis ochracea contains a new diterpene in addition to the known compounds chlorogenic acid, isochlorogenic acid, α-spinasterol and isoquercitrin. The diterpene is a geranylgeraniol derivative with a butenolide ring.

An HPLC method with UV detection has been developed and validated in order to quantify quadranguloside, the main saponin of the leaves of Passiflora alata. The analysis was achieved by simple isocratic elution (with acetonitrile:aqueous phosphoric acid) using a Nova-Pak phenyl column. The amount of quadranguloside present in aqueous extracts from leaves of P. alata was estimated to be 22.2% (w/w), corresponding to 0.8% (w/w) in relation to dried leaves. The assay method described is simple, rapid, accurate and sensitive, and may form part of future drug authentication protocols.

From leaves of Psychotria leiocarpa, an indole alkaloid was isolated to which the structure N,beta-D-glucopyranosyl vincosamide (1) was assigned. This represents the first report of an N-glycosylated monoterpenoid indole alkaloid. In field-grown plants highest amounts of 1 were found in the leaves (2.5% of dry wt) and fruit pulp (1.5% dry wt). Lower amounts were found in the stems (0.2% dry wt) and the seeds (0.1% of dry wt), whereas the alkaloid was not detected in the roots. The accumulation of 1 in aseptic seedlings was also restricted to the shoots and increased with plant age and light exposure, independent of the supply of sucrose in the culture medium.

A novel pair of lactone dimers, telephinone a and b, were isolated from roots of Sedum telephium . The structure of telephinone a and b was assigned as 3′,4′,-dihydro-4,4′-dimethyl-[2,3′-bifuran]-5,5′(2H,2′H)-dione. A minor lactone was identified as isosiphonodin, 3-hydroxymethyl-2(5H)-furanone.

Nodulation (root nodule formation) in legume roots is initiated by the induction of cell divisions and formation of root nodule primordia in the plant root cortex, usually in front of the protoxylem ridges of the central root cylinder. We isolated a factor from the central cylinder (stele) of pea roots which enhances hormone-induced cell proliferation in root cortex explants at positions similar to those of nodule primordia. The factor was identified as uridine. Uridine may act as a morphogen in plant roots at picomolar concentrations.

Two cell lines of Tabernaemontana divaricata derived from the same suspension culture accumulate different amounts of the terpenoid indole alkaloids O-acetylvallesamine and voaphylline. [(15)N]O-acetylvallesamine and [(15)N]voaphylline were added to the suspension cultures to investigate whether the lack of accumulating capacity of one of the cell lines was due to a low biosynthetic ability or to high turnover rates. The difference was shown to be due to the inability of the cell culture to biosynthesize both alkaloids. Both cell lines were able to metabolize O-acetylvallesamine. This metabolisation occurred mainly during the stationary phase. The alkaloids added were chemically unstable under culture conditions. Under normal batch cell culture conditions chemical breakdown is thought to play a minor role in the total amount of compound transformed.

Species of Hypericum accumulate phenolic compounds with the phloroglucinol substitution pattern, and there are many reports of such substances isolated from the aerial parts of the plants. In this study, flowers of plants grown in south Brazil were analyzed by means of HPLC, verifying the presence of benzopyrans in H. polyanthemum and dimeric phloroglucinol derivatives in H. caprifoliatum, H. connatum, H. myrianthum and H. polyanthemum. All flowers presented flavonoids, mainly quercetin derivatives, whereas hypericin and analogues were not detected.

With the aim of quantifying intra- and extracellular carbohydrates media and cell-extracts from a Tabernaemontana divaricata plant cell-suspension culture were investigated with (1)H-NMR.For suppression of the solvent peak the Meiboom-Gill modification of the Carr-Purcell (CPMG) spin-echo sequence was used after addition of a paramagnetic relaxation agent (Mn(2+)) to the sample. Several aspects of this method were optimized (the manganese concentration, the interpulse delay and the number of spin-echo cycles) so as to obtain a rapid and easy method in which no pretreatment of media or cell-extracts was needed. Besides the speed and ease of the method, also the direct identification of carbohydrates and other main components is an advantage.The exhaustion of extracellular carbohydrates was found to coincide with the maximum amount of intracellular carbohydrates. The intracellular carbohydrates, i.e. glucose and fructose, were consumed at a low rate, during several weeks.

Chemical investigation of ethanolic extracts of the leaves and stems of Baccharis illinita resulted in the isolation and identification of retusin, quercetin-3, 7, 4′-trimethyl ether, aromadendrin-7-methyl ether, 3-(4-hydroxyphenyl)-2-propenoic acid ( E and Z) hexacosyl ester, 3-(3, 4-dihydroxyphenyl)-2-propenoic acid ( E) hexacosyl ester and hexacosanoyl acid acetyl ester (all new for this genus), together with apigenin-7, 4′-dimethyl ether, kaempferol-3, 7, 4′-trimethyl ether, kaempferol-7,4′-dimethyl ether, p-coumaric acid, hexacosanol, and stigmasta-5, 22-dien-3-ol (3β, 22 E, 24 S) hexadecanoyl. Their structures were assigned by comparison of the obtained spectroscopic data with those in the literature. Extracts of the flowers, roots and stems demonstrated marked anticoagulant activity, but only the extract of the stems had any significant effect on platelet aggregation.

Treatment of suspension cultures of some Tabernaemontana species (Apocynaceae) with elicitors (e.g. cellulase, Candida albicans) result in a rapid de novo production of antimicrobial active triterpenes. The triterpenes are identified as ursene carboxylic acid derivatives. These triterpenes are not produced by an elicited cell suspension culture of Catharanthus roseus, another Apocynaceae.

Root exudate of Vicia sativa contains 7 inducers for the nodA promoter of Rhizobium leguminosarum biovar viciae. Six of these inducers are flavanones. One inducer was identified as 3,5,7,3'-tetrahydroxy-4'-methoxyflavanone, and a second inducer most likely is 7,3'-dihydroxy-4'-methoxyflavanone. The inducing activity of these compounds and the other inducers depends on the nodD gene present in the test strains, which originated either from R. leguminosarum biovars viciae or trifolii, or from R. meliloti. Three inducers are 'common', three others almost exclusively induce the nodA promoter in the presence of the R. leguminosarum biovar viciae nodD gene, and the last one is active with the noD genes of either R. leguminosarum biovar viciae or that of R. meliloti. Testing of a large number of flavonoids revealed two classes of structural features required for inducing ability: (i) features required for induction in general, and (ii), features restricting the inducing ability to (a) specific nodD gene(s). These features are discussed in relation to current models of the process of nodD-mediated transcription activation of the inducible nod genes.

An investigation of two species of Nuxia showed that this genus is characterized by the presence of the eight-carbon iridoid glucoside unedoside and/or its derivatives. From N. floribunda unedoside, nuxioside (6- O -α- l -rhamnopyranosyl-unedoside) and 2″-acetyl-3″-cinnamoyl-nuxioside were isolated, while from N. oppositifolia 2″-acetyl-3″-benzoyl-nuxioside was obtained. Both plants contained verbascoside. The biosynthesis of unedoside in N. floribunda was investigated and deoxyloganic acid was found to be a precursor, similar to what was found for the eight-carbon iridoids in Thunbergia alata earlier investigated. The taxonomic position of Nuxia and Buddlejaceae is briefly discussed.

CESAR VICENTE FERREIRA BATISTA, JAN SCHRIPSEMA,*~" ROBERT VERPOORTE,* SANDRA BEATRIZ RECH and AMELIA T. HENRIQUES+ + Graduated Course in Pharmaceutical Sciences, UFRGS, Av. Ipiranga 2752, 90.610.000, Porto Alegre, RS, Brazil; *Center for Biopharmaceutical Sciences, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands (Received in revised form 24 July 1995) Key Word Index--Rauwolfia sellowii; Apocynaceae; leaves; indole alkaloids; sellowiine. Abstraet--A new alkaloid, sellowiine (N~-demethyl-20-deethyl suaveoline), was obtained from leaves of Rauwolfia sellowii collected at two different locations in southern Brazil. Also 9btained were the known alkaloids, perakine, raucaffrinoline, vomilenine, 19ct,20~t-epoxy-akuammicine, picrinine and 12-demethoxytabernulosine. The NMR spectra of the alkaloids were assigned completely.