Cephaelis Ipecacuanha Research Articles

Molecular Cloning of anO-Methyltransferase from Adventitious Roots ofCarapichea ipecacuanha

Carapichea ipecacuanha produces various emetine-type alkaloids, known as ipecac alkaloids, which have long been used as expectorants, emetics, and amebicides. In this study, we isolated an O-methyltransferase cDNA from this medicinal plant. The encoded protein (CiOMT1) showed 98% sequence identity to IpeOMT2, which catalyzes the 7'-O-methylation of 7'-O-demethylcephaeline to form cephaeline at the penultimate step of emetine biosynthesis (Nomura and Kutchan, J. Biol. Chem., 285, 7722-7738 (2010)). Recombinant CiOMT1 showed both 7'-O-methylation and 6'-O-methylation activities at the last two steps of emetine biosynthesis. This indicates that small differences in amino acid residues are responsible for distinct regional methylation specificities between IpeOMT2 and CiOMT1, and that CiOMT1 might contribute to two sequential O-methylation steps from 7'-O-demethylcephaeline to emetine.

Is a metabolic enzyme complex involved in the efficient and accurate control of Ipecac alkaloid biosynthesis in Psychotria ipecacuanha?

Ipecac alkaloids produced in the medicinal plant Psychotria ipecacuanha such as emetine and cephaeline possess a monoterpenoid-tetrahydroisoquinoline skeleton, which is formed by condensation of dopamine and secologanin. The condensation products are deglucosylated, and the resulting aglycon is further processed to protoemetine, which is condensed with the second molecule of dopamine, followed by conversion into cephaeline and emetine. Although four hydroxy groups derived from two molecules of dopamine need to be methylated to form emetine, the order of O-methylation reactions had been veiled. We recently identified three Ipecac alkaloid O-methyltransferases (IpeOMT1-IpeOMT3) that are sufficient for catalyzing O-methylations of all four hydroxy groups. Detailed characterization of their catalytic properties with integration of that of the previously identified Ipecac alkaloid β-glucosidase (IpeGlu1) revealed a large portion of the biosynthetic pathway of Ipecac alkaloids. The results provide proof-of-concept to the significance and the usefulness of the biosynthetic pathway strategy by EST analysis coupled with recombinant enzyme characterization. At the same time, however, the results raised an intriguing question about the subcellular network between the biosynthetic enzymes and intermediates. Here, we provide additional discussion about this point, and indicate what remains to be elucidated.

ChemInform Abstract: Ipecac Alkaloids from Cephaelis acuminata

From the dried roots of Cephaelis acuminata, five ipecac alkaloids, neocephaeline, 7'-O-demethylcephaeline, 10-O-demethylcephaeline, 2'-N-(1-deoxy-1-beta-D-fructopyranosyl)cephaeline and 2'-N-(1-deoxy-1-beta-D-fructopyranosyl)neocephaeline, were isolated, along with emetine, cephaeline, psychotrine, protoemetine, 9-demethylprotoemetinol and isocephaeline. Structures were determined by spectroscopic and chemical means.

Molecular phylogeography of Carapichea ipecacuanha, an amphitropical shrub that occurs in the understory of both semideciduous and evergreen forests

The medicinal shrub Carapichea ipecacuanha (ipecac) is an amphitropic species with three disjunct areas of distribution. In the Brazilian Atlantic and Amazonian ranges, the species was associated mostly with the understory of seasonal semideciduous forests, whereas in the Central American-Colombian range, the species occurred in the understory of moist evergreen forests. We examined the phylogeographic structure of ipecac using chloroplast trnT-trnL and nuclear internal transcribed spacer (ITS) sequences from 120 and 46 specimens, respectively. To complement existing data on root alkaloid profiles, we used high-performance liquid chromatography to assess the levels of emetine and cephaeline in 33 specimens from the two Brazilian ranges. The three ranges shared neither nuclear nor chloroplast haplotypes. The phylogeographic structures showed an uneven distribution of genetic diversity, sharp breaks and high levels of genetic differentiation among ranges. Our results suggest that the extant populations are descendents of at least four distinct ancestral lineages. The Atlantic ipecacs showed higher levels of genetic diversity than ipecacs from the other two ranges; it is likely that they derive from two ancestral lineages, with long-term persistence in that region. The Amazonian ipecacs were monomorphic with respect to the ITS and cpDNA sequences, which supports the view that there was a recent expansion from a single parental source after a strong genetic bottleneck. The existence of a fourth distinct lineage is apparent from the high levels of genetic and chemical differentiation that we identified in the Central American-Columbian ipecacs.

Three New O-Methyltransferases Are Sufficient for All O-Methylation Reactions of Ipecac Alkaloid Biosynthesis in Root Culture of Psychotria ipecacuanha

The medicinal plant Psychotria ipecacuanha produces ipecac alkaloids, a series of monoterpenoid-isoquinoline alkaloids such as emetine and cephaeline, whose biosynthesis derives from condensation of dopamine and secologanin. Here, we identified three cDNAs, IpeOMT1-IpeOMT3, encoding ipecac alkaloid O-methyltransferases (OMTs) from P. ipecacuanha. They were coordinately transcribed with the recently identified ipecac alkaloid beta-glucosidase Ipeglu1. Their amino acid sequences were closely related to each other and rather to the flavonoid OMTs than to the OMTs involved in benzylisoquinoline alkaloid biosynthesis. Characterization of the recombinant IpeOMT enzymes with integration of the enzymatic properties of the IpeGlu1 revealed that emetine biosynthesis branches off from N-deacetylisoipecoside through its 6-O-methylation by IpeOMT1, with a minor contribution by IpeOMT2, followed by deglucosylation by IpeGlu1. The 7-hydroxy group of the isoquinoline skeleton of the aglycon is methylated by IpeOMT3 prior to the formation of protoemetine that is condensed with a second dopamine molecule, followed by sequential O-methylations by IpeOMT2 and IpeOMT1 to form cephaeline and emetine, respectively. In addition to this central pathway of ipecac alkaloid biosynthesis, formation of all methyl derivatives of ipecac alkaloids in P. ipecacuanha could be explained by the enzymatic activities of IpeOMT1-IpeOMT3, indicating that they are sufficient for all O-methylation reactions of ipecac alkaloid biosynthesis.

Clonal diversity and conservation genetics of the medicinal plant Carapichea ipecacuanha (Rubiaceae)

The roots of the understorey shrub Carapichea ipecacuanha (ipecac) have medicinal properties, and the uprooting of wild plants has supplied most of the world demand for this species. Although under severe population decline, C. ipecacuanha lacks legal protection. In the wild, the aerial stems of ipecac clump together to form clusters with well-defined borders. Cluster size may range from several to hundreds of aerial stems. To investigate the extent of clonality among aerial stems in ipecac clusters, we sampled 50 wild clusters (a total of 291 aerial stems) and screened them with 89 inter-simple sequence repeat (ISSR) markers. The 291 aerial stems were grouped into 42 putative clones. The clonal groups generally consisted of aerial stems from the same cluster, and there was little or no genetic differentiation among aerial stems at the cluster level. These findings suggest that strategies designed to conserve ipecac in situ should not rely upon census data, which are based on the number of aerial stems per cluster and the number of clusters per population, because such data greatly underestimate the species effective population size and genetic diversity. Our results also indicate that this species needs protection at a federal level.

Estudo ecogeográfico da poaia [Psychotria ipecacuanha (Brot.) Stokes

Este estudo foi conduzido com 13 populações amostradas nos estados RJ, MG, ES e BA para a coleta de dados ecogeográficos da poaia (Psychotria ipecacuanha). Foi conduzida a análise de freqüência de ocorrência de características ambientais, tais como: altitude, pluviosidade, temperaturas máxima, média e mínima absolutas, latitude e longitude. A análise etnobotânica consistiu no levantamento de formas terapêuticas, homeopáticas e as crendices relacionadas à utilização da poaia. Os teores dos alcalóides emetina e cefelina foram determinados, utilizando-se cromatografia líquida de alta eficiência (CLAE). Aproximadamente 87% das reboleiras ocorreram em solo distrófico. Dentre os micronutrientes analisados, Fe, Mn, e Cu apresentaram correlações significativas com número de hastes por reboleira (NH) e peso de raízes secas (PR). O comprimento, em 65% das reboleiras, foi inferior a 2 m, sendo que as dimensões médias das reboleiras foram de 1,8 x 1,3 m. A porcentagem da radiação fotossiteticamente ativa variou entre 0,14 e 17%. Dos locais onde ocorreram as reboleiras, cerca de 69% foram consideradas com pequena inclinação. A caracterização química revelou que em cerca de 65% das amostras de raízes analisadas, foram observados teores de emetina entre 1 e 2%, e o teor de cefelina, em 61% das amostras, foi inferior a 0,3%. As características morfológicas e químicas foram analisadas pela técnica de componentes principais, concluindo-se que os resultados obtidos são pouco informativos sobre a diversidade genética, possibilitando o descarte de características cuja variação é pequena e, também, aquelas altamente correlacionadas entre si.

The New β-D-Glucosidase in Terpenoid-Isoquinoline Alkaloid Biosynthesis in Psychotria ipecacuanha

Ipecac alkaloids produced in the medicinal plant Psychotria ipecacuanha such as emetine and cephaeline possess a monoterpenoid-tetrahydroisoquinoline skeleton, which is formed by condensation of dopamine and secologanin. Deglucosylation of one of the condensed products N-deacetylisoipecoside (1 alpha(S)-epimer) is considered to be a part of the reactions for emetine biosynthesis, whereas its 1 beta(R)-epimer N-deacetylipecoside is converted to ipecoside in P. ipecacuanha. Here, we isolated a cDNA clone Ipeglu1 encoding Ipecac alkaloid beta-D-glucosidase from P. ipecacuanha. The deduced protein showed 54 and 48% identities to raucaffricine beta-glucosidase and strictosidine beta-glucosidase, respectively. Recombinant IpeGlu1 enzyme preferentially hydrolyzed glucosidic Ipecac alkaloids except for their lactams, but showed poor or no activity toward other substrates, including terpenoid-indole alkaloid glucosides. Liquid chromatography-tandem mass spectrometry analysis of deglucosylated products of N-deacetylisoipecoside revealed spontaneous transitions of the highly reactive aglycons, one of which was supposed to be the intermediate for emetine biosynthesis. IpeGlu1 activity was extremely poor toward 7-O-methyl and 6,7-O,O-dimethyl derivatives. However, 6-O-methyl derivatives were hydrolyzed as efficiently as non-methylated substrates, suggesting the possibility of 6-O-methylation prior to deglucosylation by IpeGlu1. In contrast to the strictosidine beta-glucosidase that stereospecifically hydrolyzes 3 alpha(S)-epimer in terpenoid-indole alkaloid biosynthesis, IpeGlu1 lacked stereospecificity for its substrates where 1 beta(R)-epimers were preferred to 1 alpha(S)-epimers, although ipecoside (1 beta(R)) is a major alkaloidal glucoside in P. ipecacuanha, suggesting the compartmentalization of IpeGlu1 from ipecoside. These facts have significant implications for distinct physiological roles of 1 alpha(S)- and 1 beta(R)-epimers and for the involvement of IpeGlu1 in the metabolic fate of both of them.

Reproductive studies in ipecac (Psychotria ipecacuanha (Brot.) stockes; Rubiaceae): pollen development and morphology

The aim of this work was to carry out the reproductive studies on Brazilian accessions of ipecac, Psychotria ipecacuanha. It presented heterostyly, with brevistylous and longistylous flowers. The pollen development was observed from the sections of the anthers embedded in resin. Anther development was normal as usually observed in dicotyledones, displaying four layers: outer epidermis, endothecium, middle layer and inner tapetum. The pollen was bicellular and filled with starch at the microspore stage. Pollen morphology was studied using SEM, which showed pollen polymorphism within and between the two floral morphs. Five types of pollen with reticulate or perforate exine were identified. The characteristics showed that the sexual process was as important as the vegetative propagation for the reproduction of this species.

Genetic diversity and geographic differentiation of disjunct Atlantic and Amazonian populations of Psychotria ipecacuanha (Rubiaceae)

Ipecac (Psychotria ipecacuanha) is a perennial, medicinal herb that grows in the understory of semi-deciduous tropical forests in the Neotropics. Ipecacs present a widely disjunct distribution, with two of its three ranges occurring in Brazil. The Amazonian populations are at least 1600 km from the nearest Atlantic populations. This work used ISSR markers to compare the genetic diversity and structure of populations from the two Brazilian ranges. Lower genetic diversity in Amazon populations (P = 60.11%, Hs = 0.18) and higher genetic diversity in Atlantic populations (P = 73.94%, Hs = 0.20) were detected. Differentiation between ranges were high (theta (B) = 0.6838, G(ST)-B = 0.6665). AMOVA revealed that 65.3% of the total molecular variance can be attributed to regional differences between the two ranges. Principal coordinate analyses and cluster analyses organized ipecacs at either individual or population level into two exclusive groups that correspond each to one of the two disjunct ranges, without exception. The results do not support a scenario that postulates human-mediated, long-distance dispersal events as a plausible origin for the distribution of the Brazilian ipecacs, but indicate geographic isolation as a long-standing barrier to genetic exchange and connectivity among populations from different ranges. Conservation implications are discussed.

Gibberellic acid improved shoot multiplication in Cephaelis ipecacuanha

The internodes of Cephaelis ipecacuanha elongated when cultured on Gamborg B5 solid medium supplemented with 0.5 or 1 mg/L gibberellic acid (GA3). The size of the elongated internode doubled in length compared to the untreated shoots, and the adventitious shoots formed on the elongated internodes. The shoots grew easily into plantlets without the use of auxin for rooting. The ex vitro regenerates cultivated in the greenhouse showed normal characteristics. Emetic alkaloids were detected in the leaves of in vitro shoots and the roots of regenerates cultivated in the greenhouse. This method using GA3 propagated numerous plants at a rate of more than 100 times compared to the method without GA3.

Characteristics of Apoptosis Induction by the Alkaloid Emetine in Human Tumour Cell Lines

Cytotoxic and apoptosis-inducing effects of the alkaloid emetine from Psychotria ipecacuanha (Rubiaceae) were studied in human cell lines. In Jurkat T-cells emetine leads to phosphatidylserine exposure, mitochondrial depolarisation, and DNA fragmentation. Furthermore, activation of several caspases (caspase-3, -9/6, and -8) was demonstrated in a fluorescent caspase assay. Bcl-2 over-expressing cells are less sensitive to emetine while caspase-8-deficient Jurkat T-cells react similarly to wild-type cells. This indicates that apoptosis induction is mediated via the mitochondrial pathway. By using hepatoma cell lines with differing p53 expression, it was concluded that p53 does not seem to play a role in apoptosis induction by emetine. Alterations of protein profiles during emetine-induced apoptosis were analysed by 2D-PAGE and MALDI-TOF-MS. A new protein spot was apparent after treatment with emetine: It could be identified as the N-terminal fragment lamin B1, which is released after cleavage by caspase-6.

Reproductive studies on ipecac (Cephaelis ipecacuanha (Brot.) A. Rich; Rubiaceae): meiotic behavior and pollen viability

Reproductive studies were carried out on Brazilian accessions of ipecac, Cephaelis ipecacuanha. Meiotic behavior was studied using the squashing technique. Irregular chromosome segregation in meiosis I and II, many sets of chromosomes in telophase II, micronuclei, incorrect cytoplasm division, incomplete cytokinesis and anomalous post-meiotic products, mainly polyads, were observed. The mean meiotic index was lower than 72%. Pollen viability was analyzed using Alexander solution, and the percentages ranged between brevistylous and longistylous floral morphs (85.3 to 93.1%), and among different localities (82.5 to 92.6%) analyzed. The size of pollen ranged between viable and sterile, and empty and shrunken sterile. In its natural habitat, this species is known to propagate by vegetative multiplication, but sexual reproduction seems to be as important as the vegetative propagation to this species.

Lectotypification of Callicocca ipecacuanha Brot. and neotypification of Cephaelis acuminata H.Karst., with reference to the drug ipecac

AbstractThe main pharmacopoeias cite the roots of Cephaelis ipecacuanha (Brot.) A.Rich. (≡ Callicocca ipecacuanha Brot.) and Cephaelis acuminata H.Karst. as the source of the crude drug ipecac. However, C. acuminata does not occur in botanical literature, except in connection with the original description, and this falls entirely within the variation of the widespread and variable Cephaelis ipecacuanha. Callicocca ipecacuanha was described from Brazil, but no type specimen has been found and the name is here lectotypified with an illustration from the protologue. For Cephaelis acuminata, described from Colombia, no original material is extant, and a recent specimen from Colombia is here selected to serve as neotype. In the most recent revision of the group the name Carapichea ipecacuanha (Brot.) L. Andersson is used for the species.

Distyly and variation in floral traits in natural populations of Psychotria ipecacuanha (Brot.) Stokes (Rubiaceae)

Psychotria ipecacuanha is a perennial, medicinal herb that grows in clusters in the understory of humid, shady areas of the Atlantic Rain Forest of southeastern Brazil. The present study characterized the variation in floral traits among 35 clusters from three natural populations of this plant species. Field observations showed that the clusters are isomorphic, that is, a given cluster will either set long-styled or short-styled flowers. Stigmas and anthers are reciprocally placed in each morph, a dimorphism characteristic of distyly. The populations are isoplethic, that is, a given population exhibits an equilibrium 1:1 ratio of floral morphs. Morphometric analyses revealed that anther length, stigma length, corolla diameter, and pollen grain diameter were consistently greater in short-styled flowers, regardless of the population investigated. Significant differences for floral traits in the short-styled morph were found among populations. Floral traits in the long-styled morph also showed some significant differences among populations, but not for stigma height and corolla length. Controlled pollinations carried out in natural populations showed that fruit production was higher after inter-morph pollination. Nevertheless, observations of pollen tube growth in style, and also fruit production after spontaneous self-pollination and intra-morph pollination, indicated partial intramorph compatibility in this plant species.

Variation in emetine and cephaeline contents in roots of wild Ipecac ( Psychotria ipecacuanha)

Ipecac ( Psychotria ipecacuanha) is a perennial, medicinal herb that grows as clusters in the understory of humid, shady areas of the Atlantic Rain Forest of southeastern Brazil. This investigation followed the contents of emetine and cephaeline, the bioactive constituents, and assessed root attributes in roots that were sampled periodically from four clusters of Ipecac (VRB8, ITA1, ITA2, and ITA3) that were growing in natural conditions. HPLC analyses showed that the content of the two alkaloids underwent monthly fluctuations over one year period. The concentration of emetine, but not cephaeline, differed significantly among the four clusters. The highest mean content of emetine was found in VRB8 (1.44%), followed by ITA1 (1.14%), ITA2 (0.63%), and finally ITA3 (0.44%). The highest mean content of cephaeline was found in ITA2 and ITA3 (0.26%), although it was not significantly different from ITA1 and VRB8 contents (0.15%). Correlation analysis revealed that contents of emetine are significant ( P < 0.01), but negatively correlated (−0.23) with that of cephaeline. Small sized roots characterized the low-emetine clusters ITA2 and ITA3, while the high-emetine clusters ITA1 and VRB8 consistently yielded larger roots. In these four clusters, emetine contents were correlated positively with fresh root weight, and with diameter and weight of the dried root. Conversely, cephaeline contents were negatively correlated with these three attributes.

Transformation of ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes.

Transformed root cultures of ipecac (Cephaelis ipecacuanha A. Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15 834. Southern blot analysis of the established roots revealed that only the TL-DNA was integrated into the plant genome without incorporation of the TR-DNA. The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark. The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 degrees C under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants. Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark. The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.

Reproductive Studies in Ipecac (Cephaelis ipecacuanha (Brot.) A. Rich; Rubiaceae): Flower Bud and Anther Size Associated to Male Gamete Development Stages

Reproductive studies were carried out on Brazilian accessions of Cephaelis ipecacuanha, which is considered a threatened species. Meiotic behavior was studied using squashing technique with 1% acetic carmine and floral buds and anther were measured for the following male gamete development stages: meiosis I, meiosis II, tetrads, microspores and pollen. Bud and anther length showed significant differences (P<0.01) among the stages of pollen development. There was high significant correlation between bud and anther length by Pearson correlation. Tukey test results indicated that each analyzed stage could be associated to bud length mean but it was not found the same association for anther length.

A quantitative assessment of genetic erosion in ipecac (Psychotria ipecacuanha)

In this study, we present a methodology by which the threat of genetic erosion to a wild plant species growing in a given geographic region can be assessed in a quantitative manner. Evaluation of threat is carried out by scoring a checklist based on information about the ecosystem and the target species. This approach was used to assess the threat of genetic erosion faced by ipecac (Psychotria ipecacuanha), an endangered medicinal plant that is native to the Atlantic Rain Forest of southeastern Brazil. An analysis of the spatial distribution of 20 risk factors over 14 forest fragments indicated that the threat of genetic erosion is not uniformly distributed over the region. The most endangered clusters of ipecac are facing presently a threat almost three times larger than that faced by the least endangered.We were able to identify the short distance of a forest fragment to the major population centres and thepoor conservation status of the species as the factors that present the major contribution to threat. The distance of a forest fragment to new development projects, the extent of the use of a wild habitat within a forest fragment, and the susceptibility of target species to grazing animals were considered the least important factors of risk. The results obtained by using this approach allowed us to formulate an itemized description of which category of forest fragments we should direct forthcoming expeditions with the aim of collecting germplasm of wild ipecac.

Tetrahydroisoquinoline-monoterpene glycosides from Cephaelis acuminata

From the dried roots of Cephaelis acuminata, five tetrahydroisoquinoline-monoterpene glycosides, 2- O-β- d-glucopyranosyldemethylalangiside, demethylisoalangiside, 6″- O-β- d-glucopyranosylipecoside, 6″- O-α- d-glucopyranosylipecoside and (4 R)-4-hydroxyipecoside, were isolated. The structures of these glycosides were determined by spectroscopic and chemical means.