Alexandre Cardoso-Taketa

Metabolic Profiling of the Mexican Anxiolytic and Sedative Plant Galphimia glauca Using Nuclear Magnetic Resonance Spectroscopy and Multivariate Data Analysis

Galphimia glauca is popularly employed in Mexico for the treatment of central nervous system disorders. Pharmacological and phytochemical studies have resulted in the identification of the anxiolytic and sedative principle consisting of a mixture of nor-secofriedelanes, named the galphimine series (1 - 9). These active constituents were found in plants collected in the vicinity of a restricted region in Central Mexico, where this species is abundant. A metabolic profiling carried out by means of 1H-NMR spectroscopy and multivariate data analysis was applied to crude extracts from wild plant populations, collected from six different locations as a quality control assessment, in order to differentiate their chemical profile. Principal component analysis (PCA) of the 1H-NMR spectra revealed clear variations among the populations, with two populations out of the six studied manifesting differences, when the principal components PC-1 and PC-2 were analyzed. These two PCs permitted the differentiation of the various sample populations, depending on the presence of galphimines. This information consistently correlated with the corresponding HPLC analysis. The neuropharmacological effects of the crude extracts were evaluated by using ICR mice in the elevated plus maze, as well as the sodium pentobarbital-induced hypnosis models. Both assays demonstrated anxiolytic and sedative responses only among those sample populations which had previously been differentiated by PC-1. Partial least square regression-discriminant analysis (PLS-DA) also confirmed a strong correlation between the observed effects and the metabolic profiles of the plants. The overall results of this study confirm the benefits of using metabolic profiling for the in silico analysis of active principles in medicinal plants.

Isolation of jacaranone, a sedative constituent extracted from the flowers of the Mexican tree Ternstroemia pringlei

ETHNOPHARMACOLOGICAL RELEVANCE Ternstroemia pringlei represents one of the most widely employed and commercially exploited medicinal plant in Mexico, used popularly as a tranquilizer and for the treatment of insomnia. AIM OF THE STUDY To investigate the sedative constituents of the plant through a bio-guided fractionation of extracts derived from calyx and fruits. MATERIALS AND METHODS Crude extracts with different polarities (CHCl(3), AcOEt, MeOH, aqueous) were prepared and subjected to chromatographic fractionation, leading to the isolation of the sedative compound (1) from the MeOH crude extract. The identity of 1 was unequivocally established by means of 1D and 2D NMR spectroscopic analysis. The sleeping time induced by sodium pentobarbital and the elevated plus-maze models were performed on mice to determine the sedative and anxiolytic activities, respectively. Bioactivity was also investigated though in vitro GABA release experiments using mice brain slices. RESULTS The sedative compound was established as jacaranone (1), and its effect was clearly demonstrated through a dose-dependent response analysis (ED(50) = 25 mg/kg mouse weight). When tested in the elevated plus-maze model, none of the extracts from Ternstroemia pringlei displayed anxiolytic activity. GABA release experiments showed that the MeOH and aqueous crude extracts released this neurotransmitter at a ratio of 217 and 179 pmol/g protein, respectively, evidencing the presence of other bioactive constituents in the extracts apart of 1, whose activity was absent in this model. CONCLUSIONS Although 1 has been isolated and identified in a number of plant species, this is the first time that its sedative effect has been demonstrated. No previous record exists of other sedative compounds having been isolated from Ternstroemia pringlei.

A comparison on the metabolic profiling of the Mexican anxiolytic and sedative plant Galphimia glauca four years later

ETHNOPHARMACOLOGICAL RELEVANCE Galphimia glauca has a long traditional use, and continues to be used in Mexico as a natural tranquilizer for the treatment of Central Nervous System disorders as well as for other illnesses. AIM OF THE STUDY In 2005 the initial use of metabolic profiling to populations of Galphimia glauca resulted in two of the six collected populations being producers for galphimines, the markers for sedative and anxiolytic activities. The aim of this investigation was to confirm the previously established metabolic profile, as well as the previous in vivo results on mice. Additionally in this study we wanted to investigate potential anti-inflammatory properties. MATERIALS AND METHODS Four years later, we collected samples in the five localities designated for the first-stage investigation in 2005, and in two new locations. Metabolic profiling was carried out by means of (1)H NMR spectroscopy and multivariate data analysis applied to crude extracts from wild plant specimens. HPLC analysis was performed to confirm and quantify the presence of galphimines. Two neuropharmacological in vivo assays on mice were employed to study anxiolytic (elevated plus maze test) and sedative (sodium pentobarbital-induced hypnosis model) activities in the extracts. Anti-inflammatory activity was determined by using the tetradecanoylphorbol acetate-induced mouse ear inflammation model (TPA). RESULTS AND CONCLUSIONS The results for the 2009 collected species were similar to the 2005 collection, confirming the metabolic profiles and that galphimines are consistent good markers for CNS activity. Galloylquinic acid levels varied between the years without, as of yet, known effects. In vivo anti-inflammatory activity was similar for all plants and thus not linked with galphimines, requiring further studies to identify the active compound(s). Areas of collection affect neuropharmacological activities but not anti-inflammatory action.

Antibacterial activities of medicinal plants used in Mexican traditional medicine.

ETHNOPHARMACOLOGICAL RELEVANCE We provide an extensive summary of the in vitro antibacterial properties of medicinal plants popularly used in Mexico to treat infections, and we discuss the ethnomedical information that has been published for these species. MATERIALS AND METHODS We carried out a bibliographic investigation by analyzing local and international peer-reviewed papers selected by consulting internationally accepted scientific databases from 1995 to 2014. We provide specific information about the evaluated plant parts, the type of extracts, the tested bacterial strains, and the inhibitory concentrations for each one of the species. We recorded the ethnomedical information for the active species, as well as their popular names and local distribution. Information about the plant compounds that has been identified is included in the manuscript. This review also incorporates an extensive summary of the available toxicological reports on the recorded species, as well as the worldwide registries of plant patents used for treating bacterial infections. In addition, we provide a list with the top plant species with antibacterial activities in this review RESULTS: We documented the in vitro antibacterial activities of 343 plant species pertaining to 92 botanical families against 72 bacterial species, focusing particularly on Staphylococcus aureus, Mycobacterium tuberculosis, Escherichia coli and Pseudomonas aeruginosa. The plant families Asteraceae, Fabaceae, Lamiaceae and Euphorbiaceae included the largest number of active species. Information related to popular uses reveals that the majority of the plants, in addition to treating infections, are used to treat other conditions. The distribution of Mexican plants extended from those that were reported to grow in just one state to those that grow in all 32 Mexican states. From 75 plant species, 225 compounds were identified. Out of the total plant species, only 140 (40.57%) had at least one report about their toxic effects. From 1994 to July 2014 a total of 11,836 worldwide antibacterial patents prepared from different sources were recorded; only 36 antibacterial patents from plants were registered over the same time period. We offered some insights on the most important findings regarding the antibacterial effects, current state of the art, and research perspectives of top plant species with antibacterial activities in vitro. CONCLUSIONS Studies of the antibacterial in vitro activity of medicinal plants popularly used in Mexico to treat infections indicate that both the selection of plant material and the investigation methodologies vary. Standardized experimental procedures as well as in vivo pharmacokinetic studies to document the effectiveness of plant extracts and compounds are necessary. This review presents extensive information about the medicinal plants possessing antibacterial activity that has been scientifically studied and are popularly used in Mexico. We anticipate that this review will be of use for future studies because it constitutes a valuable information tool for selecting the most significant plants and their potential antibacterial properties.

DNA Barcoding of the Mexican Sedative and Anxiolytic Plant Galphimia glauca

ETHNOPHARMACOLOGY RELEVANCE Galphimia glauca (Malpighiaceae) is a Mexican plant popularly used as a tranquilizer in the treatment of nervous system disorders, although it is also used to treat other common illnesses. AIM OF THE STUDY The aim of this investigation is to find out if populations of Galphimia glauca collected in different regions and ecosystems in Mexico actually belong to the same species by using the contemporary technique of DNA barcodes. Our previous metabolic profiling study demonstrates that different collections of this plant obtained from various geographical areas exhibited diverse chemical profiles in terms of the active compounds named Galphimines. We expected the DNA barcodes apart from indicating the different species of Galphimia would indicate the active populations. MATERIALS AND METHODS We employed matK, rpoC1 and rbcL DNA barcodes to indicate the different species. Furthermore to investigate the possible impact of the several different ecosystems where the seven populations were collected, thin layer chromatography was employed to create a partial chemical profile, which was then compared with the metabolic profiles obtained by (1)H-NMR and multivariate data analysis. RESULTS AND CONCLUSIONS This study showed that the seven populations here analyzed contain at least three different species of the genus Galphimia, although each individual population is homogeneous. Interestingly our TLC analysis clearly showed that the active populations displayed a distinctively unique chemical profile. This work also showed that the use of DNA barcodes combined with chemical profile analysis is an excellent approach to solve the problems of quality control in the development of Galphimia-based medicines as well as for any breeding programs for this species.

Chemical and anti-ulcer evaluation of Jodina rhombifolia (Hook. & Arn.) Reissek extracts

Jodina rhombifolia (Hook. & Arn.) Reissek (Santalaceae) is a medicinal plant popularly used as an anti-ulcer medicine. The plant native from Southern Brazil was chemically investigated and tested for its in vivo gastric anti-ulcer property by chloride acid/ethanol model. The chromatographic analysis of the hydroethanol extract of its leaves revealed the presence of C-glycosylflavonoids. From the n-butanol fraction of the hydroethanol extract of its aerial parts, vicenin-2 was isolated as the main component and identified by spectroscopic methods; and, a direct comparison with authentic samples was made. This fraction afforded three other C-glycosylflavonoids: vitexin, orientin and swertisin; all of them identified by direct comparison with authentic samples. We found that the oral administration of aqueous and hydroethanolic extracts led to a significant decrease in the ulcer index.

Cytotoxic Activity and Chemical Composition of the Root Extract from the Mexican Species Linum scabrellum: Mechanism of Action of the Active Compound 6-Methoxypodophyllotoxin

The cytotoxic activity and the chemical composition of the dichloromethane/methanol root extract of Linum scabrellum Planchon (Linaceae) were analyzed. Using NMR spectra and mass spectrometry analyses of the extract we identified eight main constituents: oleic acid (1), octadecenoic acid (2), stigmasterol (3), α-amyrin (4), pinoresinol (5), 6 methoxypodophyllotoxin (6), coniferin (7), and 6-methoxypodophyllotoxin-7-O-β-D-glucopyranoside (8). By using the sulforhodamine B assay, an important cytotoxic activity against four human cancer cell lines, HF6 colon (IC50 = 0.57 μg/mL), MCF7 breast (IC50 = 0.56 μg/mL), PC3 prostate (IC50 = 1.60 μg/mL), and SiHa cervical (IC50 = 1.54 μg/mL), as well as toward the normal fibroblasts line HFS-30 IC50 = 1.02 μg/mL was demonstrated. Compound 6 (6-methoxypodophyllotoxin) was responsible for the cytotoxic activity exhibiting an IC50 value range of 0.0632 to 2.7433 µg/mL against the tested cell lines. Cell cycle studies with compound 6 exhibited a cell arrest in G2/M of the prostate PC3 cancer cell line. Microtubule disruption studies demonstrated that compound 6 inhibited the polymerization of tubulin through its binding to the colchicine site (binding constant K b = 7.6 × 106 M−1). A dose-response apoptotic effect was also observed. This work constitutes the first investigation reporting the chemical composition of L. scabrellum and the first study determining the mechanism of action of compound 6.

Production of podophyllotoxin from roots and plantlets of Hyptis suaveolens cultivated in vitro

Background: Hyptis suaveolens was an important source of food and medicines in pre-hispanic Mιxico and is actually used popularly to treat respiratory and skin diseases, fever, pain, and cramps, between other ailments. In 2008 the presence of podophyllotoxin (PTOX) was reported in this plant. Objective: To establish in vitro cultures of H. suaveolens able to produce PTOX. Materials and Methods: Explants of H. suaveolens were cultivated in Murashige and Skoog (MS) medium supplemented with different concentrations of the phytohormones 6-benzylaminopurine (6-BAP), 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA) and kinetin (Kin), in order to induce the production of podophyllotoxin. Root cultures without hormones were also established and the quantification of PTOX was performed by HPLC analysis. Results: The presence of growth regulators during in vitro cultivation of H. suaveolens, provoked morphological variations in explants, and induced the accumulation of different levels of PTOX. Roots grown without phytohormones accumulated PTOX at 0.013% dry weight (DW), while in three of the callus cultures cell lines growing together with roots, PTOX accumulated at concentrations of 0.003, 0.005 and 0.006% DW when NAA was combined with either Kin or BAP. In wild plant material PTOX was present in trace amounts in the aerial parts, while in the roots it was found at 0.005% DW. Conclusion: This study demonstrated that although it is possible to obtain PTOX in a variety of in vitro cultures of H. suaveolens, in vitro roots grown without the addition of growth regulators were better producers of PTOX.

A systematic updated review of scientifically tested selected plants used for anxiety disorders

The aim of this review is to provide a summary on multidisciplinary scientific information obtained from medicinal plants used worldwide to treat anxiety, focusing on phar- macological and clinical studies. The bibliographical investigation was carried out by consulting five peer-reviewed worldwide database publications for references, and patents. The information gathered on plants with attributed anxiolytic properties are presented as follows: (1) plant extracts with anxiolytic properties evaluated in animal models; (2) plants with clinical trials; (3) identi- fied active compounds in plants that have been assayed in animal models; (4) mechanism of action of anxiolytic plant extracts and compounds; and (5) registered patents for anxiolytic plant preparations. We recorded 112 plant species belonging to 63 botanical families for which the anxiolytic properties had been tested in animal models. Eleven plant species to treat general anxiety disorders as well as eleven species to treat anxiety-associated conditions, had been documented by clinical trials. Thirty-three registers for active compounds belonging to five general types of secondary metabolites had also been recorded. The mechanism of action at the central nervous system level had been determined in 33 plant species, either in their extracts or isolated compounds. Forty-seven patent registrations for plant preparations to be used for the treatment of anxiety were included. Abstract: The aim of this review is to provide a summary on multidisciplinary scientific information obtained from medicinal plants used worldwide to treat anxiety, focusing on phar- macological and clinical studies. The bibliographical investigation was carried out by consulting five peer-reviewed worldwide database publications for references, and patents. The information gathered on plants with attributed anxiolytic properties are presented as follows: (1) plant extracts with anxiolytic properties evaluated in animal models; (2) plants with clinical trials; (3) identi- fied active compounds in plants that have been assayed in animal models; (4) mechanism of action of anxiolytic plant extracts and compounds; and (5) registered patents for anxiolytic plant preparations. We recorded 112 plant species belonging to 63 botanical families for which the anxiolytic properties had been tested in animal models. Eleven plant species to treat general anxiety disorders as well as eleven species to treat anxiety-associated conditions, had been documented by clinical trials. Thirty-three registers for active compounds belonging to five general types of secondary metabolites had also been recorded. The mechanism of action at the central nervous system level had been determined in 33 plant species, either in their extracts or isolated compounds. Forty-seven patent registrations for plant preparations to be used for the Abstract: The aim of this review is to provide a summary on multidisciplinary scientific information obtained from medicinal plants used worldwide to treat anxiety, focusing on phar- macological and clinical studies. The bibliographical investigation was carried out by consulting five peer-reviewed worldwide database publications for references, and patents. The information gathered on plants with attributed anxiolytic properties are presented as follows: (1) plant extracts with anxiolytic properties evaluated in animal models; (2) plants with clinical trials; (3) identi- fied active compounds in plants that have been assayed in animal models; (4) mechanism of action of anxiolytic plant extracts and compounds; and (5) registered patents for anxiolytic plant preparations. We recorded 112 plant species belonging to 63 botanical families for which the anxiolytic properties had been tested in animal models. Eleven plant species to treat general anxiety disorders as well as eleven species to treat anxiety-associated conditions, had been documented by clinical trials. Thirty-three registers for active compounds belonging to five general types of secondary metabolites had also been recorded. The mechanism of action at the central nervous system level had been determined in 33 plant species, either in their extracts or isolated compounds. Forty-seven patent registrations for plant preparations to be used for the treatment of anxiety were included. Abstract The aim of the present work is to demonstrate the putative sedative and anxiolytic-like effects of a hydro-ethanolic extract obtained from the aerial parts ofAloysia polystachya(Verbenaceae) in male mice using several behavioural assays. Groups of male mice orally treated with doses of 1.0, 10.0 and 100.0mg/kg of the extract did not show any significant alteration of their locomotor activity, body temperature or motor coordination. The same treatment increased the duration of the sleeping time induced by 30.0mg/kg i.p. of sodium pentobarbital. However, the sleeping time induced by ethyl ether was not modified by the oral administration of the extract, not confirming the putative sedative effect of the plant. The ethanolicextractalsosignificantlyincreasedthepercentageofbothentries(1.0and100.0mg/kg)andthetimespent(10.0and100.0mg/kg)intothe

Transformed Cell Suspension Culture of Galphimia glauca Producing Sedative Nor-friedelanes

The Mexican species GALPHIMIA GLAUCA (Cav.) Kuntze (Malphigiaceae) synthesises a family of sedative and anxiolytic nor-secofriedelanes, designated as galphimines. These active principles accumulate at low concentration in the aerial parts of plants from wild populations. Transformed calluses and cell suspension cultures of this species were established in order to induce a greater production of nor-friedelanes. The cell suspension line GgBa was selected and grown over a period of two years of continuous subculturing in MS nutrient medium in the absence of growth regulators. PCR and Southern blot analyses were employed in order to confirm that the ROL A gene had been integrated into the plant genome. Batch cultures of the GgBa cell line were grown over a 32-day period and first-order growth kinetics was observed, reaching a specific growth rate (micro) of 0.13 d (-1). The production of glaucacetalin A ( 10), a triterpenoid related to the known galphimines, was quantified in the nutrient medium by HPLC. The transformed cell suspension culture GgBa also synthesised a novel nor-friedelane, given the name glaucacetalin D ( 13). High-resolution spectroscopic and spectrometric techniques were employed to elucidate the structure of 13. This triterpene has never been observed in wild plant tissues or in other IN VITRO cultures. Maslinic acid ( 14) was identified in cell biomasses. The triterpene production of the cell line GgBa was as follows: glaucacetalin A, 2.7 mg/L; glaucacetalin D, 2.9 mg/L and maslinic acid, 2.4 mg/g dry weight. The sedative activity of compounds 10 and 13 was demonstrated in ICR mice by using the sodium pentobarbital-induced hypnosis model. No cytotoxicity of 10 and 13 was exhibited against KB, MCF-7 and HF6 human cancer cell lines.