Anti-inflammatory activity and chemical profile of Galphimia glauca.

Galphimia glauca, commonly known as "flor de estrella", is a plant species used in Mexican traditional medicine for the treatment of different diseases that have an acute or chronic inflammatory process in common. Aerial parts of this plant contain nor-seco-triterpenoids with anxiolytic properties, which have been denominated galphimines. Other compounds identified in the plant are tetragalloyl-quinic acid, gallic acid, and quercetin, which are able to inhibit the bronchial obstruction induced by platelet-activating factor. The objective of this work was to evaluate the anti-inflammatory effect of crude extracts from G. glauca and, by means of bioguided chemical separation, to identify the compounds responsible for this pharmacological activity. n-Hexane, ethyl acetate, dichloromethane, and methanol extracts showed an important anti-inflammatory effect. Chemical separation of the active methanol extract allowed us to identify the nor-seco-triterpenes galphimine-A (1) and galphimine-E (3) as the anti-inflammatory principles. Analysis of structure-activity relationships evidenced that the presence of an oxygenated function in C6 is absolutely necessary to show activity. In this work, the isolation and structural elucidation of two new nor-seco-triterpenes denominated as galphimine-K (4) and galphimine-L (5), together with different alkanes, fatty acids, as well as three flavonoids (17-19), are described, to our knowledge for the first time, from Galphimia glauca.

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.

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.

Immobilization of Galphimia glauca plant cell suspensions for the production of enhanced amounts of Galphimine-B.

We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the effects of a two-stage culture system: cell immobilization in Ca2+-alginate beads and culture scale-up from shake-flask to two different types of bioreactor (stirred and airlift). In the shake-flask culture, using optimum media for cell growth (first stage) and G-B production (second stage), the G-B yield was similar in both immobilised and free cells. However, while the free cells accumulated G-B within cytoplasmatic compartments, where it could not be recovered without cell disruption, immobilized cells excreted up to 100 % of the G-B produced. Immobilized cells grown in bioreactors running for 14 days with growth medium and an additional 26 days with production medium in batch mode showed a high G-B yield. The stirred bioreactor was the most efficient with a G-B content in the culture medium of 1381 microg.L (-1) at day 24 of culture.

The effect of methyl jasmonate on triterpene and sterol metabolisms of Centella asiatica, Ruscus aculeatus and Galphimia glauca cultured plants.

Considering that exogenously applied methyl jasmonate can enhance secondary metabolite production in a variety of plant species and that 2,3-oxidosqualene is a common precursor of triterpenes and sterols in plants, we have studied Centella asiatica and Galphimia glauca (both synthesizing triterpenoid secondary compounds) and Ruscus aculeatus (which synthesizes steroidal secondary compounds) for their growth rate and content of free sterols and respective secondary compounds, after culturing with or without 100 microM methyl jasmonate. Our results show that elicited plantlets of G. glauca and to a higher degree C. asiatica (up to 152-times more) increased their content of triterpenoids directly synthesized from 2,3-oxidosqualene (ursane saponins and nor-seco-friedelane galphimines, respectively) at the same time as growth decreased. In contrast, the free sterol content of C. asiatica decreased notably, and remained practically unaltered in G. glauca. However, in the case of R. aculeatus, which synthesizes steroidal saponins (mainly spirostane type) indirectly from 2,3-oxidosqualene after the latter is converted to the plant phytosterol-precursor cycloartenol, while the growth rate and free sterol content clearly decreased, the spirostane saponine content was virtually unchanged (aerial part) or somewhat lower (roots) in presence of the same elicitor concentration. Our results suggest that while methyl jasmonate may be used as an inducer of enzymes involved in the triterpenoid synthesis downstream from 2,3-oxidosqualene in both C. asiatica and G. glauca plantlets, in those of C. asiatica and R. aculeatus it inhibited the enzymes involved in sterol synthesis downstream from cycloartenol.

Production of triterpenoids in liquid-cultivated hairy roots of Galphimia glauca.

The production of three triterpenoids from Galphimia glauca hairy root cultures, the sedative principle galphimine E (2), the recently described glaucacetalin A (3), and maslinic acid (6), was quantified by HPLC in the biomass and the culture medium. Batch cultures of the hairy root line VYT, obtained through infecting cotyledons with Agrobacterium rhizogenes ATCC 15 834, were grown for 41 days in shake flasks containing B5 medium without phytohormones. A maximum biomass of 11 g/L DW was obtained on day 33, while the doubling time was 6 days. Throughout the growth cycle fresh and dry weights as well as triterpene production were registered. Glaucacetalin A (3), excreted into the culture media, reached a maximum amount of 2.14 mg/L after 21 days while galphimine E (2) and maslinic acid (6) were recovered from the root biomasses reaching maximum concentrations of 0.11 and 0.43 mg/g, respectively, on day 39.

Genetic transformation of Glaphimia glauca by Agrobacterium rhizogenes and the production of norfriedelanes.

Transformed root cultures of Galphimia glauca (Malpighiaceae) were established by infecting cotyledons and hypocotyls with Agrobacterium rhizogenes ATCC 15 834. Cotyledon-derived cell lines were grown in liquid B5 nutrient medium without phytohormones and have shown the typical hairy roots phenotype over two years of continuous subculturing. PCR analysis was used to confirm the integration of rol A and rol C genes into the plant genome. The transformed cultures synthesized three major norfriedelanes, the new glaucacetalins A-C (1-3), which were secreted into the nutrient medium. The structural elucidation of these in vitro produced metabolites was performed by the application of high resolution NMR techniques that proved them to be triterpenoids related to the known galphimines, the sedative principles of this plant species. These results suggest the possibility of further biotechnological exploration of sedative friedelane biosynthesis by in vitro plant organ cultures.