DNA barcoding and TLC as tools to properly identify natural populations of the Mexican medicinal species Galphimia glauca Cav.

Galphimia glauca is a plant that is endemic to Mexico and has been commonly used since pre-Hispanic times to treat various illnesses, including central nervous system disorders and inflammation. The first studies investigating a natural population of G. glauca in Mexico showed that the plant has anxiolytic and sedative activities in mice and humans. The plant's bioactive compounds were isolated and identified, and they belong to a family of nor-secofriedelanes called galphimines. The integration of DNA barcoding and thin-layer chromatography analysis was performed to clarify whether the botanical classification of the populations in the study, which were collected in different regions of Mexico, as G. glauca was correct or if the populations consist of more than one species of the genus Galphimia. We employed six DNA barcodes (matK, rbcL, rpoC1, psbA-trnH, ITS1 and ITS2) that were analyzed individually and in combination and then compared each other, to indicate differences among the studied populations. In the phylogenetic analysis, ITS1 and ITS2 markers as well as the combination of all DNA regions were the most efficient for discriminating the population studied. The thin-layer chromatography analysis exhibited four principal chemical profiles, one of which corresponded to the populations that produced galphimines. DNA barcoding was consistent and enabled us to differentiate the populations that produce galphimines from those that do not. The results of this investigation suggest that the studied populations belong to at least four different species of the genus Galphimia. The phylogenetic analysis and the thin-layer chromatography chemical profiles were convenient tools for establishing a strong relationship between the genotype and phenotype of the studied populations and could be used for quality control purposes to prepare herbal medicines from plants of the genus Galphimia.

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.

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.