Introduction.
Size-exclusion chromatographic separation with Sephadex gels is a widely used technique for dereplication and screening methodologies of natural products. Sephadex G-10 resin is a polymeric network composed of dextran units cross-linked by epichlorohydrin and allows using physiological solutions as mobile phases for the direct coupling chromatographic separation to study living tissues or organs. Thus, this type of system enables real-time monitoring of active compounds, an application of significant importance in organic chemistry and pharmacology.
Methods.
Medium-pressure liquid chromatography separation (MPLC) with Sephadex G-10 coupled directly to biological detection using perfused organs was carried out for bio-guided study of hydro-ethanolic extracts from Nicotiana glauca Graham (Solanaceae). Sephadex G-10 provides the lowest fractionation range of gels type Sephadex, allowing the separation of substances with molecular weights below 700 Da, typically within the range of 100–1000 Da. Furthermore, this resin enabled the use of Krebs-HEPES as an elution medium, a medium that kept the studied organs in optimal conditions.
Perfused rat organs were used: Rings of aorta artery and trachea, and portions from the deferent conduct and ileum.
Results.
Two natural alkaloids, anabasine and nornicotine, were readily isolated and identified as the compounds responsible for the contractile activity observed in the smooth muscle of rat ileum and trachea.
The coupling of this chromatographic system with Sephadex G-10 to biological detection in living organs simplified the bioassay-guided: it carried out by a single person, with a physiological and non-contaminating medium, minimizing the number of animals slaughtered, with chemical characterization by mass spectrometry analysis, and reducing the study time.
Conclusions.
This application of Sephadex G-10 chromatography rationalizes the bio-guided identification of isolated natural products and advances its use toward green chemistry dereplication methodologies as well as in future automated or robotic approaches.
