A rapid method combining microwave-assisted extraction and high-speed counter-current chromatography was applied for preparative separation of six bioactive compounds including loganic acid (I), isoorientin-4'-O-glucoside (II), 6'-O-β-d-glucopyranosyl gentiopicroside (III), swertiamarin (IV), gentiopicroside (V), sweroside (VI) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. Microwave-assisted extraction parameters were predicted by central composite design-response surface methodology. That is, 5.0 g dried roots of G. Crassicaulis was extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by high-speed counter-current chromatography with n-butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail-to-head elution mode. 16.3 mg, 8.8 mg, 12.8 mg, 25.1 mg, 40.7 mg and 21.8 mg of compounds I–VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPCL with photo diode array detection and quadrupole time-of-flight MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, microwave-assisted extraction coupled with high-speed counter-current chromatography could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.
The aim of our present study is to explore the anti-arthritic potential effect of total steroid saponins (TSSN) extracted from the rhizome of Dioscorea zingiberensis C.H.Wright (DZW) and to investigate the underlying mechanisms. This work was performed using adjuvant-induced arthritis (AIA) rats in vivo and lipopolysaccharide (LPS) simulated 264.7 macrophage cells in vitro. In AIA-induced arthritic rats, TSSN significantly alleviated the arthritic progression through evaluating arthritic score, immune organ indexes, paw swelling, and body weight. This phenomenon was well correlated with significant suppression of the overproduction of inflammation cytokines (IL-1, IL-1β, IL-6, and TNF-α), oxidant stress makers (MDA and NO), eicosanoids (LTB4 and PGE2), and inflammatory enzymes (5-LOX and COX-2) versus the AIA rats without treatment. On the contrary, the release of SOD and IL-10 was profoundly increased. What’s more, TSSN could obviously ameliorate the translocation of NF-κB to the nucleus through phosphorylation of the p65 and IκBα in vivo and vitro. The current findings demonstrated that TSSN could protect the injured ankle joint from further deterioration and exert its satisfactory anti-arthritis properties through anti-inflammatory and anti-oxidant effects via inactivating NF-κB signal pathway. This research implies that DZW may be a useful therapeutic agent for the treatment of human arthritis.
In this study, a fingerprint of steroid saponins, the major bioactive
constituents in the crude extracts from Dioscorea zingiberensis
C. H. Wright (DZW), has been established for the first time by high-performance
liquid chromatography coupled with evaporative light scattering detector
(HPLC-ELSD) and the simultaneous characterization of the steroid saponins by
high-performance liquid chromatography coupled with electrospray ionization-mass
spectrometry and quadrupole tandem time-of-fight mass analyzers detection
(HPLC-ESI-Q/TOF). These HPLC analyses were both carried out on a Welchrom
C18 column (250 mm × 4.6 mm I.D., 5 μm) with a
mobile phase composed of water and acetonitrile under gradient elution. There
were 68 common characteristic peaks in the fingerprints, in which 12 of them
were confirmed by comparing their mass spectra and retention times with those of
the reference compounds. In order to identify the other unknown peaks, their
fragmentation behaviors characteristic for the major groups of steroid saponins
from DZW with six types of aglycone skeletons were discussed in detail, and
possible MS/MS fragmentation pathways were proposed for aiding the structural
identification of these components. According to the summarized fragmentation
patterns, these peaks were tentatively assigned by matching their empirical
molecular formula with those of the published compounds, or by elucidating their
quasi-molecular ions and fragment ions referring to available literature
information when the reference standards were unavailable. As a result, 22
steroid saponins were found in DZW for the first time. In addition, the
quantitative analysis of the 12 known peaks was accomplished at the same time
which indicated that there was a great variability in the amount of these active
compounds in different batches in the crude extracts. This approach could
demonstrate that the fingerprint could be considered to be a suitable tool to
comprehensively improve the quality control of DZW, and the identification and
structural elucidation of the peaks in the fingerprint may provide important
experimental data for further pharmacological and clinical researches.
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