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For wild natural medicine, unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials, which affects the efficacy and safety of clinical medication. DNA barcoding as an effective species identification tool is limited by its low sample throughput nature. In this study, combining DNA mini-barcode, DNA metabarcoding and species delimitation method, a novel biological sources consistency evaluation strategy was proposed, and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as "Guang Dilong" and 25 batches of proprietary Chinese medicines. Besides Amynthas aspergillum as the authentic source, 8 other Molecular Operational Taxonomic Units (MOTUs) were elucidated. Significantly, even the subgroups within A. aspergillum revealed here differ significantly on chemical compositions and biological activity. Fortunately, this biodiversity could be controlled when the collection was limited to designated areas, as proved by 2796 "decoction pieces" samples. This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control, and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.
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Inherent complexity of plant metabolites necessitates the use of multi-dimensional information to accomplish comprehensive profiling and confirmative identification. A dimension-enhanced strategy, by offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spec-trometry (2D-LC/IM-QTOF-MS) enabling four-dimensional separations (2D-LC, IM, and MS), is proposed. In combination with in-house database-driven automated peak annotation, this strategy was utilized to characterize ginsenosides simultaneously from white ginseng (WG) and red ginseng (RG). An offline 2D-LC system configuring an Xbridge Amide column and an HSS T3 column showed orthogonality 0.76 in the resolution of ginsenosides. Ginsenoside analysis was performed by data-independent high-definition MSE (HDMSE) in the negative ESI mode on a Vion TM IMS-QTOF hybrid high-resolution mass spectrometer, which could better resolve ginsenosides than MSE and directly give the CCS information. An in-house ginsenoside database recording 504 known ginsenosides and 58 reference compounds, was estab-lished to assist the identification of ginsenosides. Streamlined workflows, by applying UNIFI TM to auto-matedly annotate the HDMSE data, were proposed. We could separate and characterize 323 ginsenosides (including 286 from WG and 306 from RG), and 125 thereof may have not been isolated from the Panax genus. The established 2D-LC/IM-QTOF-HDMSE approach could also act as a magnifier to probe differ-entiated components between WG and RG. Compared with conventional approaches, this dimension-enhanced strategy could better resolve coeluting herbal components and more efficiently, more reli-ably identify the multicomponents, which, we believe, offers more possibilities for the systematic exposure and confirmative identification of plant metabolites.
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Traditional Chinese medicine (TCM) has played a pivotal role in maintaining the health of Chinese people and is now gaining increasing acceptance around the global scope. However, TCM is confronting more and more concerns with respect to its quality. The intrinsic "multicomponent and multitarget" feature of TCM necessitates the establishment of a unique quality and bioactivity evaluation system, which is different from that of the Western medicine. However, TCM is investigated essentially as "herbal medicine" or "natural product", and the pharmacopoeia quality monographs are actually chemical-markers-based, which can ensure the consistency only in the assigned chemical markers, but, to some extent, have deviated from the basic TCM theory. A concept of "quality marker" (Q-marker), following the "property-effect-component" theory, is proposed. The establishment of Q-marker integrates multidisciplinary technologies like natural products chemistry, analytical chemistry, bionics, chemometrics, pharmacology, systems biology, and pharmacodynamics, etc. Q-marker-based fingerprint and multicomponent determination conduce to the construction of more scientific quality control system of TCM. This review delineates the background, definition, and properties of Q-marker, and the associated technologies applied for its establishment. Strategies and approaches for establishing Q-marker-based TCM quality control system are presented and highlighted with a few TCM examples.
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To differentiate traditional Chinese medicines (TCM) derived from congeneric species in TCM compound preparations is usually challenging. The roots of(PG),(PQ) and(PN) are used as popular TCM. They contain similar triterpenoid saponins (ginsenosides) as the major bioactive constituents. Thus far, only a few chemical markers have been discovered to differentiate these three species. Herein we present a multiple marker detection approach to effectively differentiate the threespecies, and to identify them in compound preparations. Firstly, 85 batches of crude drug samples (including 32 PG, 30 PQ, and 23 PN) were analyzed by monitoring 40 major ginsenosides in the extracted ion chromatograms (EICs) using a validated LC-MS fingerprinting method. Secondly, the samples were clustered into different groups by pattern recognition chemometric approaches using PLS-DA and OPLS-DA models, and 17 diagnostic chemical markers were discovered. Aside from the previously known Rf and p-F, ginsenoside Rscould be a new marker to differentiate PG from PQ. Finally, the above multiple chemical markers were used to identify thespecies in 60 batches of TCM compound preparations.
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Objective To investigate the influence of pullulan molecular weight and spacer length on the properties of modified pullulan carriers including morphologies,sizes and in vitro release behaviours of drug-loading carriers.Methods Using cholesterol as hydrophobic ligand,succinic anhydride and 1,6-hexyldiisocyanate as spacers,hydrophobic modified pullulans with different molecular weights were prepared.Self-assembled nanoparticles were then formed in the aqueous solution,and drug-loaded nanoparticles were prepared by dialysis method.The influence of pullulan molecular weight and spacer length on the loading-content,morphologies and in vitro release behaviours of drug-loading nanoparticles were then investigated in detail.Results Self-assembled nanoparticles could be formed by the cholesterol-modified pullulan,and doxorubicin and mitoxantrone could be loaded into cholesterol-modified pullulan to form nanoparticles.Pullulan molecular weight and spacer length show influences on sizes,morphologies and stabilities of pullulan nanoparticles and drug-loaded nanocarriers.Conclusions Before drug loading,nanoparticles with larger moleculare weight and shorter spacer length are more stable in solution,while after drug loading,the influences of these two factors on the nanoparticles are drug-type depended.
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Millettia nitida var.hirsutissima is a Chinese herbal medicine used for the treatment of gynecological diseases.An HPLC/DAD/ESI-MSn method was established for the rapid separation and characterization of bioactive flavonoids in M.nitida var.hirsutissima.A total of 32 flavonoids were detected,of which 14 compounds were unambiguously characterized by comparing their retention time,UV,and MS spectra with those of the reference standards,and the others were tentatively identified based on their tandem mass spectrometry fragmentation data obtained in the negative ionization mode on line.Nineteen of these compounds characterized were reported from this plant for the first time.