Huiqin Pan

An integrated strategy for the systematic characterization and discovery of new indole alkaloids from Uncaria rhynchophylla by UHPLC/DAD/LTQ-Orbitrap-MS

AbstractThe exploration of new chemical entities from herbal medicines may provide candidates for the in silico screening of drug leads. However, this significant work is hindered by the presence of multiple classes of plant metabolites and many re-discovered structures. This study presents an integrated strategy that uses ultrahigh-performance liquid chromatography/linear ion-trap quadrupole/Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap-MS) coupled with in-house library data for the systematic characterization and discovery of new potentially bioactive molecules. Exploration of the indole alkaloids from Uncaria rhynchophylla (UR) is presented as a model study. Initially, the primary characterization of alkaloids was achieved using mass defect filtering and neutral loss filtering. Subsequently, phytochemical isolation obtained 14 alkaloid compounds as reference standards, including a new one identified as 16,17-dihydro-O-demethylhirsuteine by NMR analyses. The direct-infusion fragmentation behaviors of these isolated alkaloids were studied to provide diagnostic structural information facilitating the rapid differentiation and characterization of four different alkaloid subtypes. Ultimately, after combining the experimental results with a survey of an in-house library containing 129 alkaloids isolated from the Uncaria genus, a total of 92 alkaloids (60 free alkaloids and 32 alkaloid O-glycosides) were identified or tentatively characterized, 56 of which are potential new alkaloids for the Uncaria genus. Hydroxylation on ring A, broad variations in the C-15 side chain, new N-oxides, and numerous O-glycosides, represent the novel features of the newly discovered indole alkaloid structures. These results greatly expand our knowledge of UR chemistry and are useful for the computational screening of potentially bioactive molecules from indole alkaloids. Graphical AbstractA four-step integrated strategy for the systematic characterization and efficient discovery of new indole alkaloids from Uncaria rhynchophylla

Method development and application of offline two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry-fast data directed analysis for comprehensive characterization of the saponins from Xueshuantong Injection.

Xueshuantong Injection (XSTI), derived from Notoginseng total saponins, is a popular traditional Chinese medicine injection for the treatment of thrombus-resultant diseases. Current knowledge on its therapeutic basis is limited to five major saponins, whereas those minor ones are rarely investigated. We herein develop an offline two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry-fast data directed analysis (offline 2D LC/QTOF-Fast DDA) approach to systematically characterize the saponins contained in XSTI. Key parameters affecting chromatographic separation in 2D LC (including stationary phase, mobile phase, column temperature, and gradient elution program) and the detection by QTOF MS (involving spray voltage, cone voltage, and ramp collision energy) were optimized in sequence. The configured offline 2D LC system showed an orthogonality of 0.84 and a theoretical peak capacity of 8976. Total saponins in XSTI were fractionated into eleven samples by the first-dimensional hydrophilic interaction chromatography, which were further analyzed by reversed-phase UHPLC/QTOF-Fast DDA in negative ion mode. The fragmentation features evidenced from 36 saponin reference standards, high-accuracy MS and Fast-DDA-MS(2) data, elemental composition (C<80, H<120, O<50), double-bond equivalent (DBE 5-15), and searching an in-house library of Panax notoginseng, were simultaneously utilized for structural elucidation. Ultimately, 148 saponins were separated and characterized, and 80 have not been isolated from P. notoginseng. An in-depth depiction of the chemical composition of XSTI was achieved. The results obtained would benefit better understanding of the therapeutic basis and significant promotion on the quality standard of XSTI as well as other homologous products.

Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla.

HIGHLIGHTSSFC was used to separate and isolate two pairs of 7‐epimers of SOAs.Acetonitrile stabilized two pairs of epimers and was used as modifier in SFC.Two achiral UPC2 methods were established on the Torus 1‐AA and Diol columns.Preparative SFC enabled isolation of four SOA compounds with the purity >95%.SFC provides a solution to preparation of high‐purity reference standards. ABSTRACT Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high‐purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro‐Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7‐epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of ultra‐high performance SFC (ultra‐performance convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods were developed on the achiral Torus 1‐AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2‐EP OBD column and acetonitrile‐0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed‐phase HPLC, SFC, by use of water‐free mobile phase (inert CO2 and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.

Selective and comprehensive characterization of the quinochalcone C-glycoside homologs in Carthamus tinctorius L. by offline comprehensive two-dimensional liquid chromatography/LTQ-Orbitrap MS coupled with versatile data mining strategies

Quinochalcone C-glycosides (QCGs) are a series of pharmacologically bioactive components chemotaxonomic for Carthamus tinctorius L. The low abundance and ubiquitous interference from flavonoid O-glycosides (FOGs) frequently hinder the systematic exposure and characterization of these QCG homologs. We herein present an offline comprehensive two-dimensional liquid chromatography/linear ion-trap quadrupole/Orbitrap mass spectrometry (2D LC/LTQ-Orbitrap MS) approach coupled with versatile data mining strategies, to systematically characterize the QCGs from C. tinctorius. Initially, an offline 2D LC system, with an orthogonality of 71% and a theoretical peak capacity of 7654, was established by combining an Acchrom XAmide column and a BEH Shield RP-18 column. Subsequently, the water extract of C. tinctorius was separated by first dimensional hydrophilic interaction liquid chromatography (HILIC) yielding twelve fractions, which were further analyzed by reversed-phase ultra-high performance liquid chromatography/LTQ-Orbitrap MS using high energy C-trap dissociation (HCD) and collision-induced dissociation (CID) in the negative ion mode. The characteristic product ion filtering of m/z 119.05 (C8H7O−) in the HCD spectra, ring double bond equivalent (RDB 10–30), characteristic UV absorption around 405 nm, preferred 0,2X0 cleavage for C-glycosides, and diagnostic product ions analysis, were simultaneously employed for the structural elucidation of QCGs. Ultimately, 163 QCQ homologs were putatively characterized, and 149 are potential new ones. Particularly, nine dimers of QCG-FOG have not been previously reported. The obtained results have greatly expanded the knowledge on the structural diversity of QCGs, demonstrating the potency of the offline comprehensive 2D LC/LTQ-Orbitrap MS approach in separation and characterization of minor herbal components.

The Role of Nonphotosynthetic Microbes in the Recovery of Biological Soil Crusts in the Gurbantunggut Desert, Northwestern China

The mechanisms regulating the recovery of biological soil crusts (BSCs) due to the presence of nonphotosynthetic microbes were investigated using a soil scalping technique. Microscopic examinations identified the “glue” and “rope” action of bacteria and fungi at the initial stages of recovery of BSCs prior to the appearance of cyanobacteria. Extracellular polymeric substances (EPS) excreted by bacteria principally contained glucose and mannose. The optimum conditions for EPS production included the availability of glucose as the carbon source, the presence of CaCO 3 (2 g/L), KH 2 PO 4 (0.3 g/L), and MgSO 4 (0.1 g/L), a pH of 7 and incubation at 37°C for 72 h. Crust-forming tests in the laboratory and in the field demonstrated that inoculation of bare sand with oligotrophic bacteria was effective in accelerating the recovery of BSCs. The number of nonphotosynthetic microbes (especially actinomycetes and fungi) recorded in both the crust layer (0–2 cm) and subsurface layer (2–5 cm) was higher after 3 years than after 1 year. Microbial spatial variability of BSCs was related to nutrient status, especially available N.

An enhanced targeted identification strategy for the selective identification of flavonoid O-glycosides from Carthamus tinctorius by integrating offline two-dimensional liquid chromatography/linear ion-trap-Orbitrap mass spectrometry, high-resolution diagnostic product ions/neutral loss filtering and liquid chromatography-solid phase extraction-nuclear magnetic resonance

Targeted identification of potentially bioactive molecules from herbal medicines is often stymied by the insufficient chromatographic separation, ubiquitous matrix interference, and pervasive isomerism. An enhanced targeted identification strategy is presented and validated by the selective identification of flavonoid O-glycosides (FOGs) from Carthamus tinctorius. It consists of four steps: (i) enhanced separation and detection by offline two-dimensional liquid chromatography/LTQ-Orbitrap MS (offline 2D-LC/LTQ-Orbitrap MS) using collision-induced dissociation (CID) and high-energy C-trap dissociation (HCD); (ii) improved identification of the major aglycones by acid hydrolysis and LC-SPE-NMR; (iii) simplified spectral elucidation by high-resolution diagnostic product ions/neutral loss filtering; and (iv) more convincing structural identification by matching an in-house library. An offline 2D-LC system configuring an Acchrom XAmide column and a BEH Shield RP-18 UPLC® column enabled much better separation of the easily co-eluting components. Combined use of CID and HCD could produce complementary fragmentation information. The intensity ratios of the aglycone ion species ([Y0-H]-/Y0- and [Y0-2H]-/Y0-) in the HCD-MS2 spectra were found diagnostic for discriminating the aglycone subtypes and characterizing the glycosylation patterns. Five aglycone structures (kaempferol, 6-hydroxykaempferol, 6-methoxykaempferol, carthamidin, and isocarthamidin) were identified based on the 1H-NMR data recorded by LC-SPE-NMR. Of the 107 characterized flavonoids, 80 FOGs were first reported from C. tinctorius. Unknown aglycones, pentose, and novel acyl substituents were discovered. A new compound thereof was isolated and fully identified, which could partially validate the MS-oriented identification. This integral strategy can improve the potency, efficiency, and accuracy in the detection of new compounds from medicinal herbs and other natural sources.

An efficient and target-oriented sample enrichment method for preparative separation of minor alkaloids by pH-zone-refining counter-current chromatography

An efficient and target-oriented sample enrichment method was established to increase the content of the minor alkaloids in crude extract by using the corresponding two-phase solvent system applied in pH-zone-refining counter-current chromatography. The enrichment and separation of seven minor indole alkaloids from Uncaria rhynchophylla (Miq.) Miq. ex Havil(UR) were selected as an example to show the advantage of this method. An optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v) was used in this study, where triethylamine (TEA) as the retainer and hydrochloric acid (HCl) as the eluter were added at the equimolar of 10mM. Crude alkaloids of UR dissolved in the corresponding upper phase (containing 10mM TEA) were extracted twice with lower phase (containing 10mM TEA) and lower phase (containing 10mM HCl), respectively, the second lower phase extract was subjected to pH-zone-refining CCC separation after alkalization and desalination. Finally, from 10g of crude alkaloids, 4g of refined alkaloids was obtained and the total content of seven target indole alkaloids was increased from 4.64% to 15.78%. Seven indole alkaloids, including 54mg isocorynoxeine, 21mg corynoxeine, 46mg isorhynchophylline, 35mg rhynchophylline, 65mg hirsutine, 51mg hirsuteine and 27mg geissoschizine methylether were all simultaneously separated from 2.5g of refined alkaloids, with the purity of 86.4%, 97.5%, 90.3%, 92.1%, 98.5%, 92.3%, and 92.8%, respectively. The total content and purities of the seven minor indole alkaloids were tested by HPLC and their chemical structures were elucidated by ESI-HRMS and (1)H NMR.

UHPLC-Q-TOF-MS-based metabolomics approach to compare the saponin compositions of Xueshuantong injection and Xuesaitong injection.

Various traditional Chinese medicine preparations developed from Notoginseng total saponins, including Xueshuantong injection and Xuesaitong injection, are extensively used in China to treat cardiocerebrovascular diseases. However, the difference of their saponin compositions remains unknown. An ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry based metabolomics approach was developed to probe the saponin discrimination between Xueshuantong and Xuesaitong and the related factors by large sample analysis. A highly efficient chromatographic separation was achieved on an HSS T3 column within 20 min with the holistic metabolites information recorded in the negative MSE mode. A six-step data pretreatment procedure mainly based on Progenesis QI and mass defect filtering was established. Pattern recognition chemometrics was used to discover the potential saponin markers. The saponin composition of Wuzhou Xueshuantong showed distinct discrimination from the other products. Wuzhou Xueshuantong contains more abundant protopanaxatriol-type noto-R1 , Rg1 , Re, and protopanaxadiol-type Rb1 , but less Rd and other low-polarity protopanaxadiol-type ginsenosides. These differences could not directly correlate to the use of different parts of Panax notoginseng, but possibly to the different preparation techniques employed by different manufacturers. These results are beneficial to the establishment of pharmacopoeia standards and the assessment of the efficacy and adverse drug reactions for these homologous products.

Mass defect filtering-oriented classification and precursor ions list-triggered high-resolution mass spectrometry analysis for the discovery of indole alkaloids from Uncaria sinensis

Discovery of new natural compounds is becoming increasingly challenging because of the interference from those known and abundant components. The aim of this study is to report a dereplication strategy, by integrating mass defect filtering (MDF)-oriented novelty classification and precursor ions list (PIL)-triggered high-resolution mass spectrometry analysis, and to validate it by discovering new indole alkaloids from the medicinal herb Uncaria sinensis. Rapid chromatographic separation was achieved on a Kinetex® EVO C18 column (<16min). An in-house MDF algorithm, developed based on the informed phytochemistry information and molecular design, could more exactly screen the target alkaloids and divide them into three novelty levels: Known (KN), Unknown-but-Predicted (UP), and Unexpected (UN). A hybrid data acquisition method, namely PIL-triggered collision-induced dissociation-MS2 and high-energy C-trap dissociation-MS3 with dynamic exclusion on a linear ion trap/Orbitrap mass spectrometer, facilitated the acquisition of diverse product ions sufficient for the structural elucidation of both indole alkaloids and the N-oxides. Ultimately, 158 potentially new alkaloids, including 10 UP and 108 UN, were rapidly characterized from the stem, leaf, and flower of U. sinensis. Two new alkaloid compounds thereof were successfully isolated and identified by 1D and 2D NMR analyses. The varied ring E and novel alkaloid-acylquinic acid conjugates were first reported from the whole Uncaria genus. Conclusively, it is a practical chemical dereplication strategy that can enhance the efficiency and has the potential to be a routine approach for the discovery of new natural compounds.

Ultra-performance liquid chromatography of amino acids for the quality assessment of pearl powder

Pearls have been widely used as a traditional medicine, in cosmetics, and as a health food supplement in China since ancient times. However, the identification and quality assessment of pearl powder have been challenging tasks because of the similar morphological features and chemical composition of its common adulterants, especially conch powders. In this study, ultra-performance liquid chromatography was combined with pre-column derivatization to rapidly quantify 14 amino acids in pearl powder and its analogues. Based upon the quantification results, a quality criterion of a total amino acid content of not less than 1.10% was proposed for pearl powder. Principal component analysis indicated that leucine and phenylalanine were the amino acids characteristic for distinguishing between pearls and nacres. The area ratio of leucine to phenylalanine was demonstrated to be an effective diagnostic marker to discriminate freshwater cultured pearls, natural seawater pearls, and nacres. The proposed method, involving both the qualitative and quantitative aspects, was subsequently applied to quality assessment of pearl powders purchased commercially in various parts of China; eight out of 18 batches were deemed authentic and unadulterated. In the future, this analytical process should play a significant role in standardizing and providing quality control to the pearl powder market.