Machine learning prediction for constructing a universal multidimensional information library of Panax saponins (ginsenosides).

Accurate characterization of Panax herb ginsenosides is challenging because of the isomers and lack of sufficient reference compounds. More structural information could help differentiate ginsenosides and their isomers, enabling more accurate identification. Based on the VionTM ion-mobility high-resolution LC-MS platform, a multidimensional information library for ginsenosides, namely GinMIL, was established by predicting retention time (tR) and collision cross section (CCS) through machine learning. Robustness validation experiments proved tR and CCS were suitable for database construction. Among three machine learning models we attempted, gradient boosting machine (GBM) exhibited the best prediction performance. GinMIL included the multidimensional information (m/z, molecular formula, tR, CCS, and some MS/MS fragments) for 579 known ginsenosides. Accuracy in identifying ginsenosides from diverse ginseng products was greatly improved by a unique LC-MS approach and searching GinMIL, demonstrating a universal Panax saponins library constructed based on hierarchical design. GinMIL could improve the accuracy of isomers identification by approximately 88%.

Characterization and comparison of cardiomyocyte protection activities of non-starch polysaccharides from six ginseng root herbal medicines.

Ginseng is rich of polysaccharides, however, the evidence supporting polysaccharides to distinguish various ginseng species is rarely reported. Focusing on six root ginseng (e.g., Panax ginseng-PG, P. quinquefolius-PQ, P. notoginseng-PN, red ginseng-RG, P. japonicus-PJ, and P. japonicus var. major-PJM), the contained non-starch polysaccharides (NPs) were structurally characterized and compared by both the chemical and biological evaluation. Holistic fingerprinting at three levels (the NPs and the acid hydrolysates involving oligosaccharides and monosaccharides) utilized various chromatography methods, and the treatment of H9c2 cells with the NPs by OGD and H2O2-induced injury models was used to assess the protective effect. NPs from six Panax herbal medicines occupied about 20 % of the total polysaccharides, which were of the highest content in RG and the lowest in PN. NPs from six ginseng exhibited weak differentiations in the molecular weight distribution, while marker oligosaccharides were found to distinguish PN and RG from the others. Glc and GalA were more abundant in the NPs for PG and RG, respectively. NPs from PQ (100/200 µg/mL) showed significant cardiomyocyte protection effect by regulating the mitochondrial functions. This work further testifies the role of polysaccharides in quality control of herbal medicine, with new markers discovered beneficial to distinguish the ginseng.

Multiple heart-cutting two-dimensional liquid chromatography/charged aerosol detector assay of ginsenosides for quality evaluation of ginseng from diverse Chinese patent medicines.

For quality control of Chinese patent medicines (CPMs) containing the same herbal medicine or different herbal medicines that have similar chemical composition, current ″one standard for one species″ research mode leads to poor universality of the analytical approaches unfavorable to discriminate easily confused species. Herein, we were aimed to elaborate a multiple heart-cutting two-dimensional liquid chromatography/charged aerosol detector (MHC-2DLC/CAD) approach to quantitatively assess ginseng from multiple CPMs. Targeting baseline resolution of 16 ginsenosides (noto-R1/Rg1/Re/Rf/Ra2/Rb1/Rc/Ro/Rb2/Rb3/Rd/Rh1/Rg2/Rg3/Rg3(R)/24(R)-p-F11), experiments were conducted to optimize key parameters and validate its performance. A Poroshell 120 EC-C18 column and an XBridge Shield RP18 column were separately utilized in the first-dimensional (1D) and the second-dimensional (2D) chromatography. Eight consecutive cuttings could achieve good separation of 16 ginsenosides within 85 min. The developed MHC-2DLC/CAD method showed good linearity (R2 > 0.999), repeatability (RSD < 6.73%), stability (RSD < 5.63%), inter- and intra-day precision (RSD < 5.57%), recovery (93.76-111.14%), and the limit of detection (LOD) and limit of quantification (LOQ) varied between 0.45-2.37 ng and 0.96-4.71 ng, respectively. We applied it to the content determination of 16 ginsenosides simultaneously from 28 different ginseng-containing CPMs, which unveiled the ginsenoside content difference among the tested CPMs, and gave useful information to discriminate ginseng in the preparation samples, as well. The MHC-2DLC/CAD approach exhibited advantages of high specificity, good separation ability, and relative high analysis efficiency, which also justified the feasibility of our proposed ″Monomethod Characterization of Structure Analogs″ strategy in quality evaluation of diverse CPMs that contained different ginseng.

A practical strategy enabling more reliable identification of ginsenosides from Panax quinquefolius flower by dimension-enhanced liquid chromatography/mass spectrometry and quantitative structure-retention relationship-based retention behavior prediction.

To accurately identify the metabolites is crucial in a number of research fields, and discovery of new compounds from the natural products can benefit the development of new drugs. However, the preferable phytochemistry or liquid chromatography/mass spectrometry approach is time-/labor-extensive or receives unconvincing identifications. Herein, we presented a strategy, by integrating offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS), exclusion list-containing high-definition data-dependent acquisition (HDDDA-EL), and quantitative structure-retention relationship (QSRR) prediction of the retention time (tR), to facilitate the in-depth and more reliable identification of herbal components and thus to discover new compounds more efficiently. Using the saponins in Panax quinquefolius flower (PQF) as a case, high orthogonality (0.79) in separating ginsenosides was enabled by configuring the XBridge Amide and CSH C18 columns. HDDDA-EL could improve the coverage in MS2 acquisition by 2.26 folds compared with HDDDA (2933 VS 1298). Utilizing 106 reference compounds, an accurate QSRR prediction model (R2 = 0.9985 for the training set and R2 = 0.88 for the validation set) was developed based on Gradient Boosting Machine (GBM), by which the predicted tR matching could significantly reduce the isomeric candidates identification for unknown ginsenosides. Isolation and establishment of the structures of two malonylginsenosides by NMR partially verified the practicability of the integral strategy. By these efforts, 421 ginsenosides were identified or tentatively characterized, and 284 thereof were not ever reported from the Panax species. The current strategy is thus powerful in the comprehensive metabolites characterization and rapid discovery of new compounds from the natural products.

An efficient approach addressing the chemical complexity of Jiawei Fangji Huangqi decoction by integrating ultra-high-performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry and intelligent data processing workflows.

A challenge in the quality control of traditional Chinese medicine is the systematic multicomponent characterization of the compound formulae. Jiawei Fangji Huangqi, a modified form of Fangji Huangqi, is a prescription comprising seven herbal medicines. To address the chemical complexity of the Jiawei Fangji Huangqi decoction, we integrated ion mobility-quadrupole time-of-flight high-definition MSE coupled to ultra-high-performance liquid chromatography and intelligent data processing workflows available in the UNIFI software package. Good chromatographic separation was achieved on CORTECS UPLC T3 column within 52 min, and high-accuracy MS2 data were acquired using high-definition MSE in the negative and positive modes. A chemical library of 1250 compounds was created and incorporated into the UNIFI software to enable automatic peak annotation of the high-definition MSE data. We identified or tentatively characterize 430 compounds in the Jiawei Fangji Huangqi decoction. The potential superiority of high-definition MSE over conventional MS data acquisition approaches was revealed in its spectral quality (MS2 ), differentiation of isomers, separation of coeluting compounds, and target mass coverage. The multiple components of the Jiawei Fangji Huangqi decoction were elucidated, offering insight into its improved pharmacological action compared with that of the Fangji Huangqi formula.

Comparative Characterization of the Ginsenosides from Six Panax Herbal Extracts and Their In Vitro Rat Gut Microbial Metabolites by Advanced Liquid Chromatography-Mass Spectrometry Approaches.

Ginseng extracts are extensively used as raw materials for food supplements and herbal medicines. This study aimed to characterize ginsenosides obtained from six Panax plant extracts (Panax ginseng, red ginseng, Panax quinquefolius, Panax notoginseng, Panax japonicus, and Panax japonicus var. major) and compared them with their in vitro metabolic profiles mediated by rat intestinal microbiota. Ultrahigh-performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) with scheduled multiple reaction monitoring (sMRM) quantitation methods were developed to characterize and compare the ginsenoside composition of the different extracts. After in vitro incubation, 248 ginsenosides/metabolites were identified by UHPLC/IM-QTOF-MS in six biotransformed samples. Deglycosylation was determined to be the main metabolic pathway of ginsenosides, and protopanaxadiol-type and oleanolic acid-type saponins were easier to be easily metabolized. Compared with the ginsenosides in plant extracts, those remaining in six biotransformed samples were considerably fewer after biotransformation for 8 h. However, the compositional differences in four subtypes of the ginsenosides among the six Panax plants became more distinct.

Three-dimensional characteristic chromatogram by online comprehensive two-dimensional liquid chromatography: Application to the identification and differentiation of ginseng from herbal medicines to various Chinese patent medicines.

One bottleneck problem in the quality control of traditional Chinese medicine (TCM) is the accurate identification of easily confused herbal medicines from Chinese patent medicine (CPM). Ginseng products derived from the multiple parts (e.g., root/rhizome, leaf, and flower bud) of multiple Panax species (P. ginseng, P. quinquefolius, P. notoginseng, P. japonicus, and P. japonicus var. major) are globally popular; however, their authentication is very challenging. Using online comprehensive two-dimensional liquid chromatography (LC × LC), we propose the concept of a three-dimensional characteristic chromatogram (3D CC) by integrating enhanced LC × LC separation and a contour plot that visualizes the stereoscopic chromatographic peaks and examine its performance in authenticating various ginseng products. Targeted at the resolution of 17 ginsenoside markers, an online LC × LC/UV system with a 56 min analysis time was constructed: a CORTECS UPLC Shield RP 18 column running at 0.1 mL/min for the first-dimensional chromatography and a Poroshell SB-Aq column at 2.0 mL/min in shift gradient mode in the second dimension of separation. In particular, ginsenosides Rg1/Re and Rc/Ra1 were well resolved. According to the presence/absence of stereo peaks consistent with the main ginsenoside markers in the 3D CC and the depth of shade (depending on peak volume), it was feasible to use a single method to identify and distinguish among 12 different ginseng species as the drug materials and the use of ginseng simultaneously from 21 CPMs. Conclusively, a practical solution enabling the accurate identification of easily confused TCMs was provided, covering both the drug materials and the compound preparations.

Collision Cross Section Prediction Based on Machine Learning.

Ion mobility-mass spectrometry (IM-MS) is a powerful separation technique providing an additional dimension of separation to support the enhanced separation and characterization of complex components from the tissue metabolome and medicinal herbs. The integration of machine learning (ML) with IM-MS can overcome the barrier to the lack of reference standards, promoting the creation of a large number of proprietary collision cross section (CCS) databases, which help to achieve the rapid, comprehensive, and accurate characterization of the contained chemical components. In this review, advances in CCS prediction using ML in the past 2 decades are summarized. The advantages of ion mobility-mass spectrometers and the commercially available ion mobility technologies with different principles (e.g., time dispersive, confinement and selective release, and space dispersive) are introduced and compared. The general procedures involved in CCS prediction based on ML (acquisition and optimization of the independent and dependent variables, model construction and evaluation, etc.) are highlighted. In addition, quantum chemistry, molecular dynamics, and CCS theoretical calculations are also described. Finally, the applications of CCS prediction in metabolomics, natural products, foods, and the other research fields are reflected.

Pseudotargeted Metabolomics Approach Enabling the Classification-Induced Ginsenoside Characterization and Differentiation of Ginseng and Its Compound Formulation Products.

The use of diversified ginseng extracts in health-promoting foods is difficult to differentiate, as they share bioactive ginsenosides among different Panax species (e.g., P. ginseng, P. quinquefolius, P. notoginseng, and P. japonicus) and different parts (e.g., root, leaf, and flower). This work was designed to develop a pseudo-targeted metabolomics approach to discover ginsenoside markers facilitating the precise authentication of ginseng and its use in compound formulation products (CFPs). Versatile mass spectrometry experiments on the QTrap mass spectrometer achieved classified characterization of the neutral, malonyl, and oleanolic acid-type ginsenosides, with 567 components characterized. A pseudo-targeted metabolomics approach by multiple reaction monitoring (MRM) of 262 ion pairs could assist to establish key identification points for 12 ginseng species. The simultaneous detection of 14 markers enabled the identification of ginseng from 15 ginseng-containing CFPs. The pseudo-targeted metabolomics strategy enabled better performance in differentiating among multiple ginseng, compared with the full-scan high-resolution mass spectrometry approach.

Multicomponent Characterization of the Flower Bud of Panax notoginseng and Its Metabolites in Rat Plasma by Ultra-High Performance Liquid Chromatography/Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry.

The flower bud of Panax notoginseng (PNF) consumed as a tonic shows potential in the prevention and treatment of cardiovascular diseases. To identify the contained multi-components and, in particular, to clarify which components can be absorbed and what metabolites are transformed, unveiling the effective substances of PNF is of vital significance. A unique ultrahigh-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) profiling approach and efficient data processing by the UNIFITM bioinformatics platform were employed to comprehensively identify the multi-components of PNF and the related metabolites in the plasma of rats after oral administration (at a dose of 3.6 g/kg). Two MS2 data acquisition modes operating in the negative electrospray ionization mode, involving high-definition MSE (HDMSE) and data-dependent acquisition (DDA), were utilized aimed to extend the coverage and simultaneously ensure the quality of the MS2 spectra. As a result, 219 components from PNF were identified or tentatively characterized, and 40 thereof could be absorbed. Moreover, 11 metabolites were characterized from the rat plasma. The metabolic pathways mainly included the phase I (deglycosylation and oxidation). To the best of our knowledge, this is the first report that systematically studies the in vivo metabolites of PNF, which can assist in better understanding its tonifying effects and benefit its further development.

Integrating Enhanced Profiling and Chemometrics to Unveil the Potential Markers for Differentiating among the Leaves of Panax ginseng, P. quinquefolius, and P. notoginseng by Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry.

The leaves of Panax species (e.g., Panax ginseng-PGL, P. quinquefolius-PQL, and P. notoginseng-PNL) can serve as a source for healthcare products. Comprehensive characterization and unveiling of the metabolomic difference among PGL, PQL, and PNL are critical to ensure their correct use. For this purpose, enhanced profiling and chemometrics were integrated to probe into the ginsenoside markers for PGL/PQL/PNL by ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS). A hybrid scan approach (HDMSE-HDDDA) was established achieving the dimension-enhanced metabolic profiling, with 342 saponins identified or tentatively characterized from PGL/PQL/PNL. Multivariate statistical analysis (33 batches of leaf samples) could unveil 42 marker saponins, and the characteristic ginsenosides diagnostic for differentiating among PGL/PQL/PNL were primarily established. Compared with the single DDA or DIA, the HDMSE-HDDDA hybrid scan approach could balance between the metabolome coverage and spectral reliability, leading to high-definition MS spectra and the additional collision-cross section (CCS) useful to differentiate isomers.

Systematic Qualitative and Quantitative Analyses of Wenxin Granule via Ultra-High Performance Liquid Chromatography Coupled with Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry and Triple Quadrupole-Linear Ion Trap Mass Spectrometry.

Wenxin granule (WXG) is a popular traditional Chinese medicine (TCM) preparation for the treatment of arrhythmia disease. Potent analytical technologies are needed to elucidate its chemical composition and assess the quality differences among multibatch samples. In this work, both a multicomponent characterization and quantitative assay of WXG were conducted using two liquid chromatography-mass spectrometry (LC-MS) approaches. An ultra-high performance liquid chromatography-ion mobility quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) approach combined with intelligent peak annotation workflows was developed to characterize the multicomponents of WXG. A hybrid scan approach enabling alternative data-independent and data-dependent acquisitions was established. We characterized 205 components, including 92 ginsenosides, 53 steroidal saponins, 14 alkaloids, and 46 others. Moreover, an optimized scheduled multiple reaction monitoring (sMRM) method was elaborated, targeting 24 compounds of WXG via ultra-high performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC/QTrap-MS), which was validated based on its selectivity, precision, stability, repeatability, linearity, sensitivity, recovery, and matrix effect. By applying this method to 27 batches of WXG samples, the content variations of multiple markers from Notoginseng Radix et Rhizoma (21) and Codonopsis Radix (3) were depicted. Conclusively, we achieved the comprehensive multicomponent characterization and holistic quality assessment of WXG by targeting the non-volatile components.

Effects of Herba Erigerontis injection on pharmacodynamics and pharmacokinetics of warfarin in rats in vivo.

Herba Erigerontis injection (HEI) is an aqueous solution derived from whole plants of Erigeron breviscapus, which may be co-administered with warfarin to treat cardiovascular and cerebrovascular disorders. This research was conducted to make sure whether HEI would affect anticoagulation of warfarin to guarantee reasonable medication. The pharmacodynamic study was designed to measure prothrombin time (PT) and activated partial thromboplastin time (APTT) values, and international normalized ratio (INR) values were calculated. For pharmacokinetic study, ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) technology was applied to measure plasma concentrations of warfarin enantiomers. The influence of HEI on plasma protein binding rate of warfarin was assessed by ultrafiltration. Pharmacodynamic study demonstrated that both HEI alone and co-administered with warfarin could increase PT and INR values significantly (P < .01), whereas the APTT values were unaffected (P > .05). Pharmacokinetic study manifested that Cmax , AUC and t1/2 prolonged significantly (P < .01) for R/S-warfarin in presence of HEI. Low (3.6 mL/kg), medium (7.2 mL/kg) and high (10.8 mL/kg) doses of HEI could decrease plasma protein binding rate of warfarin significantly (P < .01). The results mean that HEI can potentiate the anticoagulant response of warfarin through both pharmacodynamics and pharmacokinetics.