Intelligent chemical profiling of 73 edible flowers by liquid chromatography-high resolution mass spectrometry combined with HRMS database and their authentication based on large-scale fingerprints.

A commercial high-resolution MS database "TCM-PCDL" was innovatively introduced to automatically identify multi-components in 73 edible flowers rapidly and accurately by liquid chromatography-high resolution mass spectrometry, which can be time-consuming and labor-intensive in traditional manual method. The database encompasses over 2565 natural products with various energy levels. Unknown compounds can be identified through direct matching and scoring MS2 spectra with database. A total of 870 compounds were identified from 73 flowers, with polyphenols constituting up to 75%. Focusing on polyphenols, a high performance liquid chromatography (HPLC) method was developed to generate fingerprints from 510 batches, establishing an "HPLC database" that enabled accurate authentication using similarity scores and rankings. This method demonstrated an accuracy rate of 100% when applied to 30 unknown samples. For flowers prone to confusion, additional statistical analysis methods could be employed as aids in authentication. This study provides valuable insights for large-scale sample chemical profiling and authentication.

Comprehensive HRMS Screening and Risk Assessments of Aristolochic Acid Analogues in Asari Radix et Rhizoma and Related Commercial Health Products.

Aristolochic acid analogues (AAAs) are well-known toxins. We performed the first comprehensive screening on AAAs in Asari Radix et Rhizoma (underground part of Asarum heterotropoides Schmidt), the only Aristolochiaceae plant widely used in clinical practice. LC-HRMS revealed 70 trace AAAs using polygonal mass defect filtering and precursor ion list strategies, 38 of which were newly discovered in A. heterotropoides. UHPLC-QTrap-MS/MS was then utilized for quantitative/semiquantitative analysis of 26 abundant compounds. Seventeen AAAs were detected from 91 batches of A. heterotropoides and 20 AAAs from 166 consumable products. For 141 Asari-containing proprietary products, aristolactam I and aristolactam II-glucoside exhibited the widest distribution, present in 98% products. AA IVa was the most abundant, detected in 91%. Notably, 60% of the products contained AA I (0.03-0.79 ppm). The safety was assessed using linear extrapolation, permitted daily exposure, cumulative amount, and the margin of exposure. It is recommended that AA I content be limited to 3 ppm.

Integrated UPLC/Q-TOF-MS/MS Analysis and Network Pharmacology to Reveal the Neuroprotective Mechanisms and Potential Pharmacological Ingredients of Aurantii Fructus Immaturus and Aurantii Fructus.

Aurantii Fructus (AF) and Aurantii Fructus Immaturus (AFI) have been used for thousands of years as traditional Chinese medicine (TCM) with sedative effects. Modern studies have shown that Citrus plants also have protective effects on the nervous system. However, the effective substances and mechanisms of action in Citrus TCMs still remain unclear. In order to explore the pharmacodynamic profiles of identified substances and the action mechanism of these herbs, a comprehensive approach combining ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS) analysis and network pharmacology was employed. Firstly, UNIFI 2.1.1 software was used to identify the chemical characteristics of AF and AFI. Secondly, the SwissTargetPrediction database was used to predict the targets of chemical components in AF and AFI. Targets for neuroprotection were also collected from GeneCards: The Human Gene Database (GeneCards-Human Genes|Gene Database|Gene Search). The networks between targets and compounds or diseases were then constructed using Cytoscape 3.9.1. Finally, the Annotation, Visualization and Integrated Discovery Database (DAVID) (DAVID Functional Annotation Bioinformatics Microarray Analysis) was used for GO and pathway enrichment analysis. The results showed that 50 of 188 compounds in AF and AFI may have neuroprotective biological activities. These activities are associated with the regulatory effects of related components on 146 important signaling pathways, derived from the KEGG (KEGG: Kyoto Encyclopedia of Genes and Genomes), such as neurodegeneration (hsa05022), the Alzheimer's disease pathway (hsa05010), the NF-kappa B signaling pathway (hsa04064), the hypoxia-inducible factor (HIF)-1 signaling pathway (hsa04066), apoptosis (hsa04210), the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance signaling pathway (hsa01521), and others, by targeting 108 proteins, including xanthine dehydrogenase (XDH), glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B), and glucose-6-phosphate dehydrogenase (G6PD), among others. These targets are thought to be related to inflammation, neural function and cell growth.

Energy preservation for skeletal muscles: Shenqi Fuzheng injection prevents tissue wasting and restores bioenergetic profiles in a mouse model of chemotherapy-induced cachexia.

BACKGROUND: Energy deficiency is the characteristic of chemotherapy-induced cachexia (CIC) which is manifested by muscle wasting. glycolysis, tricarboxylic acid (TCA) cycle, and lipid metabolism are central to muscle bioenergy production, which is vulnerable to chemotherapy during cancer treatment. Recent investigations have spotlighted the potential of Shenqi Fuzheng injection (SQ), a Chinese proprietary medicine comprising Radix Codonopsis and Radix Astragali, in alleviating CIC. However, the specific effects of SQ on muscle energy metabolism remains less explored. PURPOSE AND METHODS: Here, we integrated transcriptomics, spatial metabolomics, gas chromatography-mass spectrometry targeted quantitative analysis, and transmission electron microscopy techniques, combined with Seahorse live-cell metabolic analysis to reveal the changes in genes and pathways related to energy metabolism in the CIC model and SQ's protective effects at molecular and functional levels. RESULTS: Our data showed that chemotherapeutic agents caused glycolysis imbalance, which further leads to metabolic derangements of TCA cycle intermediates. SQ maintained glycolysis balance by facilitating pyruvate fluxing to mitochondria for more efficient bioenergy production, which involved a dual effect on promoting functions of mitochondrial pyruvate dehydrogenase complexes and inhibiting lactate dehydrogenase for lactate production. As a result of the sustained pyruvate level achieved by SQ administration, glycolysis balance was maintained, which further led to the preservation of mitochondrial integrity and function of electron transport chain, thereby, ensuring the normal operation of the TCA cycle and the proper synthesis of adenosine triphosphate (ATP). The above results were further validated using the Seahorse live-cell assay. CONCLUSION: In conclusion, our study highlights SQ as a promising strategy for CIC management, emphasizing its ability to harmonize the homeostasis of the muscle bioenergetic profile. Beyond its therapeutic implications, this study also offers a novel perspective for the development of innovative treatments in the realm of herbal medicine.

Species discrimination of multiple botanical origins of Fritillaria species based on infrared spectroscopy, thin layer chromatography-image analysis and untargeted metabolomics.

BACKGROUND: Fritillaria Bulbus (FB), a precious medicinal herb renowned for its heat-clearing, lung-moistening, cough-relieving and phlegm-eliminating effects. In pursuit of profits, unscrupulous merchants have engaged in the substitution or adulteration of valuable varieties with cheaper alternatives. It is, therefore, urgent to develop effective technical approaches to identify FBs from adulterants. METHODS: This paper employed infrared spectroscopy (IR), thin layer chromatography-image analysis (TLC-IA), and untargeted metabolomics techniques to discriminate ten species of FBs. RESULTS: Five species of FBs were successfully differentiated using mid-infrared spectroscopy. Furthermore, the power of TLC-IA technology allowed the differentiation of five species of FBs and two origins of FCBs (Fritillariae Cirrhosae Bulbus). Remarkably, through the application of untargeted metabolomics technique, the precise discrimination of five species of FBs, as well as three origins of FCBs were accomplished. Moreover, a comprehensive identification of 101 markers that reliably distinguished diverse FBs was achieved through the employment of untargeted metabolomics technique. CONCLUSION: The investigation presented powerful means of detection for assuring the quality control of Fritillaria herbs.

Systematical targeted multicomponent characterization and comparison of Arnebiae Radix and its three confusing species by offline two-dimensional liquid chromatography/LTQ-Orbitrap mass spectrometry.

Arnebiae Radix, commonly known as "Zicao," can be easily confused with other compounding species, posing challenges for its clinical use. Here, we developed a comprehensive strategy to systematically characterize the diverse components across Arnebiae Radix and its three confusing species. First, an offline two-dimensional liquid chromatography (2D-LC) system integrating hydrophilic interaction chromatography (HILIC) and reverse phase (RP) separations was established, enabling effective separation and detection of more trace constituents. Second, a polygonal mass defect filtering (MDF) workflow was implemented to screen target ions and generate a precursor ion list (PIL) to guide multistage mass (MSn) data acquisition. Third, a three-step characterization strategy utilizing diagnostic ions and neutral losses was developed for rapid determination of molecular formulas, structure classes, and compound identification. This approach enabled systematic characterization of Arnebiae Radix and its three confusing species, with 437 components characterized including 112 shikonins, 22 shikonfurans, 144 phenolic acids, 131 glycosides, 18 flavonoids, and 10 other compounds. Additionally, 361, 230, 340, and 328 components were identified from RZC, YZC, DZC, and ZZC, respectively, with 142 common components and 30 characteristic components that may serve as potential markers for distinguishing the four species. In summary, this is the first comprehensive characterization and comparison of the phytochemical profiles of Arnebiae Radix and its three confusing species, advancing our understanding of this herbal medicine for quality control.

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%.

MATLAB language assisted data acquisition and processing in liquid chromatography Orbitrap mass spectrometry: Application to the identification and differentiation of Radix Bupleuri from its adulterants.

Comprehensive and rapid analysis of secondary metabolites like saponins remains challenging. This study aimed to establish a semi-automated workflow for filtration, identification, and characterization of saikosaponins in six Bupleurum species. Radix Bupleuri, a high-sales herbal medicine, is often adulterated, restricting its quality control and applications. Two authentic Radix Bupleuri species and four major adulterants were analyzed through UHPLC-LTQ-Orbitrap-MS for targeted saikosaponin analysis. To reveal trace saikosaponins and obtain quality fragment data, a MATLAB-based process automatically enumerating "sugar chain + aglycone + side chain" combinations and deduplicating generated a predicted saikosaponin database covering all possible saikosaponins as a precursor ion list for comprehensive targeted acquisition. To focus on informative ions and reduce MS analysis workload, we utilized MATLAB to automatically filtrate the false positive ions by MS1 and MS2 spectrometry. The newly established MATLAB-assisted data acquisition approach exhibited 50 % improvement in characterization of targeted saikosaponins. Furthermore, positive and negative ionization workflows were designed for accurate saikosaponins characterization based on fragmentation rules. In total, 707 saikosaponins were characterized, including over 500 potential new compounds and previously unreported C29 aglycones. We identified 25 saikosaponins present in both authentic species but absent in adulterants as potential markers. This unprecedented comprehensive multi-origin species differentiation demonstrates the promise of MATLAB-assisted acquisition and processing to advance saponin identification and standardize the Radix Bupleuri market.

Asponchimides A-E: new enantiomeric N-acetyldopamine trimers from Aspongopus chinensis.

Five new racemic N-acetyldopamine (NADA) trimers, asponchimides A-E (1-5), were isolated from Aspongopus chinensis, a prominent traditional Chinese medicinal insect employed for alleviating pain, treating indigestion, and addressing kidney ailments. Compounds 1-5 were successfully resolved by chiral high-performance liquid chromatography (HPLC), yielding five pairs of enantiomers: (+)- and (-)-asponchimides A-E (1a/1b-5a/5b). Their structural identities were discerned by extensive spectroscopic analyses, including high-resolution mass spectrometry (HRMS), ultraviolet-visible (UV-Vis) spectroscopy, infrared (IR) spectroscopy, and nuclear magnetic resonance (NMR), and their absolute configurations were determined by electronic circular dichroism (ECD) calculations. Compounds 1-5 are pioneering instances of NADA trimers featuring a Δ7 double bond. When subjected to a series of bioassays, a majority of the compounds exhibited weak inhibitory activity against nitric oxide (NO) production in LPS-induced RAW 264.7 cells.

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics.

Pheretima, also called "earthworms", is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines (CPMs) in Chinese Pharmacopoeia (2020 edition). However, its zoological origin is unclear, both in the herbal market and CPMs. In this study, a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms (restricted search, open search, and de novo) was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima, including Pheretima aspergillum (PA), Pheretima vulgaris (PV), and Metaphire magna (MM). We identified 10,477 natural peptides in the PA, 7,451 in PV, and 5,896 in MM samples. Five specific signature peptides were screened and then validated using synthetic peptides; these demonstrated robust specificity for the authentication of PA, PV, and MM. Finally, all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills, revealing the inconsistent Pheretima species used in these CPMs. In conclusion, our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines, especially non-model species with poorly annotated protein databases.

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.

Targeted trace ingredients coupled with chemometric analysis for consistency evaluation of Panax notoginseng saponins injectable formulations.

Evaluating the consistency of herb injectable formulations could improve their product quality and clinical safety, particularly concerning the composition and content levels of trace ingredients. Panax notoginseng Saponins Injection (PNSI), widely used in China for treating acute cardiovascular diseases, contains low-abundance (10%-25%) and trace saponins in addition to its five main constituents (notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd). This study aimed to establish a robust analytical method and assess the variability in trace saponin levels within PNSI from different vendors and formulation types. To achieve this, a liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method employing multiple ions monitoring (MIM) was developed. A "post-column valve switching" strategy was implemented to eliminate highly abundant peaks (NR1, Rg1, and Re) at 26 min. A total of 51 saponins in PNSI were quantified or relatively quantified using 18 saponin standards, with digoxin as the internal standard. This study evaluated 119 batches of PNSI from seven vendors, revealing significant variability in trace saponin levels among different vendors and formulation types. These findings highlight the importance of consistent content in low-abundance and trace saponins to ensure product control and clinical safety. Standardization of these ingredients is crucial for maintaining the quality and effectiveness of PNSI in treating acute cardiovascular diseases.

A comprehensive analysis of the Bencao (herbal) small RNA Atlas reveals novel RNA therapeutics for treating human diseases.

Cross-kingdom herbal miRNA was first reported in 2012. Using a modified herbal extraction protocol, we obtained 73,677,287 sequences by RNA-seq from 245 traditional Chinese Medicine (TCM), of which 20,758,257 were unique sequences. We constructed a Bencao (herbal) small RNA (sRNA) Atlas ( http://bencao.bmicc.cn ), annotated the sequences by sequence-based clustering, and created a nomenclature system for Bencao sRNAs. The profiles of 21,757 miRNAs in the Atlas were highly consistent with those of plant miRNAs in miRBase. Using software tools, our results demonstrated that all human genes might be regulated by sRNAs from the Bencao sRNA Atlas, part of the predicted human target genes were experimentally validated, suggesting that Bencao sRNAs might be one of the main bioactive components of herbal medicines. We established roadmaps for oligonucleotide drugs development and optimization of TCM prescriptions. Moreover, the decoctosome, a lipo-nano particle consisting of 0.5%-2.5% of the decoction, demonstrated potent medical effects. We propose a Bencao (herbal) Index, including small-molecule compounds (SM), protein peptides (P), nucleic acid (N), non-nucleic and non-proteinogenic large-molecule compounds (LM) and elements from Mendeleev's periodic table (E), to quantitatively measure the medical effects of botanic medicine. The Bencao sRNA Atlas is a resource for developing gene-targeting oligonucleotide drugs and optimizing botanical medicine, and may provide potential remedies for the theory and practice of one medicine.

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.

Enantiomeric N-acetyldopamine trimers from Cicadae Periostracum and their absolute configurations.

Six previously undescribed N-acetyldopamine (NADA) trimmer racemates, percicamides A-F (1-6), were isolated from a 70% ethanol extract of Cicadae Periostracum. Subsequent chiral-phase separation afforded six pairs of enantiomers, (+)- and (-)-percicamides A-F (1a/1b-6a/6b). Their structures including absolute configurations were elucidated by combined extensive spectroscopic data and quantum chemical calculations. Compounds 1-6 represent the first examples of NADA trimmers with a cis-relationship of H-7'/H-8' or H-7''/H-8''. Bioassays verified that all isolated compounds showed weak inhibitory effects on nitric oxide production in RAW 264.7 cells.

A MS-feature-based medicinal plant database-driven strategy for ingredient identification of Chinese medicine prescriptions.

Identification of the individual herbs that constitute the Chinese medicine prescription (CMP) is a key step to control the quality and ensure the efficacy of traditional Chinese medicine (TCM), but also a challenging task for analysts from all over the world. In this study, a MS-feature-based medicinal plant database-driven strategy was proposed for quick and automatic interpretation of CMP ingredients. The single herb database consisting of stable ions of sixty-one common TCM medicinal herbs was first constructed. And then, the data of CMP was imported into a self-built searching program to achieve quick and automatic identification with four steps including level 1 candidate herb screening based on stable ions (step 1), level 2 candidate herb screening based on unique ions (step 2), difficult-to-distinguish herb differentiation (step 3) and results integration (step 4). The identification model was optimized and validated with homemade Shaoyaogancao Decoction, Mahuang Decoction, Banxiaxiexin Decoction, and their related negative prescriptions and homemade fakes. Another nine batches of homemade and commercial CMPs were applied to this new approach and most of composed herbs in the corresponding CMPs were correctly identified. This work provided a promising and universal strategy for the clarification of CMP ingredients.

Characterization of the natural peptidome of four leeches by integrated proteogenomics and pseudotargeted peptidomics.

Animal-derived drugs are an indispensable part of folk medicine worldwide. However, their chemical constituents are poorly approached, which leads to the low level of the quality standard system of animal-derived drugs and further causes a chaotic market. Natural peptides are ubiquitous throughout the organism, especially in animal-derived drugs. Thus, in this study, we used multi-source leeches, including Hirudo nipponica (HN), Whitmania pigra (WP), Whitmania acranulata (WA), and Poecilobdella manillensis (PM), as a model. A strategy integrating proteogenomics and novel pseudotargeted peptidomics was developed to characterize the natural peptide phenotype and screen for signature peptides of four leech species. First, natural peptides were sequenced against an in-house annotated protein database of closely related species constructed from RNA-seq data from the Sequence Read Archive (SRA) website, which is an open-sourced public archive resource. Second, a novel pseudotargeted peptidomics integrating peptide ion pair extraction and retention time transfer was established to achieve high coverage and quantitative accuracy of the natural peptides and to screen for signature peptides for species authentication. In all, 2323 natural peptides were identified from four leech species whose databases were poorly annotated. The strategy was shown to significantly improve peptide identification. In addition, 36 of 167 differential peptides screened by pseudotargeted proteomics were identified, and about one-third of them came from the leucine-rich repeat domain (LRR) proteins, which are widely distributed in organisms. Furthermore, six signature peptides were screened with good specificity and stability, and four of them were validated by synthetic standards. Finally, a dynamic multiple reaction monitoring (dMRM) method based on these signature peptides was established and revealed that one-half of the commercial samples and all of the Tongxinluo capsules were derived from WP. All in all, the strategy developed in this study was effective for natural peptide characterization and signature peptide screening, which could also be applied to other animal-derived drugs, especially for modelless species that are less studied in protein database annotation.

Systematical characterization of gypenosides in Gynostemma pentaphyllum and the chemical composition variation of different origins.

Gynostemma pentaphyllum (Thunb.) Makino is an herbaceous plant of Cucurbitaceae family, which has been widely used as an herbal tea and traditional Chinese medicine. Since its saponins are similar to ginsenosides and have a wide range of activities, it has attracted wide interest. However, there are still a large number of unknown saponins that have not been isolated, especially some trace gypenosides. In the present study, a HILIC × RP offline two-dimensional liquid separation combined with a multimode data acquisition was developed for the systematical characterization of gypenosides. On top of the negative mode information, considering that saponins are prone to in-source fragmentations in positive ion mode, a precursor ion list data acquisition method was used for the targeted acquisition of multistage positive data. Reference herbal drug was taken as a golden sample to probe the chemical composition of G. pentaphyllum. The mixed sample of commercially available samples were also analyzed in parallel. Furthermore, the chemical compositions of commercially available samples from different sources were compared. In total, 1108 saponins were characterized, among which 588 were accurately characterized, with 574 identified in the reference herbal drug and 700 in the mixed commercially available samples. The commercially available samples showed great composition variation. These findings clarified the material basis and provided clues for quality control of G. pentaphyllum.