The "depict" strategy for discovering new compounds in complex matrices: Lycibarbarspermidines as a case.

The comprehensive detection and identification of active ingredients in complex matrices is a crucial challenge. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is the most prominent analytical platform for the exploration of novel active compounds from complex matrices. However, the LC-HRMS-based analysis workflow suffers from several bottleneck issues, such as trace content of target compounds, limited acquisition for fragment information, and uncertainty in interpreting relevant MS2 spectra. Lycibarbarspermidines are vital antioxidant active ingredients in Lycii Fructus, while the reported structures are merely focused on dicaffeoylspermidines due to their low content. To comprehensively detect the new structures of lycibarbarspermidine derivatives, a "depict" strategy was developed in this study. First, potential new lycibarbarspermidine derivatives were designed according to the biosynthetic pathway, and a comprehensive database was established, which enlarged the coverage of lycibarbarspermidine derivatives. Second, the polarity-oriented sample preparation of potential new compounds increased the concentration of the target compounds. Third, the construction of the molecular network based on the fragmentation pathway of lycibarbarspermidine derivatives broadened the comprehensiveness of identification. Finally, the weak response signals were captured by data-dependent scanning (DDA) followed by parallel reaction monitoring (PRM), and the efficiency of acquiring MS2 fragment ions of target compounds was significantly improved. Based on the integrated strategy above, 210 lycibarbarspermidine derivatives were detected and identified from Lycii Fructus, and in particular, 170 potential new compounds were structurally characterized. The integrated strategy improved the sensitivity of detection and the coverage of low-response components, and it is expected to be a promising pipeline for discovering new compounds.

A funnel-type stepwise filtering strategy for identification of potential Q-markers of traditional Chinese medicine formulas.

Quality marker (Q-marker) serves as an important driver for the standardization of quality control in traditional Chinese medicine (TCM) formulas. However, it is still challenging to discover comprehensive and representative Q-markers. This study aimed to identify Q-markers of Hugan tablet (HGT), a famous TCM formula with ideal clinical effects in liver diseases. Here, we proposed a funnel-type stepwise filtering strategy that integrated secondary metabolites characterization, characteristic chromatogram, quantitative analysis, literature mining, biotransformation rules and network analysis. Firstly, the strategy of "secondary metabolites-botanical drugs-TCM formula" was applied to comprehensively identify the secondary metabolites of HGT. Then, the secondary metabolites with specificity and measurability in each botanical drug were identified by HPLC characteristic chromatogram, biosynthesis pathway and quantitative analysis. Based on literature mining, the effectiveness of botanical metabolites that met the above conditions was evaluated. Furthermore, the metabolism of the above metabolites in vivo was studied to reveal their biotransformation forms, which were used for network analysis. At last, according to biotransformation rules of the prototype drugs in vivo, the secondary metabolites were traced and preliminarily chosen as Q-markers. As a result, 128 plant secondary metabolites were identified in HGT, and 11 specific plant secondary metabolites were screened out. Then, the content of specific plant secondary metabolites in 15 batches of HGT was determined, which confirmed their measurability. And the results of literature mining showed that eight secondary metabolites had therapeutic effects in treating liver disease at the in vivo level, and three secondary metabolites inhibited liver disease-related indicators at the in vitro level. After that, 26 compounds absorbed into the blood (11 specific plant metabolites and their 15 metabolites in vivo) were detected in rats. Moreover, 14 compounds, including prototype components and their metabolites, were selected as Q-marker candidates by the "TCM formula-botanical drugs-compounds-targets-pathways" network. Finally, 9 plant secondary metabolites were defined as comprehensive and representative Q-markers. Our study not only provides a scientific basis for the improvement and secondary development of the quality standard of HGT, but also proposes a reference method for discovering and identifying Q-markers of TCM preparations.

Feature-Based Molecular Networking Facilitates the Comprehensive Identification of Differential Metabolites in Diabetic Cognitive Dysfunction Rats.

Cognitive dysfunction is a frequent complication of type 2 diabetes mellitus (T2DM), usually accompanied by metabolic disorders. However, the metabolic changes in diabetic cognitive dysfunction (DCD) patients, especially compared to T2DM groups, are not fully understood. Due to the subtle differences in metabolic alterations between DCD groups and T2DM groups, the comprehensive detection of the untargeted metabolic profiles of hippocampus and urine samples of rats was conducted by LC-MS, considering the different ionization modes and polarities of the examined compounds, and feature-based molecular networking (FBMN) was performed to help identify differential metabolites from a comprehensive perspective in this study. In addition, an association analysis of the differential metabolites in hippocampus and urine was conducted by the O2PLS model. Finally, a total of 71 hippocampal tissue differential metabolites and 179 urine differential metabolites were identified. The pathway enrichment results showed that glutamine and glutamate metabolism, alanine, aspartate, and glutamate metabolism, glycerol phospholipid metabolism, TCA cycle, and arginine biosynthesis in the hippocampus of DCD animals were changed. Seven metabolites (AUC > 0.9) in urine appeared as key differential metabolites that might reflect metabolic changes in the target tissue of DCD rats. This study showed that FBMN facilitated the comprehensive identification of differential metabolites in DCD rats. The differential metabolites may suggest an underlying DCD and be considered as potential biomarkers for DCD. Large samples and clinical experiments are needed for the subsequent elucidation of the possible mechanisms leading to these alterations and the verification of potential biomarkers.

Integrated Metabolomics and Network Pharmacology Study on the Mechanism of Kangfuxiaoyan Suppository for Treating Chronic Pelvic Inflammatory Disease.

Kangfuxiaoyan suppository (KFXYS) is a commonly used traditional Chinese medicine (TCM) preparation for the treatment of chronic pelvic inflammatory disease (CPID) clinically, and its safety and effectiveness have been well verified. However, the potential mechanism remains unclear. The integrated strategy of metabolomics and network pharmacology was employed in the study to reveal the potential mechanism of KFXYS in the treatment of CPID. Our research consists of five steps. First, the effect of KFXYS in reversing uterine inflammation indexes was verified. Second, based on the comprehensive characterization of 123 chemical ingredients of KFXYS, the ingredients of KFXYS absorbed into blood were identified by UPLC-Q-TOF/MS, then ADME research was carried out on the main ingredients. Third, the differential metabolites with significant correlation to inflammatory indexes were discovered by metabolomics and correlation analysis. Fourth, the potential targets and pathways of KFXYS in treating CPID were predicted by network pharmacology based on the ingredients which had good ADME behavior. Fifth, the proteins in common pathways of metabolomics and network pharmacology were used to screen the key targets from the potential targets of network pharmacology, and the potential mechanism of KFXYS in treating CPID was clarified. As a result, KFXYS significantly reversed the uterine inflammation indexes, including IL-1 and IL-6. The ingredients absorbed into blood including matrine, sophocarpine, aloin, esculetin-O-glucuronide, 7,4'-dihydroxyisoflavone-O-glucuronide, and 4'-methoxyisoflavone-7-O-glucuronide had good ADME behavior in vivo. Among the differential metabolites, Leukotriene A4, 5-Hydroxyindoleacetic acid, Ornithine, Arginine, and PC (20:1 (11Z)/20:4 (8Z,11Z,14Z,17Z)) were significant correlation to inflammation indexes. The integration analysis of metabolomics and network pharmacology shows that KFXYS may regulate the key targets including ARG1, NOS2, NOS3, etc. We speculate that ingredients of KFXYS, such as matrine, sophocarpine, aloin etc. act on the key proteins including ARG1, NOS2, and NOS3, to exert anti-inflammatory effect.

BH+/MH+-matching method for discovery of cis-diol-containing modified nucleosides in urine by ribose-targeted solid phase extraction followed by dual-mass spectrometry platform identification.

Profiling of new modified nucleosides from urine plays an important role in exploring biomarkers for cancer. However, limitations from the nature of the compound, bio-sample, instrumentation, and analytical method pose great challenges to achieving a comprehensive analysis of urinary nucleosides. Herein, a method of BH+/MH+-matching (BH+, protonated nucleobase ion; MH+, protonated precursor ion) was developed to discover novel modified nucleosides from human urine samples based on solid phase extraction targeted toward specific modified nucleosides combined with ultra-performance liquid chromatography coupled with dual-mass spectrometry platforms. Firstly, nucleosides containing 2,3-diol structure on ribose were effectively enriched by PBA (Phenylboronic Acid) cartridges. Secondly, a novel method, "BH+/MH+-matching" was established to achieve rapid screening of modified nucleosides. Based on the in-source fragmentation pattern of nucleosides, a series of putative modified nucleosides were rationally designed and characterized by matching the daughter ion BH+ and its parent ion MH+ in UPLC-MSE spectra. Finally, as a complement to UPLC Q-TOF/MS, UPLC Q-Trap/MS was employed to validate the structure of putative compounds by MRM-IDA-EPI mode. Using the strategy, 12 new cis-diol-containing nucleoside analogs were successfully characterized, which were formed by modified base (m1A, m6A, m2,2,7G, ac4C) and modified ribose containing C5'-O-formylation or C5'-O-methylation. Taken together, the results demonstrated our strategy could efficiently support the rapid discovery of cis-diol-containing nucleosides with modifications on either ribose or base moiety (or both), which exhibited a promising perspective in the future application of biochemical analysis and clinical diagnosis.

Ethnobotanical survey of medicinal plants in Gaomi, China.

ETHNOPHARMACOLOGICAL RELEVANCE: The uses of medicinal plants have a long history and become one of the important sources of the health cares in Gaomi City, Shandong Province, China. However, limited studies have been done to identify these medicinal plant species and to scientifically document their associated traditional knowledge. Many species used by indigenous people could potentially represent a novel resource of medicine. The study can aid in further investigations of modern pharmacology and planning of the wild species conservation. AIM OF THE STUDY: The study aimed to investigate and record the medicinal plant taxa and their associated traditional knowledge in Gaomi City, China. MATERIALS AND METHODS: Field study was conducted from March 2018 to May 2019 with 184 residents of Gaomi City. Traditional medicinal plant specimens were collected from the field with the help of these residents and were identified and authenticated in the Herbarium of the School of Pharmacy, Binzhou Medical University. Ethnobotanical knowledge was collected by semi-structured face-to-face interviews. The quantitative data were analyzed by using the informant consensus factor (ICF) method and the number of citations. RESULTS: A total of 181 species belonging to 137 genera and 65 families were collected in Gaomi City. Asteraceae was the predominant family and Fabaceae took the second place. River basins and the southern hills in Gaomi were rich in vegetation. However, the cultivated area of medicinal plants only accounted for 10% of agricultural acreage. The main preparation method was decocting (170, 94.48%) and the most frequent mode of administration was oral (177, 97.97%). The highest numerical ICF value was recorded for treating endocrine, metabolic, and nutritional (ICF: 0.85) conditions. Seven of the medicinal plant species used by the people in Gaomi have not been reported previously in China. Verbena officinalis L. was found in Gaomi City, which is a new distribution record for this species. CONCLUSIONS: People in Gaomi hold valuable knowledge about the use of medicinal plants; however, their knowledge has not been comprehensively documented. The therapeutic uses of the documented medicinal plants will provide a basis for further pharmacological and phytochemical investigations. Additionally, the result of this study indicated that the elder people in Gaomi have more traditional knowledge of plant medicines than the younger ones.