Biosynthetic pathway analysis combined with feature-based molecular networking to comprehensively characterize the chemical constituents in seeds of Sterculia lychnophora.

INTRODUCTION: The seeds of Sterculia lychnophora Hance, commonly known as Pangdahai (PDH) in Chinese, have found extensive use in both culinary and traditional medicinal practices. However, a comprehensive understanding of the chemical composition of PDH has been lacking. OBJECTIVES: This study proposes a strategy that integrates biosynthetic pathway analysis with feature-based molecular networking (FBMN), aiming for a thorough and global characterization of the chemical compositions of PDH. METHODOLOGY: The FBMN map reveals potential compounds with structural similarity, and the MS/MS fragments could be annotated based on library matches, which could predict the plausible biosynthetic pathways in PDH, accomplishing the annotation of compounds clustered in FBMN by integrating biosynthetic pathways. RESULTS: Consequently, 126 compounds were plausibly or unambiguously identified, including 37 phenolic acids and glycosides, 20 flavonoids and glycosides, 12 procyanidins, 21 alkaloids, 22 lipids, and 14 others. Leveraging the information, 40 compounds, including 1 unique isoquinoline alkaloid and 2 rare linear furocoumarins, were isolated and confirmed. CONCLUSIONS: This study not only demonstrates a highly effective approach for identifying compounds within complex herbal mixtures but also establishes a robust foundation for the further development of PDH.

Determination of 44 major components and chemical profiling of saccharide in Chinese medicinal formula Lanqin Oral Liquid.

INTRODUCTION: Lanqin Oral Liquid (LQL) is a traditional Chinese medicine preparation (TCMP) containing five herbal medicines and has been commonly used for the treatment of pharyngitis and hand-foot-and-mouth disease in clinic. The material basis of LQL has been reported in our previous study, but the contents of the major components and the features of saccharide in LQL are still unclear. OBJECTIVES: This study aimed to establish accurate and rapid methods for the quantification of the major components and profiling of saccharide in LQL. The quantitative results combined with similarity evaluation were applied to improve the quality control of LQL. METHODOLOGY: An ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-QQQ-MS) method was utilised to determine 44 major components. Cosine similarity was used to evaluate the similarities among 20 batches of LQL based on the quantitative results of 44 major components. The physicochemical properties, structure, composition, and contents of saccharide in LQL were detected by a combination of chemical and instrumental analysis. RESULTS: A total of 44 compounds, including flavonoids, iridoid glycosides, alkaloids, and nucleosides, were accurately determined. The 20 batches of LQL were remarkably similar (> 0.95). In addition, d-glucose, galactose, d-glucuronic acid, arabinose, and d-mannose were detected in saccharide of LQL. The contents of saccharide in LQL were 13.52-21.09 mg/ml. CONCLUSIONS: The established methods can be applied for the comprehensive quality control of LQL, including characterisation of saccharide and quantification of representative components. Our study will provide a robust chemical foundation for disclosing the quality markers of its therapeutic effect.

An unexpected hydratase synthesizes the green light-absorbing pigment fucoxanthin.

The ketocarotenoid fucoxanthin and its derivatives can absorb blue-green light enriched in marine environments. Fucoxanthin is widely adopted by phytoplankton species as a main light-harvesting pigment, in contrast to land plants that primarily employ chlorophylls. Despite its supreme abundance in the oceans, the last steps of fucoxanthin biosynthesis have remained elusive. Here, we identified the carotenoid isomerase-like protein CRTISO5 as the diatom fucoxanthin synthase that is related to the carotenoid cis-trans isomerase CRTISO from land plants but harbors unexpected enzymatic activity. A crtiso5 knockout mutant in the model diatom Phaeodactylum tricornutum completely lacked fucoxanthin and accumulated the acetylenic carotenoid phaneroxanthin. Recombinant CRTISO5 converted phaneroxanthin into fucoxanthin in vitro by hydrating its carbon-carbon triple bond, instead of functioning as an isomerase. Molecular docking and mutational analyses revealed residues essential for this activity. Furthermore, a photophysiological characterization of the crtiso5 mutant revealed a major structural and functional role of fucoxanthin in photosynthetic pigment-protein complexes of diatoms. As CRTISO5 hydrates an internal alkyne physiologically, the enzyme has unique potential for biocatalytic applications. The discovery of CRTISO5 illustrates how neofunctionalization leads to major diversification events in evolution of photosynthetic mechanisms and the prominent brown coloration of most marine photosynthetic eukaryotes.

Discovery of a potent allosteric activator of DGKQ that ameliorates obesity-induced insulin resistance via the sn-1,2-DAG-PKCε signaling axis.

sn-1,2-diacylglycerol (sn-1,2-DAG)-mediated activation of protein kinase Cε (PKCε) is a key pathway that is responsible for obesity-related lipid metabolism disorders, which induces hepatic insulin resistance and type 2 diabetes. No small molecules have been previously reported to ameliorate these diseases through this pathway. Here, we screened and identified the phytochemical atractylenolide II (AT II) that reduces the hepatic sn-1,2-DAG levels, deactivates PKCε activity, and improves obesity-induced hyperlipidemia, hepatosteatosis, and insulin resistance. Furthermore, using the ABPP strategy, the diacylglycerol kinase family member DGKQ was identified as a direct target of AT II. AT II may act on a novel drug-binding pocket in the CRD and PH domains of DGKQ to thereby allosterically regulate its kinase activity. Moreover, AT II also increases weight loss by activating DGKQ-AMPK-PGC1α-UCP-1 signaling in adipose tissue. These findings suggest that AT II is a promising lead compound to improve obesity-induced insulin resistance.

Time-Series-Dependent Global Data Filtering Strategy for Mining and Profiling of Xenobiotic Metabolites in a Dynamic Complex Matrix: Application to Biotransformation of Flavonoids in the Extract of Ginkgo biloba by Gut Microbiota.

Efficient characterization of xenobiotic metabolites and their dynamics in a changing complex matrix remains difficult. Herein, we proposed a time-series-dependent global data filtering strategy for the rapid and comprehensive characterization of xenobiotic metabolites and their dynamic variation based on metabolome data. A set of data preprocessing methods was used to screen potential xenobiotic metabolites, considering the differences between the treated and control groups and the fluctuations over time. To further identify metabolites of the target, an in-house accurate mass database was constructed by potential metabolic pathways and applied. Taking the extract of Ginkgo biloba (EGB) co-incubated with gut microbiota as an example, 107 compounds were identified as flavonoid-derived metabolites (including 67 original from EGB and 40 new) from 7468 ions. Their temporal metabolic profiles and regularities were also investigated. This study provided a systematic and feasible method to elucidate and profile xenobiotic metabolism.

A Streptomyces P450 enzyme dimerizes isoflavones from plants.

Dimerization is a widespread natural strategy that enables rapid structural diversification of natural products. However, our understanding of the dimerization enzymes involved in this biotransformation is still limited compared to the numerous reported dimeric natural products. Here, we report the characterization of three new isoflavone dimers from Streptomyces cattleya cultured on an isoflavone-containing agar plate. We further identified a cytochrome P450 monooxygenase, CYP158C1, which is able to catalyze the dimerization of isoflavones. CYP158C1 can also dimerize plant-derived polyketides, such as flavonoids and stilbenes. Our work represents a unique bacterial P450 that can dimerize plant polyphenols, which extends the insights into P450-mediated biaryl coupling reactions in biosynthesis.

A database-guided integrated strategy for comprehensive chemical profiling of traditional Chinese medicine.

A comprehensive chemical profiling of traditional Chinese medicine is the basic issue for further pharmacological research and quality assessment. To facilitate chemical identification and potential components discovery, the present study proposed an integrated identification strategy guided by a self-built component database constructed from literatures to carry out the global profiling of complex matrixes. Lanqin Oral Liquid was applied as example to validate the feasibility of this strategy. Based on LQL Component Database containing 710 compounds, modified MDF windows was established to extract the interested analogues, isoquinoline alkaloids, flavonoids and iridoid glycosides, according to their regular integral masses and mass defect. For compounds with characteristic substructures, such as quinic acids, crocins and some glycoside derivatives, the associated neutral losses and diagnostic fragment ions were collected to assist in profiling. Directly matching the m/z or formulas in database was proposed to components with limited regularity of accurate masses and substructures, like indole alkaloids, sesquiterpenes and some nucleosides. Eventually, 170 ions of 1038 precursor ions were identified or temporarily deduced, including 59 alkaloids, 36 flavonoids, 48 terpenoids, 24 organic acids and their derivatives, 2 oligosaccharides, and 1 lignans. Among them, 52 putative compounds were confirmed by chemical standards. The results indicated that the database-oriented identification strategy could locate potential components quickly and eliminate interfering ions, which have the potential for in-depth analysis of compounds.

A validated UHPLC-MS/MS method for pharmacokinetic and brain distribution studies of twenty constituents in rat after oral administration of Jia-Wei-Qi-Fu-Yin.

A rapid ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ MS/MS) approach with high sensitivity and selectivity was developed for the quantification of twenty compounds, including 9 saponins, 8 flavonoids, 2 oligosaccharide esters and 1 phenolic acid, in rat plasma and brain, which was administrated intragastrically with Jia-Wei-Qi-Fu-Yin (JWQFY), Mass spectrometric detection was operated under multiple reaction monitoring (MRM) mode. All calibration curves possessed good linearity with correlation coefficients ( r2) higher than 0.9916 in their respective linear ranges. For intra- and inter-day precision, all the relative standard deviations (RSDs) at different levels were less than 14.68 %. Based on the UHPLC-QqQ MS/MS quantitative results, pharmacokinetic study and brain distribution of multiple components in JWQFY was then successfully performed. As a result, constituents with discrepancy structures showed diverse pharmacokinetic and distribution characteristics. For instance, ferulic acid (phenolic acid), 3, 6'-disinapoyl sucrose and tenuifoliside A (oligosaccharide esters) showed short Tmax (< 10 min), whereas the Tmax of ginsenosides Rb1, Rb2 and Rc (ppd-type terpenoid saponins) were much longer (> 4 h). Besides, ferulic acid, epimedin C, icariin, glycyrrhizin, ginsenoside Rb1 and ginsenoside Rg1 were considered as the potential effective ingredients of JWQFY because of their relatively high exposure to blood and brain. Our study would provide relevant information for discovery of pharmacodynamic ingredients, as well as further action mechanisms investigations of JWQFY.

Comprehensive chemical profiling of Jia-Wei-Qi-Fu-Yin and its network pharmacology-based analysis on Alzheimer's disease.

Jia-Wei-Qi-Fu-Yin (JWQFY) is a newly developed anti-Alzheimer's disease (AD) prescription modified from a classical traditional Chinese medicine formula, Qi-Fu-Yin (QFY). However, a systematic understanding of its chemical constituents and molecular mechanisms is still elusive. To address this problem, comprehensive chemical profiling followed by network pharmacology-based analysis of JWQFY was performed. Firstly, a total of 136 compounds were characterized by high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF MS), 17 of them were specifically identified in JWQFY comparing with QFY. Seventy compounds were further quantified via a validated HPLC coupled with triple quadrupole tandem mass spectrometry (QQQ MS) method. Then the protein targets of the seventy compounds were gathered from public databases for network construction. As a result, fifty-seven compounds were filtered, which interacted with 655 targets. Thirty-four of them were mapped into the KEGG pathway of AD, indicating JWQFY might exert anti-AD effects by anti-inflammation, neuronal apoptosis intervening, Aß production inhibition and phosphorylating tau protein moderating. Furthermore, in the compound-target-AD network, a list of hub compounds and hub targets was identified based on their topological features, including the degree, node betweenness and closeness. Four of the hub compounds were specifically originated from JWQFY, supporting the modification rationality of this formula. This study provided a scientific basis for understanding the bioactive compounds and the multi-target mechanism of JWQFY.

Generation and Reactivity of Amidyl Radicals: Manganese-Mediated Atom-Transfer Reaction.

A simple and efficient protocol to generate amidyl radicals from amine functionalities through a manganese-mediated atom-transfer reaction has been developed. This approach employs an earth-abundant and inexpensive manganese complex, Mn2 (CO)10 , as the catalyst and visible light as the energy input. Using this strategy, site-selective chlorination of unactivated C(sp3 )-H bonds of aliphatic amines and intramolecular/intermolecular chloroaminations of unactivated alkenes were readily realized under mild reaction conditions, thus providing efficient access to a range of synthetically valuable alkyl chlorides, chlorinated pyrrolidines, and vicinal chloroamine derivatives. These practical reactions exhibit a broad substrate scope and tolerate a wide array of functional groups, and complex molecules including various marketed drug derivatives.

Visible-Light-Promoted Manganese-Catalyzed Atom Transfer Radical Cyclization of Unactivated Alkyl Iodides.

An expedient visible-light-promoted atom transfer radical cyclization (ATRC) reaction of unactivated alkyl iodides facilitated by earth-abundant and inexpensive manganese catalysis is described. The practical protocol shows a broad substrate scope and good functional-group tolerance, allowing for the preparation of synthetically valuable alkenyl iodides and diquinanes under simple and mild reaction conditions. Notably, the method provides a net redox-neutral strategy for ATRC reactions that avoids classic hydrogen atom transfer mechanism.

A strategy for screening bioactive components from natural products based on two-dimensional cell membrane chromatography and component-knockout approach.

Cell membrane chromatography (CMC) is a bioaffinity chromatographic method used to screen active compounds from natural products. However, since the receptor capacity of CMC column is limited, high content/affinity compounds may cause column overloading and thus lead to ignorance of other positive candidates. For avoiding this effect and comprehensively discovering bioactive components, a strategy based on two-dimensional CMC and component-knockout approach was proposed. As an illustrative case study, red yeast rice (RYR), a rice product with good myocardial protective effect in clinical studies, was selected as the model experimental sample. For discovering its potential cardioprotective compounds, a CMC model with H9c2 rat cardiac myoblasts (H9c2/CMC) with good selectivity, stability and reproducibility was established. By using two-dimensional H9c2/CMC-HPLC coupled with QTOF MS system, three components were firstly screened out. After knocking out high content/affinity compound, another four bioactive compounds were then found. By this two-round screening, column overloading caused by high concentration or infinity compounds was avoided, and trace compounds were enriched. As a result, one pigment and six monacolins from RYR were fished out. The results indicate the proposed strategy might be used to discover active compounds from complex matrix.

A fast and accurate method for the identification of peroxidase inhibitors from Radix Salvia Miltiorrhizae by on-flow biochemical assay coupled with LC/Q-TOF-MS: comparison with ultrafiltration-based affinity selection.

Development of fast and accurate methods to discover lead compounds for drug candidates is highly important. In this study, a reliable and effective post-column on-flow biochemical assay (POBA) was established to screen potent peroxidase inhibitors from complex chemical mixtures (e.g., natural product extracts). Multiple factors such as flow rate, organic phase, detection wavelength, and reaction coil were carefully investigated. To better understand the features of POBA, another emerging technology of ultrafiltration LC-MS was used for comparison. The result showed that POBA had advantages in saving time, avoiding false positives, and improving the accuracy. To illustrate the practicality of the method, Radix Salvia Miltiorrhizae, a traditional herb for cardiovascular disease treatment, was applied as the research objective. Finally, six compounds including tanshinol, protocatechuic aldehyde, salvianolic acid D, rosmarinic acid, lithospermic acid, and salvianolic acid B were determined as novel peroxidase inhibitors. Their bioactivities were validated by microplate-based assay, molecular docking, and pharmacophore modeling. This study demonstrates a great potential of POBA in the efficient and accurate discovery of drug candidates. Graphical abstract Compared with a classical method of ultrafiltration LC-MS, the newly developed method of on-flow bioassay shows advantages in saving time, avoiding false positives and improving the accuracy.