A comprehensive study of Chuanwang Xiaoyan capsule based on characterization of chemical constituents in vitro and in vivo using ultra-high-performance liquid chromatography-quadrupole-Exactive Orbitrap mass spectrometry and pharmacokinetic study of multiple components in rats using ultra-high-performance liquid chromatography-triple quadrupole-tandem mass spectrometry.

Chuanwang Xiaoyan (CWXY) capsule is primarily used to treat a variety of acute and chronic inflammations, including acute and chronic pharyngitis and tonsillitis. However, a systematic study of its chemical constituents is still not available. This study evaluated the chemical constituents in vitro and metabolite profiles in vivo of CWXY using ultra-high-performance liquid chromatography (UHPLC) coupled with Q-Exactive Orbitrap mass spectrometry, and the pharmacokinetic behaviors of the nine main components in rats were detected using ultra-high-performance liquid chromatography-triple quadrupole-mass spectrometry (UPLC-QQQ-MS/MS). A total of 92 chemical constituents in CWXY were preliminarily identified in vitro. After oral administration to rats, 56 prototype components and 128 metabolites of CWXY were detected in the biological samples of rat plasma, urine, bile, and feces. Of these prototype components and metabolites, seven new compounds, namely M15, M16, M25, M30, M31, M71, and M128, were detected for the first time. The quantitation method of nine components in rat plasma was developed using a pharmacokinetic study. To the best of our knowledge, this study was the first to investigate the pharmacokinetic behavior of triumbelletin.

Integrating serum metabolomics analysis and network pharmacology to reveal the potential mechanism of Shengmai Jianghuang San in the treatment of nasopharyngeal carcinoma.

Shengmai Jianghuang San (SMJHS) is a traditional Chinese herbal compound reported to inhibit Nasopharyngeal Carcinoma (NPC) progression and enhance radiosensitivity. However, the specific active ingredients and regulatory mechanisms of SMJHS against NPC, particularly under hypoxic conditions, remain unclear. In this study, Sprague-Dawley (SD) rats were gavaged with Shengmai Jianghuang San (SMJHS), and their blood was collected from the abdominal aorta. UHPLC-Q-Exactive orbitrap MS/MS was used to identify the metabolite profiles of SMJHS drug-containing serum. A molecular network of the active compositions in SMJHS targeting NPC was constructed through network pharmacology and molecular docking. The HIF-1α/VEGF pathway was in key positions. The effects of SMJHS on the proliferation, migration, and radiosensitivity of hypoxic NPC cells were assessed by in vitro experiments. NPC cell lines stably overexpressing HIF-1α were established using a lentivirus to investigate the regulation of HIF-1α/VEGF signaling in hypoxic NPC cells by SMJHS. Through a combination of network pharmacological analysis, cellular biofunctional validation, and molecular biochemical experiments, our study found that SMJHS had an anti-proliferative effect on NPC cells cultured under hypoxic conditions, inhibiting their migration and increasing their radiosensitivity. Additionally, SMJHS suppressed the expression of HIF-1α and VEGFA, exhibiting potential as an effective option for improving NPC treatment.

Effect of particle size on dissolution of different chemical components in Codonopsis pilosula.

Codonopsis pilosula (Franch.) Nannf. is a traditional herb for treating immunosuppression. C. pilosula boiling powder (CP-BP) contains particles of a small size made from C. pilosula decoction pieces (CP-DP). It is still unclear how changes in particle size during the decoction process affect the dissolution of various chemical components in C. pilosula. Herein, an ultra-high-performance liquid chromatography-quadrupole-Exactive Orbitrap mass spectrometry technique was established to characterize the components of CP-BP and CP-DP decoctions. The contents of the components were evaluated based on the relative peak area, extract yield, and alcohol solubility rate. A total of 71 compounds were finally identified, and their content in the CP-BP decoction was generally higher than that in the CP-DP decoction. Alkaloids had the highest average content, whereas terpenoids were the most affected by changes in particle size. In addition, immunosuppression was used as model to investigate whether these changes have practical significance. The results of network pharmacology suggested that the phosphoinositide 3-kinase (PI3K)-Akt pathway may be a potential pathway of C. pilosula for treating immunosuppression. The results of molecular docking indicated that compounds with large content variations have good docking affinity with key targets (epidermal growth factor receptor [EGFR], prostaglandin-endoperoxide synthase 2 [PTGS2], and peroxisome proliferator-activated receptor gamma [PPARG]). These results provide an important reference for further development and use of C. pilosula.

Proposing anti-counterfeiting pharmacopoeia quality markers for Shenlingbaizhu granule based on UHPLC-Q-orbitrap-MS identification.

INTRODUCTION: Shenlingbaizhu granule, a Traditional Chinese Medicine prescription comprising Renshen, Gancao, and Shanyao, is widely consumed in China nowadays. OBJECTIVE: The study tries to propose pharmacopoeia quality markers (Q-markers) to prevent counterfeiting involving Renshen, Gancao, and Shanyao. METHODOLOGY: A novel strategy, that is, library-based ultra-high-performance liquid chromatography-quadrupole-orbitrap mass spectrometry, was used to analyse the lyophilised aqueous powder of Shenlingbaizhu granule. Subsequently, quantum chemistry calculation and UV-vis spectra scanning were also performed through theoretical or experimental approaches. RESULT: Thirty-two isomers have been strictly distinguished, especially positional isomeric isochlorogenic acid B versus isochlorogenic acid C, positional isomeric schaftoside versus isoschaftoside, positional isomeric ginsenoside Rg2 versus 20S-ginsenoside Rg3, and stereoisomeric 20S-ginsenoside Rg3 versus 20R-ginsenoside Rg3. Seventeen compounds were unexpectedly observed, particularly scoparone and pectolinarigenin, while a total of 76 bioactive compounds have been putatively identified in the study. The quantum chemistry calculation and UV-vis spectra scanning results revealed that glycyrrhizic acid, ginsenoside Re, ginsenoside Rb1, and diosgenin displayed different dipole moment values and maximum absorption wavelengths from each other. CONCLUSION: The study recommends glycyrrhizic acid, ginsenoside Re, ginsenoside Rb1, and diosgenin as four anti-counterfeiting Q-markers for the pharmacopoeia. The anti-counterfeiting Q-markers can be detected using conventional HPLC. The observation of 17 unexpected compounds updates our knowledge regarding the bioactives of Shenlingbaizhu granule.

Renal toxicity of Aconitum plants? A study based on a new mass spectrometry scanning strategy and computer virtual screening.

BACKGROUND: Radix Aconiti Lateralis (Fuzi), a mono-herbal preparation of Aconitum herbs in the genus Aconitum, is commonly used in traditional Chinese medicine (TCM) to treat critical illnesses. The curative effect of Fuzi is remarkable. However, the toxic effects of Fuzi are still a key clinical focus, and the substances inducing nephrotoxicity are still unclear. Therefore, this study proposes a research model combining "in vitro and in vivo component mining-virtual multi-target screening-active component prediction-literature verification" to screen potential nephrotoxic substances rapidly. METHOD: The UHPLC-Q-Exactive-Orbitrap MS analysis method was used for the correlation analysis of Fuzi's in vitro-in vivo chemical substance groups. On this basis, the key targets of nephrotoxicity were screened by combining online disease databases and a protein-protein interaction (PPI) network. The computer screening technique was used to verify the binding mode and affinity of Fuzi's components with nephrotoxic targets. Finally, the potential material basis of Fuzi-induced nephrotoxicity was screened. RESULTS: Eighty-one Fuzi components were identified. Among them, 35 components were absorbed into the blood. Based on the network biology method, 21 important chemical components and three potential key targets were screened. Computer virtual screening revealed that mesaconine, benzoylaconine, aconitine, deoxyaconitine, hypaconitine, benzoylhypaconine, benzoylmesaconine, and hypaconitine may be potential nephrotoxic substances of Fuzi. CONCLUSIONS: Fuzi may interact with multiple components and targets in the process of inducing nephrotoxicity. In the future, experiments can be designed to explore further. This study provides a reference for screening Fuzi nephrotoxic components and has certain significance for the safe use of Fuzi.

Online identification of chemical constituents in Mongolian medicine Zhachong-13 pills by UHPLC-Q-exactive Orbitrap MS.

Zhachong-13 pills (ZC-13), as a traditional prescription of Mongolian medicine, are often used in the clinical practice of Mongolian hospitals for the treatment of stroke and rheumatic arthritis. In this experiment, UHPLC-Q-Exactive Orbitrap MS was used to explore the chemical composition of ZC-13. The results showed that 315 compounds were identified or inferred, including 56 alkaloids, 77 2-(2-phenylethyl)chromones, 61 flavonoids, 31 tannins, 8 coumarins, 16 lignans, 21 terpenoids, 5 amino acids, 19 organic acids, and 21 other components. In addition, the pharmacological activities related to anti-cerebral ischemia of these components were summarized. This result laid a foundation for further study on the pharmacodynamic material basis of ZC-13 and provided a scientific basis for the formulation of ZC-13 quality specifications.

Utilizing Integrated UHPLC-Q-Exactive Orbitrap-MS, Multivariate Analysis, and Bioactive Evaluation to Distinguish between Wild and Cultivated Niudali (Millettia speciosa Champ.).

Millettia speciosa Champ. (MSCP) enjoys widespread recognition for its culinary and medicinal attributes. Despite the extensive history of MSCP cultivation, the disparities in quality and bioactivity between wild and cultivated varieties have remained unexplored. In this study, 20 wild and cultivated MSCP samples were collected from different regions in China. We embarked on a comprehensive investigation of the chemical constituents found in both wild and cultivated MSCP utilizing UHPLC-Q-Exactive Orbitrap-MS technology and multivariate analysis such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). In total, 62 chemical components were unequivocally identified or tentatively characterized. Via the multivariate statistical analysis, we successfully pinpointed nine compounds with the potential to serve as chemical markers, enabling the differentiation between wild and cultivated MSCP varieties. Moreover, both genotypes exhibited substantial antioxidant and anti-fatigue properties. The bioactivities of wild MSCP were marginally higher when compared to their cultivated counterparts. This study illuminates the impressive antioxidant and anti-fatigue potential present in both wild and cultivated MSCP genotypes, further augmenting the allure of this species and opening new avenues for the economic valorization of MSCP. Hence, this study provides a valuable method for the identification and quality control of MSCP and a method in chemistry and pharmacology to assess an alternative possibility for cultivated MSCP.

Detection of Adulterated Naodesheng Tablet (Naodesheng Pian) via In-Depth Chemical Analysis and Subsequent Reconstruction of Its Pharmacopoeia Q-Markers.

Naodesheng Tablet (Naodesheng Pian), a traditional Chinese medicine formula for stroke treatment, is made up of five herbal medicines, i.e., Sanqi, Gegen, Honghua, Shanzha, and Chuanxiong. However, the current Pharmacopoeia quality-marker (Q-marker) system cannot detect possible adulteration. Our study tried to use a new strategy, i.e., standards-library-dependent ultra-high-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS/MS) putative identification, to reconstruct the Q-marker system. Through the strategy, 30 isomers were successfully differentiated (such as 2'-hydroxygenistein, luteolin, and kaempferol; ginsenoside Rg2 and ginsenoside Rg3; ginsenoside Rf and ginsenoside Rg1). In particular, 11 compounds were unexpectedly found in Naodesheng, including 2'-hydroxygenistein, 7,4'-dihydroxyflavone, pectolinarigenin, 7-methoxy-4'-hydroxyisoflavone, scoparone, matrine, 3,3',4',5,6,7,8-heptamethoxyflavone, 5-hydroxyflavone, diosgenin, chloesteryl acetate, and (+)-4-cholesten-3-one. In total, 68 compounds were putatively identified and fully elucidated for their MS spectra. Subsequently, relevant compounds were further investigated using UV-vis scanning experiments, semi-quantitative analysis, and quantum chemical calculation. Finally, five adulterated Naodesheng Tablets were used for validation experiments. The experiment successfully detected five adulterated ones via a lower-version LC-MS analysis. On this basis, three new candidates (hydroxy safflor yellow A (HSYA), citric acid, and levistilide A), along with puerarin and notoginsenoside R1, are re-nominated as the Q-markers for LC-MS analysis. The LC-MS analysis of puerarin, notoginsenoside R1, HSYA, citric acid, and levistilide A can clearly detect adulteration regarding all five herbal medicines mentioned above. Therefore, the reconstructed Q-markers are described as a "perfect" quality control system to detect adulteration in Naodesheng and will offer a valuable recommendation for the Pharmacopoeia Commission.

A Systematic Methodology for the Identification of the Chemical Composition of the Mongolian Drug Erdun-Uril Compound Utilizing UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

The traditional Mongolian medicine Erdun-Uril is a conventional combination of 29 herbs commonly used for the treatment of cerebrovascular ailments. It has the effects of reducing inflammation, counteracting oxidative stress, and averting strokes caused by persistent cerebral hypoperfusion. Prior research on Erdun-Uril has predominantly concentrated on its pharmacodynamics and mechanism of action; however, there has been a lack of systematic and comprehensive investigation into its chemical constituents. Therefore, it is crucial to establish an efficient and rapid method for evaluating the chemical constituents of Erdun-Uril. In this study, Erdun-Uril was investigated using UHPLC-Q-Exactive Orbitrap MS combined with parallel reaction monitoring for the first time. Eventually, a total of 237 compounds, including 76 flavonoids, 68 phenolic compounds, 19 alkaloids, 7 amino acids, etc., were identified based on the chromatographic retention time, bibliography data, MS/MS2 information, neutral loss fragments (NLFs), and diagnostic fragment ions (DFIs). And of these, 225 were reported for the first time in this study. This new discovery of these complex components would provide a reliable theoretical basis for the development of pharmacodynamics and quality standards of the Mongolian medicine Erdun-Uril.

Enhancing anthocyanin extraction efficiency in vegetables and fruits: a high-speed shear homogenization technology.

BACKGROUND: To extract anthocyanins with high efficiency, a hypothesis for high-speed shear homogenization extraction (HSHE) method was established through a combination of solvent and ultrasonic-assisted extractions. The efficacy of this hypothesis was demonstrated by performing qualitative and quantitative analyses of 16 anthocyanins extracted from five northern vegetables, and five berry fruits using ultra-high-performance Q-Exactive Orbitrap tandem mass spectrometry. Single-factor experiments were conducted by varying ethanol concentration, temperature, pH and extraction cycles to determine the optimal conditions for this method. RESULTS: Optimal extraction conditions (ethanol 70-80%, 40-50 °C, pH 3-4, performed twice) were determined using an HSHE (5 min, 10 000 rpm, 25 °C) assisted shaker (60 min) and ultrasonication (40 kHz, 160 W cm-2, 30 min, 25 °C) procedure. Compared to the traditional non-HSHE method, the total anthocyanin content obtained through HSHE extraction showed a significant increase, ranging from 1.0 to 3.9 times higher, with purple cabbage exhibiting the most pronounced enhancement in content. More types of anthocyanins were detected in blueberry (9), black bean (7) and raspberry (5), of which malvidin was the major anthocyanin (0.426 g kg-1) in blueberry, having an amount five times than previously obtained. CONCLUSION: The established HSHE method has been proven to be a superior technique for anthocyanin extraction, with higher extraction efficiency and concentrations. This technique also provides a new avenue for extracting bioactive compounds from diverse food sources, with potential applications in improving the functional properties of food products. © 2024 Society of Chemical Industry.

Combination of UHPLC-Q Exactive-Orbitrap MS, Bioinformatics and Molecular Docking to Reveal the Mechanism of Huan-Lian-Jie-Du Decoction in the Treatment of Diabetic Encephalopathy.

Diabetic encephalopathy (DE) is a serious complication of diabetes, which affects patients' quality of life. We aimed to explore HLJDD in the treatment of DE by LC/MS and bioinformatics. UPLC-Q Exactive-Orbitrap MS was employed to clarify the compounds. The modules and hub targets of DE were gained from WGCNA. Subsequently, an Herb-Compound-Target network was constructed and enrichment analysis was used. In addition, a protein-protein interaction (PPI) network was constructed and molecular docking was used to verify the above analysis. As result, 138 compounds and 10 prototypes in brain were identified. In network pharmacology, 8 modules and 5692 hub targets were obtained from WGCNA. An Herb-Compound-Target network was constructed by 4 herbs, 10 compounds and 56 targets. The enrichment analysis showed that the treatment of DE with HLJDD involve oxidative stress and neuroprotection. Beside, SRC, JUN, STAT3, MAPK1 and PIK3R1 were identified and as hub targets of HLJDD in treating DE. Moreover, Molecular docking showed that five hub targets had strong affinity with the corresponding alkaloids. Therefore, we explored the underlying mechanisms of HLJDD in the treatment of DE and to provide the theoretical and scientific basis for subsequent experimental studies and clinical applications.

The Metabolites and Mechanism Analysis of Genistin against Hyperlipidemia via the UHPLC-Q-Exactive Orbitrap Mass Spectrometer and Metabolomics.

Genistin, an isoflavone, has been reported to have multiple activities. However, its improvement of hyperlipidemia is still unclear, and the same is true with regard to its mechanism. In this study, a high-fat diet (HFD) was used to induce a hyperlipidemic rat model. The metabolites of genistin in normal and hyperlipidemic rats were first identified to cause metabolic differences with Ultra-High-Performance Liquid Chromatography Quadrupole Exactive Orbitrap Mass Spectrometry (UHPLC-Q-Exactive Orbitrap MS). The relevant factors were determined via ELISA, and the pathological changes of liver tissue were examined via H&E staining and Oil red O staining, which evaluated the functions of genistin. The related mechanism was elucidated through metabolomics and Spearman correlation analysis. The results showed that 13 metabolites of genistin were identified in plasma from normal and hyperlipidemic rats. Of those metabolites, seven were found in normal rat, and three existed in two models, with those metabolites being involved in the reactions of decarbonylation, arabinosylation, hydroxylation, and methylation. Three metabolites, including the product of dehydroxymethylation, decarbonylation, and carbonyl hydrogenation, were identified in hyperlipidemic rats for the first time. Accordingly, the pharmacodynamic results first revealed that genistin could significantly reduce the level of lipid factors (p < 0.05), inhibited lipid accumulation in the liver, and reversed the liver function abnormalities caused by lipid peroxidation. For metabolomics results, HFD could significantly alter the levels of 15 endogenous metabolites, and genistin could reverse them. Creatine might be a beneficial biomarker for the activity of genistin against hyperlipidemia, as revealed via multivariate correlation analysis. These results, which have not been reported in the previous literature, may provide the foundation for genistin as a new lipid-lowering agent.

[Rapid identification of components in Wuzhuyu Decoction using UHPLC Q-Exactive Orbitrap MS~n and molecular network technology].

Wuzhuyu Decoction, the classical formula recorded in the Treatise on Febrile Diseases(Shang Han Lun), has been included in the Catalogue of Ancient Classic Prescriptions(the First Batch). Consisting of Euodiae Fructus, Ginseng Radix et Rhizoma, Zingiberis Rhizoma Recens, and Jujubae Fructus, it is effective in warming the middle, tonifying deficiency, dispelling cold, and descending adverse Qi, and is widely applied clinically with remarkable efficacies. For a classical formula, the chemical composition is the material basis and an important premise for quantity value transfer. This study aimed to establish a rapid identification method of chemical components in Wuzhuyu Decoction by high-resolution mass spectrometry(HR-MS) and molecular network. AQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) was used for sample separation, and acetonitrile-0.1% formic acid in water was used as mobile phases for gradient elution. Q-Exactive Orbitrap MS data were collected in positive and negative ion modes, and GNPS molecular network was plotted according to the similarity of MS/MS fragmentation modes. Cytoscape 3.6.1 was used to screen molecular clusters with similar structures. Finally, the chemical components of Wuzhuyu Decoction were rapidly identified according to the controls, as well as the information of retention time, accurate relative molecular weight of HR-MS, and MS/MS multistage fragments. A total of 105 chemical components were identified in Wuzhuyu Decoction. This study can provide data for the follow-up quality control, standard substance research, and pharmacodynamic material research on Wuzhuyu Decoction, as well as references for the rapid qualitative analysis of the chemical components of Chinese medicine.

[Comparison of alkaloids in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata based on UHPLC-Q-Exactive Orbitrap MS/MS].

UHPLC-Q-Exactive Orbitrap MS/MS was used to systematically analyze and compare the alkaloids in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata. After the samples were pretreated in the solid-phase extraction cartridges, 0.1% ammonium hydroxide(A)-acetonitrile(B) was used for gradient elution. The LC-MS method for characterization of alkaloids in the three herbal medicines was established in ESI positive ion mode to collect high resolution MS data of reference substances and samples. On the basis of the information of reference substance cracking behavior, retention time, accurate molecular mass, and related literature, a total of 155 alkaloids were identified in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Prae-parata. Specifically, 130, 127, and 92 alkaloids were identified in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata, respectively. Monoester alkaloids and amino-alcohol alkaloids were dominant in the three herbal medicines, and the alkaloids in Aconiti Kusnezoffii Radix and Aconiti Radix were similar. This paper can provide a reference for elucidating the pharmacological effects and clinical application differences of the three herbal medicines produced from plants of Aconitum.

Stepwise Diagnostic Product Ions Filtering Strategy for Rapid Discovery of Diterpenoids in Scutellaria barbata Based on UHPLC-Q-Exactive-Orbitrap-MS.

Diterpenoids are considered the major bioactive components in Scutellaria barbata to treat cancer and inflammation, but few comprehensive profiling studies of diterpenoids have been reported. Herein, a stepwise diagnostic product ions (DPIs) filtering strategy for efficient and targeted profiling of diterpenoids in Scutellaria barbata was developed using UHPLC-Q-Exactive-Orbitrap-MS. After UHPLC-HRMS/MS analysis of six diterpenoid reference standards, fragmentation behaviors of these references were studied to provide DPIs. Then, stepwise DPIs filtering aimed to reduce the potential interferences of matrix ions and achieve more chromatographic peaks was conducted to rapidly screen the diterpenoids. The results demonstrated that stepwise DPIs were capable of simplifying the workload in data post-processing and the effective acquisition of low abundance compounds. Subsequently, DPIs and MS/MS fragment patterns were adopted to identify the targeted diterpenoids. As a result, 381 diterpenoids were unambiguously or tentatively identified, while 141 of them with completely new molecular weights were potential new diterpenoids for Scutellaria barbata. These results demonstrate that the developed stepwise DPIs filtering method could be employed as an efficient, reliable, and valuable strategy to screen and identify the diterpenoid profile in Scutellaria barbata. This might accelerate and simplify target constituent profiling from traditional Chinese medicine (TCM) extracts.

Systematic Screening of Chemical Constituents in the Traditional Chinese Medicine Arnebiae Radix by UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

Arnebiae Radix (dried root of Arnebia euchroma (Royle) Johnst.) has been used in traditional Chinese medicine (TCM) to treat macular eruptions, measles, sore throat, carbuncles, burns, skin ulcers, and inflammation. Previous studies have shown that shikonins and shikonofurans are two of their main bioactive ingredients. However, systematic investigations of their constituents have rarely been conducted. It is necessary to establish a rapid and effective method to identify the chemical constituents of Arnebiae Radix. This will help to further improve the effective resource utilization rate of this plant. In this study, a rapid and effective UHPLC-Q-Exactive Orbitrap mass spectrometry method was established to simultaneously analyze chemical ingredients in Arnebiae Radix within a short period of time. Based on the results of a full scan MS, the MS2 database (mzVault and mzCloud), the diagnostic fragment ions, the retention time, and the bibliography, a total of 188 compounds were identified, with 114 of those being reported from Arnebiae Radix for the first time. The results of this study lay the foundation for obtaining a thorough understanding of the active ingredients in Arnebiae Radix and its quality control. This method may be widely used for the chemical characterization of different samples.

Antioxidant Effects of Roasted Licorice in a Zebrafish Model and Its Mechanisms.

Licorice (Gan-Cao, licorice) is a natural antioxidant and roasted licorice is the most common processing specification used in traditional Chinese medicine prescriptions. Traditional Chinese medicine theory deems that the honey-roasting process can promote the efficacy of licorice, including tonifying the spleen and augmenting "Qi" (energy). The antioxidant activity and mechanisms underlying roasted licorice have not yet been reported. In this study, we found that roasted licorice could relieve the oxidative stress injury induced by metronidazole (MTZ) and could restrain the production of excessive reactive oxygen species (ROS) induced by 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) in a zebrafish model. It was further found that roasted licorice could exert its oxidative activity by upregulating the expression of key genes such as heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), glutamate-cysteine ligase modifier subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC) in the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway both in vivo and in vitro. Furthermore, consistent results were obtained showing that rat serum containing roasted licorice was estimated to reduce cell apoptosis induced by H2O2. Then, the UHPLC-Q-Exactive Orbitrap MS analysis results elucidated the chemical composition of rat plasma containing roasted licorice extracts, including ten prototype chemical components and five metabolic components. Among them, six compounds were found to have binding activity with Kelch-like ECH-associated protein 1 (KEAP1), which plays a crucial role in the transcriptional activity of NRF2, using a molecular docking simulation. The results also showed that liquiritigenin had the strongest binding ability with KEAP1. Immunofluorescence further confirmed that liquiritigenin could induce the nuclear translocation of NRF2. In summary, this study provides a better understanding of the antioxidant effect and mechanisms of roasted licorice, and lays a theoretical foundation for the development of a potential antioxidant for use in clinical practice.

A Systematic Method for the Identification of Aporphine Alkaloid Constituents in Sabia schumanniana Diels Using UHPLC-Q-Exactive Orbitrap/Mass Spectrometry

Sabia schumanniana Diels (SSD) is a plant whose stems are used in traditional folk medicine for the treatment of lumbago and arthralgia. Previous studies have revealed chemical constituents of SSD, including triterpenoids and aporphine alkaloids. Aporphine alkaloids contain a variety of active components, which might facilitate the effective treatment of lumbago and arthralgia. However, only 5-oxoaporphine (fuseine) has been discovered in SSD to date. In this study, we sought to systematically identify the aporphine alkaloids in SSD. We established a fast and reliable method for the detection and identification of these aporphine alkaloids based on ultra-high-performance liquid chromatography (UHPLC)-Q-Exactive-Orbitrap/mass spectrometry combined with parallel reaction monitoring (PRM). We separated all of the analyzed samples using a Thermo Scientific Hypersil GOLD™ aQ C18 column (100 mm × 2.1 mm, 1.9 µm). Finally, we identified a total of 70 compounds by using data such as retention times and diagnostic ions. No fewer than 69 of these SSD aporphine alkaloids have been reported here for the first time. These findings may assist in future studies concerning this plant and will ultimately contribute to the research and development of new drugs.

Surface Functionalized Magnetic Nanoparticles as a Selective Sorbent for Affinity Fishing of PPAR-γ Ligands from Choerospondias axillaris.

Coronary heart disease (CHD), which has developed into one of the major diseases, was reported to be treated by the target of peroxisome proliferators-activate receptor γ (PPAR-γ). As a natural medicine long used in the treatment of CHD, there are few studies on how to screen the target active compounds with high specific activity from Choerospondias axillaris. To advance the pace of research on target-specific active compounds in natural medicines, we have combined magnetic ligand fishing and functionalized nano-microspheres to investigate the active ingredients of PPAR-γ targets in Choerospondias axillaris. The PPAR-γ functionalized magnetic nano-microspheres have been successfully synthesized and characterized by vibrating sample magnetometer (VSM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The specificity, reusability, and reproducibility of the nano-microspheres were investigated with the help of the specific binding of rosiglitazone to PPAR-γ. In addition, the incubation temperature and the pH of the buffer solution in the magnetic ligand fishing were optimized to improve the specific adsorption efficiency of the analytes. Finally, with the aid of ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS/MS), the 16 active ligands including 9 organic acids, 5 flavonoids, and 2 phenols were found in the ethanolic extracts of Choerospondias axillaris. Therefore, the study can provide a successful precedent for realizing the designated extraction and rapid isolation of target-specific active ingredient groups in the complex mixtures.

Rapid Identification of Constituents in Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. Using UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. (CT) belongs to the Rubiaceae family. Its dried leaves are widely used in traditional Chinese medicine to treat enteritis, dysentery, toothache, furuncles, swelling, traumatic injury, fracture, bleeding, and scalding. In order to further clarify the unknown chemical composition of CT, a rapid strategy based on UHPLC-Q-exactive orbitrap was established for this analysis using a Thermo Scientific Hypersil GOLDTM aQ (100 mm × 2.1 mm, 1.9 µm) chromatographic column. The mobile phase was 0.1% formic acid water-acetonitrile, with a flow rate of 0.3 mL/min and injection volume of 2 µL; for mass spectrometry, an ESI ion source in positive and negative ion monitoring modes was adopted. A total of 135 chemicals comprising 67 chlorogenic acid derivatives, 48 flavonoids, and 20 anthocyanin derivatives were identified by comparing the mass spectrum information with standard substances, public databases, and the literature, which were all discovered for the first time in this plant. This result broadly expands the chemical composition of CT, which will contribute to understanding of its effectiveness and enable quality control.