Screening and identification of reactive metabolic compounds of Cortex Periplocae based on glutathione capture-mass spectrometry.

As a traditional Chinese medicine (TCM), Cortex Periplocae (CP) has a wide range of pharmacological effects, as well as toxic side effects. The main toxic components of it are cardiac glycosides, which tend to cause cardiotoxicity. Currently, it has also been reported in studies to cause hepatotoxicity, but it is not clear whether the hepatotoxicity is related to the toxicity caused by the reactive metabolites. This study aims to investigate the target components of CP that generate reactive metabolic toxicity. The fluorescent probe method was used to detect glutathione (GSH)-trapped reactive metabolites in a co-incubation system of CP extract with rat liver microsomes. Identification of GSH conjugates was performed by LC-MS/MS and that of the possible precursor components that produce reactive metabolites was conducted by UPLC-Q-TOF/MS. Cell viability assays were performed on HepG2 and L02 cells to determine the cytotoxicity of the target components. The findings of our study demonstrate that the extract derived from CP has the ability to generate metabolites that exhaust the intracellular GSH levels, resulting in the formation of GSH conjugates and subsequent cytotoxic effects. Through the utilization of the UPLC-Q-TOF/MS technique, we were able to accurately determine the molecular weight of the precursor compound in CP to be 355.1023. The primary evidence to determining the GSH conjugetes relies on the appearance of characteristic product ions resulting from central neutral loss (CNL) scanning of 129 Da and product scanning of m/z 660 in the positive MS/MS spectrum. Through analysis, it was ultimately ascertained that the presence of chlorogenic acid (CGA) and its isomers, namely neochlorogenic acid (NCGA) and cryptochlorogenic acid (CCGA), could lead to the production of GSH conjugates, resulting in cytotoxicity at elevated levels. Taking these findings into consideration, the underlying cause for the potential hepatotoxicity of CP was initially determined.

Convolutional Neural Network-Based Pattern Recognition of Partial Discharge in High-Speed Electric-Multiple-Unit Cable Termination.

Partial discharge detection is considered a crucial technique for evaluating insulation performance and identifying defect types in cable terminals of high-speed electric multiple units (EMUs). In this study, terminal samples exhibiting four typical defects were prepared from high-speed EMUs. A cable discharge testing system, utilizing high-frequency current sensing, was developed to collect discharge signals, and datasets corresponding to these defects were established. This study proposes the use of the convolutional neural network (CNN) for the classification of discharge signals associated with specific defects, comparing this method with two existing neural network (NN)-based classification models that employ the back-propagation NN and the radial basis function NN, respectively. The comparative results demonstrate that the CNN-based model excels in accurately identifying signals from various defect types in the cable terminals of high-speed EMUs, surpassing the two existing NN-based classification models.

The Molecular Basis of the Anti-Inflammatory Property of Astragaloside IV for the Treatment of Diabetes and Its Complications.

Astragali Radix is a significant traditional Chinese medication, and has a long history of clinical application in the treatment of diabetes mellitus (DM) and its complications. AS-IV is an active saponin isolated from it. Modern pharmacological study shows that AS-IV has anti-inflammatory, anti-oxidant and immunomodulatory activities. The popular inflammatory etiology of diabetes suggests that DM is a natural immune and low-grade inflammatory disease. Pharmacological intervention of the inflammatory response may provide promising and alternative approaches for the prevention and treatment of DM and its complications. Therefore, this article focuses on the potential of AS-IV in the treatment of DM from the perspective of an anti-inflammatory molecular basis. AS-IV plays a role by regulating a variety of anti-inflammatory pathways in multiple organs, tissues and target cells throughout the body. The blockade of the NF-κB inflammatory signaling pathway may be the central link of AS-IV's anti-inflammatory effect, resulting in a reduction in the tissue structure and function damage stimulated by inflammatory factors. In addition, AS-IV can delay the onset of DM and its complications by inhibiting inflammation-related oxidative stress, fibrosis and apoptosis signals. In conclusion, AS-IV has therapeutic prospects from the perspective of reducing the inflammation of DM and its complications. An in-depth study on the anti-inflammatory mechanism of AS-IV is of great significance for the effective use of Chinese herbal medicine and the promotion of its status and influence on the world.

Comprehensive investigation on the metabolism of emodin both in vivo and in vitro.

Emodin is a natural anthraquinone, which displays numerous pharmacological activities, including anti-tumor, anti-inflammation and immunosuppression. However, there was no comprehensive study on its absorption, metabolism, distribution, and excretion. In order to further evaluation on the possibility of drug development of emodin, both in vivo and in vitro experiments were fulfilled in this study. The results showed that the absolute bioavailability of emodin is approximately 3.2%. Furthermore, about 56% of emodin was unabsorbed and mainly excreted into feces as prototype. The absorb constituent could be rapidly metabolized as hydroxylated and glucuronidated metabolites. Both prototype and metabolites of emodin absorbed into the body circulation were predominantly distributed in kidney. Hydroxyed metabolites were predominantly excreted via urine and feces and glucuronidated metabolites were predominantly excreted via urine and bile. CYP1A2, CYP2E1, UGT1A1, UGT1A9, and UGT2B7 played a key role in the metabolism of emodin in liver microsomes of rats. To the best of our knowledge, this is the first comprehensive study on the absorption, metabolism, distribution, and excretion of emodin, and our results could help to understand both pharmaceutical and toxicological effects of emodin greatly.

Studies on Chemical Characterization of Ginkgo Amillaria Oral Solution and Its Drug-Drug Interaction With Piceatannol 3'-O-ß-D-Glucopyranoside for Injection.

Ginkgo Amillaria oral solution (GAO) is commonly used for the treatment of cardiovascular and cerebrovascular diseases in China. Piceatannol-3'-O-ß-D-glucopyranoside for injection (PGI) is mainly used for the prevention and treatment of ischemic cerebrovascular diseases. With the spread of cerebrovascular disease, the possibility of combining the two drugs has increased; however, there is no research on the drug-drug interaction (DDI) between these two medicines. In this paper, an ultrahigh-performance liquid chromatography/quadrupole-orbitrap mass spectrometry (UHPLC/Q-Orbitrap MS) method was established to characterize the chemical constituents of GAO first; 62 compounds were identified or tentatively identified based on their retention time (RT), MS, and MS/MS data. Nine main compounds were determined by ultrahigh-performance liquid chromatography/triple quadrupole mass spectrometry (UPLC-QQQ-MS). Furthermore, incubation with liver microsomes in vitro was fulfilled; the results showed that GAO had a significant inhibitory effect on UGT1A9 and UGT2B7 (p < 0.05), and PGI was mainly metabolized by UGT1A9. The identification results of in vivo metabolites of PGI showed that PGI mainly undergoes a phase II binding reaction mediated by UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) in vivo. Therefore, pharmacokinetic studies were performed to investigate the DDI between GAO and PGI. The results showed that the AUC (p < 0.05) and T1/2 (p < 0.05) of PGI in vivo were significantly increased when administered together with GAO, whereas the CL was significantly decreased (p < 0.05). The exploration of in vitro and in vivo experiments showed that there was a DDI between GAO and PGI.

Characterization of volatile organic compounds in Polygonum multiflorum and two of its processed products based on multivariate statistical analysis for processing technology monitoring.

Herein we describe a comprehensive analysis of the volatile organic compounds (VOCs) of raw Polygonum multiflorum Thunb. (PM) and two of its processed products, as well as an effective and simple method based on volatile markers to determine to which extent the PM had been processed. Sixty-five VOCs were identified by headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), along with headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). Principal component analysis (PCA) of the HS-SPME-GC-MS spectra and fingerprint analysis of the HS-GC-IMS spectra allowed the identification of raw PM from its processed products based the VOCs identified. Furthermore, the content and distribution of VOCs in the samples were easily analyzed visually based on clustering-kernel density estimation (Cluster-KDE). Finally, exploratory factor analysis (EFA) allowed the screening of significant markers to identify the processing method and consequently distinguish the three studied groups of PM.

Biodistribution and pharmacokinetic profile of berberine and its metabolites in hepatocytes.

BACKGROUND: Berberine has been shown in clinical studies to have many health benefits, including anti-inflammatory and antioxidant properties, along with gut-flora balancing properties. However, its clinical efficacy is hindered by its low oral bioavailability and rapid metabolism. PURPOSE: This study aims to identify the berberine metabolites' forms and characterize their biodistribution patterns in and out of HepG2 cells. METHODS: The qualitative analysis of metabolites of berberine in HepG2 cells was performed using the LC/MSn-IT-TOF method. Subsequent cellular pharmacokinetics characterization of intracellular and extracellular berberine and its metabolites was performed by LC-MS/MS analysis. RESULTS: Berberine's metabolites of phase I metabolism were demethyleneberberine, jatrorrhizine, columbamine, berberrubine, etc., while its phase II metabolites were sulfate and glucuronide conjugates of phase I metabolites. Among the phase I metabolites of berberine, jatrorrhizine+columbamine accounted for over two-thirds of the total, followed by demethyleneberberine, which accounted for about a quarter. The intracellular demethyleneberberine is 25.14 times more enriched than extracellular demethyleneberberine. On the other hand, jatrorrhizine+columbamine and berberrubine were primarily distributed extracellularly, and their extracellular concentrations were 7.13 times and 15.61 times of their intracellular concentrations, respectively. Berberine metabolites produced in phase II metabolism are predominantly sulfate conjugates. CONCLUSION: Our results show that demethyleneberberine is highly concentrated intracellularly in HepG2, possibly because it is an essential metabolite of berberine that likely contributes to berberine's efficacy. In light of our findings, berberine's poor plasma concentration-effectiveness characteristics have been partially explained.

The interaction of alkaloids in Coptis chinensis Franch -Tetradium ruticarpum (A. Juss.) T.G. Hartley with hOCT1 and hOCT2.

ETHNOPHARMACOLOGICAL RELEVANCE: Zuojin Pill, a traditional poly-herbal drug, comprises Coptis chinensis Franch - Tetradium ruticarpum (A. Juss.) T.G. Hartley (6:1). The significant quantity of alkaloids found in the participating herbs is a key aspect of the Zuojin Pill. According to traditional Chinese medicine (TCM), these numerous alkaloidal compounds within Zuojin Pill have various essential therapeutic effects. AIM OF THE STUDY: The alkaloids in Tetradium are mainly indole alkaloids, while the alkaloids in Coptis are mostly isoquinoline alkaloids with low bioavailability. Alkaloids and their metabolites are nitrogen-containing compounds or weakly alkaline substances that can be partially ionized under physiological pH conditions. Fortunately, organic cation transporters (OCTs) play a crucial role in the cellular uptake of weakly alkaline compounds. Therefore, we speculated that the alkaloidal compounds might interact with liver cation transporters hOCT1 and kidney cation transporters hOCT2 to alter cell drug disposal. In order to clarify our hypothesis, a series of alkaloids-OCTs interaction experiments were conducted. MATERIALS AND METHODS: HEK293 cells stably expressing hOCT1 and hOCT2 were modeled and evaluated. Afterward, high-content screening (HCS) was conducted to analyze whether the main alkaloids and their metabolites of Coptis - Tetradium were inhibitors of hOCT1 and hOCT2 transporters. Meanwhile, LC-MS/MS was used to investigate whether the alkaloidal compounds were substrates of hOCT1 and hOCT2 transporters. Finally, drug interactions at the cellular level were assessed by LC-MS/MS after co-administration of berberine and rutacorine. RESULTS: Berberine, jateorhizine, coptisine, epiberberine, columbamine, demethyleneberberine, and berberrubine could significantly inhibit hOCT1 and hOCT2 activity. Isoquinoline alkaloids, including berberine, jateorhizine, coptisine, epiberberine, columbamine, and palmatine, were substrates of hOCT1 and hOCT2, but not the indole alkaloids evodiamine and rutaecarpine. Furthermore, evodiamine at a concentration of 20 µmol/L had a trivial effect on berberine accumulation in HEK293-hOCT2 cells. CONCLUSIONS: These results support the idea that alkaloidal compounds within Coptis and Tetradium have hOCT1 and hOCT2 inhibitory activity or be their substrates, and the increased oral bioavailability of berberine in vivo was closely related to the potential interactions of small molecules in Coptis- Tetradium. Overall, our study provides a framework for investigating the potential interactions of small molecules in Coptis- Tetradium.

An integrated strategy for the systematic chemical characterization of Salvianolate lyophilized injection using four scan modes based on the ultra-high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry.

Salvianolate lyophilized injection (SLI), a freeze-dried powder injection derived from aqueous extract of S. miltiorrhiza, is therapeutically used to treat the syndrome of blood stasis and collateral blockage during the recovery period after stroke. To date, it has remained a significant challenge to comprehensively characterize the compounds of SLI, particularly the minor components with potential bioactivities, in one sample injection analysis. Using an integrative four scan modes approach coupled with ultra-high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (UHPLC-QTRAP-MS/MS), we propose a novel, sensitive, and simple strategy for systematic and rapid profiling of the chemical components of SLI. First, an in-house database of constituents from the water-soluble extract of Danshen was created. Second, the fragmentation behaviors of the representative components in SLI were obtained using the untargeted scan mode enhanced MS (EMS)-information dependent acquisition (IDA)-enhanced product ion (EPI). The specific fragments acquired were then utilized to conduct precursor ion (Prec) and neutral loss (NL)-IDA-EPI scans. Following that, a sensitive predictive multiple reaction monitoring (pMRM)-IDA-EPI scan method with 454 transitions was developed based on the prominent fragment ions and plausible predictions. A total of 171 compounds were tentatively identified from SLI. Among them, 27 minor components have not been previously reported. This strategy allows most isomeric compounds at trace levels to be readily distinguished and annotated. Finally, 15 batches of 13 representative components in SLI selected by the qualitative results were accurately quantified. Salvianolic acid A (Sal A), Sal B, Sal D, lithospermic acid (LA), and rosmarinic acid (RA) were proved to be the predominant constituents. Sal B had the highest amount (195.08-350.46 µg·mg-1), followed by LA, Sal A, Sal D, and RA. Moreover, these 15 batches of samples showed good uniformity, and no abnormal batches existed. These results suggest that this novel strategy can accelerate the identification of undiscovered chemical components and serve as an alternative method for in-depth profiling of compounds in other traditional Chinese medicines (TCMs).