Glycyrrhetinic acid blocks SARS-CoV-2 infection by activating the cGAS-STING signalling pathway.

BACKGROUND AND PURPOSE: Traditional Chinese medicine (TCM) played an important role in controlling the COVID-19 pandemic, but the scientific basis and its active ingredients are still weakly studied. This study aims to decipher the underlying anti-SARS-CoV-2 mechanisms of glycyrrhetinic acid (GA). EXPERIMENTAL APPROACH: GA's anti-SARS-CoV-2 effect was verified both in vitro and in vivo. Homogeneous time-resolved fluorescence assays, biolayer interferometry technology, and molecular docking were employed to examine interactions of GA with human stimulator of interferon genes (hSTING). Immunofluorescence staining, western blot, and RT-qPCR were used to investigate nuclear translocation of interferon regulatory factor 3 (IRF3) and levels of STING target genes. Pharmacokinetics of GA was studied in mice. KEY RESULTS: GA could directly bind to Ser162 and Tyr240 residues of hSTING, thus up-regulating downstream targets and activation of the STING signalling pathway. Such activation is crucial for limiting the replication of SARS-CoV-2 Omicron in Calu-3 cells and protecting against lung injury induced by SARS-CoV-2 Omicron infection in K18-ACE2 transgenic mice. Immunofluorescence staining and western blot indicated that GA increased levels of phosphorylated STING, phosphorylated TANK-binding kinase-1, and cyclic GMP-AMP synthase (cGAS). Importantly, GA increased nuclear translocation of IRF3. Pharmacokinetic analysis of GA in mice indicated it can be absorbed into circulation and detected in the lung at a stable level. CONCLUSION AND IMPLICATIONS: Activation of the cGAS-STING pathway through the GA-STING-IRF3 axis is essential for the antiviral activity of GA in mice, providing new insights into the potential translation of GA for treating SARS-CoV-2 in patients.

Improved Pharmacokinetics of Icariin (ICA) within Formulation of PEG-PLLA/PDLA-PNIPAM Polymeric Micelles.

Icariin (ICA) is a major flavonoid that contains the active compound Epimedii Folium. However, ICA's pharmacokinetic characteristics remain unsatisfactory due to its low bioavailability, and hence limited drugability. In order to improve its pharmacokinetics and achieve prolonged blood circulation time, a novel polymeric micelle, made of the self-assembled micelle between poly (ethylene glycol)-poly (L-lactic acid) (PEG-PLLA) and poly (D-lactic acid)-poly(N-isopropylacrylamide) (PDLA-PNIPAM), was designed to encapsulate ICA. Our experimental results showed that this polymeric micelle formulation of ICA exhibited uniform nano-size distribution and high stability within 48 h. The new formulation also allowed sustained ICA release in an in vitro drug release study. Furthermore, in vivo experiments revealed that ICA bioavailability in the PEG-PLLA/PDLA-PNIPAM polymeric micelle formulation was significantly higher compared to ICA alone, or ICA in the traditional Pluronic F127 micelle formulation. Finally, we show that metabolite analysis confirmed that ICA within the PEG-PLLA/PDLA-PNIPAM polymeric micelle formulation provided better drug protection, reduced drug metabolites production, and decreased undesired first-pass effects. Overall, these data show that ICA within PEG-PLLA/PDLA-PNIPAM polymeric micelle formulation exhibit advantages, in terms of improved physicochemical properties, sustained release of ICA in vitro, and improved bioavailability of ICA in vivo, which represent a feasible approach for improving the drugability of pharmaceutical small molecules with low bioavailability or poor stability.

Relationship between Tissue Distributions of Modified Wuzi Yanzong Prescription () in Rats and Meridian Tropism Theory.

OBJECTIVE: To investigate the relationship between tissue distributions of modified Wuzi Yanzong prescription (, MWP) in rats and meridian tropism theory. METHODS: A high-performance liquid chromatography with Fourier transform-mass spectrometry (HPLC-FT) method was used to identify the metabolites of MWP in different tissues of rats after continued oral administration of MWP for 7 days. The relationship between MWP and meridian tropism theory was studied according to the tissue distributions of the metabolites of MWP in rats and the relevant literature. RESULTS: Nineteen metabolites, mainly flavanoid compounds, were detected in the different rat tissues and classified to each herb in MWP. Further, it was able to establish that the tissue distributions of the metabolites of MWP were consistent with the descriptions of meridian tropism of MWP available in literature, this result might be useful in clarifying the mechanism of MWP on meridian tropism. In the long run, these data might provide scientific evidence of the meridian tropism theory to further promote the reasonable, effective utilization, and modernization of Chinese medicine. CONCLUSION: The tissue distributions of MWP in vivo were consistent with the descriptions of meridian tropism of MWP.

Serum sphingolipids reflect the severity of chronic HBV infection and predict the mortality of HBV-acute-on-chronic liver failure.

Patients with HBV-acute-on-chronic liver failure (HBV-ACLF) have high mortality and frequently require liver transplantation; few reliable prognostic markers are available. As a class of functional lipids, sphingolipids are extensively involved in the process of HBV infection. However, their role in chronic HBV infection remains unknown. The aim of this study was to determine the serum sphingolipid profile in a population of patients with chronic HBV infection, paying special attention to exploring novel prognostic markers in HBV-ACLF. High performance liquid chromatography tandem mass spectrometry was used to examine the levels of 41 sphingolipids in 156 serum samples prospectively collected from two independent cohorts. The training and validation cohorts comprised 20 and 28 healthy controls (CTRL), 29 and 23 patients with chronic hepatitis B (CHB), and 30 and 26 patients with HBV-ACLF, respectively. Biometric analysis was used to evaluate the association between sphingolipid levels and disease stages. Multivariate analysis revealed difference of sphingolipid profiles between CHB and HBV-ACLF was more drastic than that between CTRL and CHB, which indicated that serum sphingolipid levels were more likely to associate with the progression HBV-ACLF rather than CHB. Furthermore, a 3-month mortality evaluation of HBV-ACLF patients showed that dhCer(d18 : 0/24 : 0) was significantly higher in survivors than in non-survivors (including deceased patients and those undergoing liver transplantation, p < 0.05), and showed a prognostic performance similar to that of the MELD score. The serum sphingolipid composition varies between CTRL and chronic HBV infection patients. In addition, dhCer(d18 : 0/24 : 0) may be a useful prognostic indicator for the early prediction of HBV-ACLF.

Metabolic studies of four soy isoflavones in rats by HPLC-HR-MS.

In this paper, the metabolites of four soy isoflavones, daidzein, daidzin, genistein, and genistin, on perfused rat intestine-liver model were investigated by high-performance liquid chromatography coupled with high-resolution mass spectrometer/tandem mass spectrometer. Totally 16 metabolites were detected and identified based on accurate mass, fragmentation patterns, and multiple-stage mass data (MS(n)). The metabolic site of dadzein-7-methyl ether (D-7-M) was further confirmed by nuclear magnetic resonance. Methylation, glucuronide conjugation, and sulfate conjugation were the primary metabolic processes. Among them, six metabolites, daidzin-4',7-diglucoside, genistein-4'-glucoside, D-7-M, dadzein-4',7-dimethyl ether, genistein-4'-methyl ether, and genistein-7-methyl ether were detected in rats for the first time and not reported in humans. The metabolic pathways of daidzein, daidzin genistein, and genistin in rats were postulated. The biological effects of these metabolites are worthy of further investigation.

Lipidomic profiling of plasma in patients with chronic hepatitis C infection.

Chronic hepatitis C virus (HCV) infection is a global health issue. Although its progression is reported to be closely associated with lipids, the way in which the plasma lipidome changes during the development of chronic HCV infection in humans is currently unknown. Using an improved quantitative high-throughput lipidomic platform, we profiled 284 lipids in human plasma samples obtained from healthy controls (n = 11) and patients with chronic HCV infection (n = 113). The intrahepatic inflammation grade (IG) of liver tissue was determined by biopsy. Two types of mass spectrometers were integrated into a single lipidomic platform with a wide dynamic range. Compared with previous methods, the performance of this method was significantly improved in terms of both the number of target sphingolipids identified and the specificity of the high-resolution mass spectrometer. As a result, 44 sphingolipids, one diacylglycerol, 43 triglycerides, 24 glycerophosphocholines, and 5 glycerophospho-ethanolamines were successfully identified and quantified. The lipid profiles of individuals with chronic HCV infection were significantly different from those of healthy individuals. Several lipids showed significant differences between mild and severe intrahepatic inflammation grades, indicating that they could be utilized as novel noninvasive indicators of intrahepatic IG. Using multivariate analysis, healthy controls could be discriminated from HCV patients based on their plasma lipidome; however, patients with different IGs were not well discriminated. Based on these results, we speculate that variations in lipid composition arise as a result of HCV infection, and are caused by HCV-related digestive system disorders rather than progression of the disease.

Identification of impurities in methotrexate drug substances using high-performance liquid chromatography coupled with a photodiode array detector and Fourier transform ion cyclotron resonance mass spectrometry.

RATIONALE: Methotrexate (MTX) is an antineoplastic therapeutic medicine that acts as an antimetabolite of folic acid. In this study we identified the impurities in MTX drug substances produced by different manufacturers and in different batches using high-performance liquid chromatography coupled with a photodiode array detector and Fourier transform ion cyclotron resonance mass spectrometry (HPLC-PDA/FTICR-MS). METHODS: MTX and its impurities were separated on a Restek Pinnacle II C18 column (250 × 4.6 mm, 5 µm) with a gradient elution system composed of 0.2% formic acid and acetonitrile at a flow rate of 1.0 mL/min. Ultraviolet (UV) detection was set at 305 nm. Mass detection was carried out using FTICR-MS with full-scan mass analysis at a resolving power of 100 000 coupled with multiple-stage mass analysis using a parent list of compounds. RESULTS: Fifteen impurities were detected in MTX drug substances, and their structures were predicted from using HPLC-PDA/FTICR-MS data, including their UV spectra, high-resolution mass spectrometry (HRMS), fragmentation patterns, and MS(n) spectra. Ten of the impurities detected in the MTX drug substances are reported for the first time. There was a high abundance of esterified impurities in some batches of MTX drug substances, over the identification threshold of International Conference on Harmonization (ICH) guidelines, which requires particular attention. CONCLUSIONS: This paper describes a HPLC-PDA/FTICR-MS method to profile and identify impurities in MTX drug substances. The results suggest that HPLC-PDA/FTICR-MS is a valuable analytical technique for the rapid identification of impurities.

A new strategy for the discovery of epimedium metabolites using high-performance liquid chromatography with high resolution mass spectrometry.

In this paper, a new strategy of drug metabolite discovery and identification was established using high-performance liquid chromatography with high resolution mass spectrometry (HPLC-HRMS) and a mass spectral trees similarity filter (MTSF) technique. The MTSF technique was developed as a means to rapidly discover comprehensive metabolites from multiple active components in a complicated biological matrix. Using full-scan mass spectra as the stem and data-dependent subsequent stage mass spectra to form branches, the HRMS and multiple-stage mass spectrometric data from detected compounds were converted to mass spectral trees data. Potential metabolites were discovered based on the similarity between their mass spectral trees and that known compounds or metabolites in a mass spectra trees library. The threshold value for match similarity scores was set at above 200, allowing approximately 80% of interference to be filtered out. A total of 115 metabolites of five flavonoid monomers (epimedin A, epimedin B, epimedin C, icariin, and baohuoside I) and herbal extract of epimedium were discovered and identified in rats via this new strategy. As a result, a metabolic profile for epimedium was obtained and a metabolic pathway was proposed. In addition, comparing to the widely used neutral loss filter (NLF), product ion filter (PIF), and mass defect filter (MDF) techniques, the MTSF technique was shown superior efficiency and selectivity for discovering and identifying metabolites in traditional Chinese medicine (TCM).

Sphingolipids as new biomarkers for assessment of delayed-type hypersensitivity and response to triptolide.

BACKGROUND: Hypersensitivity diseases are associated with many severe human illnesses, including leprosy and tuberculosis. Emerging evidence suggests that the pathogenesis and pathological mechanisms of treating these diseases may be attributable to sphingolipid metabolism. METHODS: High performance liquid chromatography-tandem mass spectrometry was employed to target and measure 43 core sphingolipids in the plasma, kidneys, livers and spleens of BALB/c mice from four experimental groups: control, delayed-type hypersensitivity (DTH) model, DTH+triptolide, and control+triptolide. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify potential biomarkers associated with variance between groups. Relationships between the identified biomarkers and disease markers were evaluated by Spearman correlation. RESULTS: As a treatment to hypersensitivity disease, triptolide significantly inhibit the ear swelling and recover the reduction of splenic index caused by DTH. The sphingolipidomic result revealed marked alterations in sphingolipid levels between groups that were associated with the effects of the disease and triptolide treatment. Based on this data, 23 potential biomarkers were identified by OPLS-DA, and seven of these biomarkers correlated markedly with the disease markers (p<0.05) by Spearman correlation. CONCLUSIONS: These data indicate that differences in sphingolipid levels in plasma and tissues are related to DTH and treatment with triptolide. Restoration of proper sphingolipid levels may attribute to the therapeutic effect of triptolide treatment. Furthermore, these findings demonstrate that targeted sphingolipidomic analysis followed by multivariate analysis presents a novel strategy for the identification of biomarkers in biological samples.

Identification and characterization of flavonoids from semen zizyphi spinosae by high-performance liquid chromatography/linear ion trap FTICR hybrid mass spectrometry.

Semen zizyphi spinosae (SZS) has been used to treat insomnia and anxiety for thousands of years. In this paper, a novel high-performance liquid chromatography coupled with the photodiode array detector/linear ion trap-MS(n) (HPLC-PDA/LTQ-MS(n)) method was established to separate and identify flavonoids from the extract of SZS. Separation was performed on an HYPERSIL C(18) column by gradient elution using CH(3)CN/H(2)O-CH(3)COOH as the mobile phase at a flow rate of 0.8 ml/min. UV spectral data, accurate molecular weights, and multi-stage MS/MS fragmentation information were obtained. Electrospray ionization/MS/MS fragmentation patterns were proposed. Nineteen flavonoid glycosides were identified or tentatively characterized based on their retention time, UV spectral data, accurate molecular weights, and mass fragmentation behavior. The method was useful for separation and identification of the flavonoid components from SZS and could be applied to other complex samples, especially for minor constituents.

Mass spectral fragmentation analysis of triterpene saponins from Ardisia crenata Sims by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

We used the electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) technique to study the characteristic mass fragmentation patterns of eight triterpene saponins from Ardisia crenata Sims. Eight triterpene saponins were analyzed using parent mass list-triggered data-dependent multiple-stage accurate mass analysis at a resolving power of 100,000 in the external calibration mode. The chemical formula with unsaturation numbers was calculated from accurate m/z values of precursor, and product ions were obtained and used to assign the structures of eight triterpene saponins and two trace unknown compounds. The mass accuracies obtained for all full-scan MS and MS(n) spectra were within 7 ppm (< 5 ppm in most cases) in the ESI negative-ion mode. On FTICR-MS and FTICR-MS/MS, the eight triterpene saponins showed characteristic mass fragmentation patterns that facilitated the identification of their structural types, including the individual monosaccharide types, the monosaccharide numbers, and the sequences of the substituted saccharide groups. We proposed their fragmentation mechanisms. Based on their characteristic mass fragmentation patterns and fragmentation mechanisms, two unknown trace triterpene saponins were identified in the mixture.