Metabolite profile of COVID-19 revealed by UPLC-MS/MS-based widely targeted metabolomics.

The metabolic characteristics of COVID-19 disease are still largely unknown. Here, 44 patients with COVID-19 (31 mild COVID-19 patients and 13 severe COVID-19 patients), 42 healthy controls (HC), and 42 patients with community-acquired pneumonia (CAP), were involved in the study to assess their serum metabolomic profiles. We used widely targeted metabolomics based on an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The differentially expressed metabolites in the plasma of mild and severe COVID-19 patients, CAP patients, and HC subjects were screened, and the main metabolic pathways involved were analyzed. Multiple mature machine learning algorithms confirmed that the metabolites performed excellently in discriminating COVID-19 groups from CAP and HC subjects, with an area under the curve (AUC) of 1. The specific dysregulation of AMP, dGMP, sn-glycero-3-phosphocholine, and carnitine was observed in the severe COVID-19 group. Moreover, random forest analysis suggested that these metabolites could discriminate between severe COVID-19 patients and mild COVID-19 patients, with an AUC of 0.921. This study may broaden our understanding of pathophysiological mechanisms of COVID-19 and may offer an experimental basis for developing novel treatment strategies against it.

Preclinical Pharmacokinetics and In Vitro Properties of GS-441524, a Potential Oral Drug Candidate for COVID-19 Treatment.

Preclinical pharmacokinetics (PK) and In Vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62-78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.4 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.

Comparative pharmacokinetics of 24 major bioactive components in normal and ARDS rats after oral administration of Xuanfei Baidu granules.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanfei Baidu prescription, consisting of 13 Chinese medicines, was formulated by academicians Boli Zhang and Professor Qingquan Liu based on their experience in first-line clinical treatment of COVID-19. Xuanfei Baidu granules (XFBD granules) are a proprietary Chinese medicine preparation developed based on Xuanfei Baidu prescription. It is recommended for the treatment of patients with the common wet toxin and lung stagnation syndrome of COVID-19. However, the pharmacokinetic characteristics of its major bioactive components in rats under different physiological and pathological conditions are unclear. MATERIALS AND METHODS: A rapid and sensitive analytical method, ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS), was developed and applied to 24 major bioactive components in normal and ARDS rats after oral administration of XFBD granules. We studied the metabolic process of XFBD granules in vivo to compare the differences in pharmacokinetic parameters between normal and model metabolic processes. RESULTS: This method was successfully applied to the pharmacokinetic investigation of 24 major components of XFBD granules following oral administration in normal and ARDS rats. Eight components, including ephedrine and amygdalin, were more highly absorbed and had shorter Tmax values than the model group; the absorption of six components, such as rhein, decreased in ARDS rats, and there was no significant difference in the absorption of ten components, such as verbenalin and naringin, between the normal and ARDS rats. The results showed that the peak times of other analytes were very short, and 80% of these target constituents were eliminated in both normal and ARDS rats within 6 h except for liquiritigenin and 18ß-glycyrrhetinic acid. CONCLUSIONS: In this study, a rapid and sensitive UPLC-MS/MS analytical method was developed and applied to 24 major bioactive components in normal and ARDS rats after the oral administration of XFBD granules. This will serve to form the basis for further studies on the pharmacokinetic-pharmacodynamic correlation of XFBD granules.

Mycotoxins in Maize Silage from China in 2019.

Animal feed (including forage and silage) can be contaminated with mycotoxins. Here, 200 maize silage samples from around China were collected in 2019 and analyzed for regulated mycotoxins, masked mycotoxins (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, and deoxynivalenol-3-glucoside), and emerging mycotoxins (beauvericin, enniatins, moniliformin, and alternariol). Deoxynivalenol and zearalenone were detected in 99.5% and 79.5% of the samples, respectively. Other regulated mycotoxins were detected in fewer samples. The highest deoxynivalenol and zearalenone concentrations were 3600 and 830 µg/kg, respectively. The most commonly detected masked mycotoxin was 15-acetyldeoxynivalenol, which was detected in 68.5% of the samples and had median and maximum concentrations of 61.3 and 410 µg/kg, respectively. The emerging mycotoxins beauvericin, alternariol, enniatin A, enniatin B1, and moniliformin were detected in 99.5%, 85%, 80.5%, 72.5%, and 44.5%, respectively, of the samples but at low concentrations (medians <25 µg/kg). The samples tended to contain multiple mycotoxins, e.g., the correlation coefficients for the relationships between the concentrations of beauvericin and deoxynivalenol, deoxynivalenol and zearalenone, and zearalenone and beauvericin were 1.0, 0.995, and 0.995, respectively. The results indicated that there needs to be more awareness of the presence of one or more masked and emerging mycotoxins in maize silage in China.

Bioavailability Enhancement of Cepharanthine via Pulmonary Administration in Rats and Its Therapeutic Potential for Pulmonary Fibrosis Associated with COVID-19 Infection.

Cepharanthine (CEP) has excellent anti-SARS-CoV-2 properties, indicating its favorable potential for COVID-19 treatment. However, its application is challenged by its poor dissolubility and oral bioavailability. The present study aimed to improve the bioavailability of CEP by optimizing its solubility and through a pulmonary delivery method, which improved its bioavailability by five times when compared to that through the oral delivery method (68.07% vs. 13.15%). An ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method for quantification of CEP in rat plasma was developed and validated to support the bioavailability and pharmacokinetic studies. In addition, pulmonary fibrosis was recognized as a sequela of COVID-19 infection, warranting further evaluation of the therapeutic potential of CEP on a rat lung fibrosis model. The antifibrotic effect was assessed by analysis of lung index and histopathological examination, detection of transforming growth factor (TGF)-ß1, interleukin-6 (IL-6), α-smooth muscle actin (α-SMA), and hydroxyproline level in serum or lung tissues. Our data demonstrated that CEP could significantly alleviate bleomycin (BLM)-induced collagen accumulation and inflammation, thereby exerting protective effects against pulmonary fibrosis. Our results provide evidence supporting the hypothesis that pulmonary delivery CEP may be a promising therapy for pulmonary fibrosis associated with COVID-19 infection.

Chemical profiling and unraveling of anti-COVID-19 biomarkers of red sage (Lantana camara L.) cultivars using UPLC-MS/MS coupled to chemometric analysis, in vitro study and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Red sage (Lantana camara L.) (Verbenaceae) is a widely spread plant that was traditionally used in Brazil, India, Kenya, Thailand, Mexico, Nigeria, Australia and Southeast Asia for treating several ailments including rheumatism and leprosy. Despite its historical role in relieving respiratory diseases, limited studies progressed to the plant's probable inhibition to respiratory viruses especially after the striking spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. AIM OF THE STUDY: This study aimed to investigate the inhibitory activity of different L. camara cultivars to SARS-CoV-2, that was not previously inspected, and clarify their mechanisms of action in the metabolomics viewpoint, and to determine the biomarkers that are related to such activity using UPLC-MS/MS coupled to in vitro-studies and chemometric analysis. MATERIALS AND METHODS: Chemical profiling of different cultivars was accomplished via UPLC-MS/MS. Principle component analysis (PCA) and orthogonal projection to latent structures (OPLS) models were built using SIMCA® (multivariate data analysis software). Cytotoxicity and COVID-19 inhibitory activity testing were done followed by TaqMan Real-time RT-PCR (Reverse transcription polymerase chain reaction) assay that aimed to study extracts' effects on RNA-dependent RNA polymerase (RdRp) and E-genes expression levels. Detected biomarkers from OPLS analysis were docked into potential targets pockets to investigate their possible interaction patterns using Schrodinger® suite. RESULTS: UPLC-MS/MS analysis of different cultivars yielded 47 metabolites, most of them are triterpenoids and flavonoids. PCA plots revealed that inter-cultivar factor has no pronounced effect on the chemical profiles of extracts except for L. camara, cultivar Drap d'or flowers and leaves extracts as well as for L. camara cv Chelsea gem leaves extract. Among the tested extracts, flowers and leaves extracts of L. camara cv Chelsea gem, flowers extracts of L. camara cv Spreading sunset and L. camara cv Drap d'or showed the highest selectivity indices scoring 12.3, 10.1, 8.6 and 7.8, respectively, indicating their relative high safety and efficacy. Leaves and flowers extracts of L. camara cv Chelsea gem, flowers extracts of L. camara cv Spreading sunset and L. camara cv Drap d'or were the most promising inhibitors to viral plaques exhibiting IC50 values of 3.18, 3.67, 4.18 and 5.01 µg/mL, respectively. This was incremented by OPLS analysis that related their promising COVID-19 inhibitory activities to the presence of twelve biomarkers. Inhibiting the expression of RdRp gene is the major mechanism behind the antiviral activity of most extracts at almost all concentration levels. Molecular docking of the active biomarkers against RdRp revealed that isoverbascoside, luteolin-7,4'-O-diglucoside, camarolic acid and lantoic acid exhibited higher docking scores of -11.378, -10.64, -6.72 and -6.07 kcal/mol, respectively, when compared to remdesivir (-5.75 kcal/mol), thus these four compounds can serve as promising anti-COVID-19 candidates. CONCLUSION: Flowers and leaves extracts of four L. camara cultivars were recognized as rich sources of phytoconstituents possessing anti-COVID-19 activity. Combination of UPLC-MS/MS and chemometrics is a promising approach to detect chemical composition differences among the cultivars and correlate them to COVID-19 inhibitory activities allowing to pinpoint possible biomarkers. Further in-vitro and in-vivo studies are required to verify their activity.

Study on flavonoid and bioactivity features of the pericarp of Citri Reticulatae 'Chachi' during storage

The mature pericarp of Citri Reticulatae 'Chachi' (PCRC) is one of the six traditional Chinese medicinal materials that should be used after long storage, and it was regarded that the longer the medicine was stored, the better. However, the aging mechanism of the medicine is not clear. To further investigate the effect of aging on the main active flavonoids of PCRC, ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) and metabolomics analysis were used to analyze the flavonoids of PCRC stored for different periods. In the results, 219 flavonoids were detected. 5,7,3',4',5'-pentamethoxy dihydroflavone and 2'-hydroxy-3,4,5,3'4',6'-hexamethoxychalcone were found from PCRC for the first time. According to the clustering analysis of metabolites, aging times of 0 year, 1 year, and 2 year were clustered into one group, and aging times of 3 year, 4 year, and 29 year were clustered into the other group. Quantitative analysis showed that the former group contained a greater amount of 4 flavonoids than the latter group, while the latter group contained a greater amount of 15 polymethoxyflavonoids. The newly harvested PCRC was compared with the other 5 groups of PCRC (stored for 1, 2, 3, 4, and 29 years). Eight flavonoids, tectochrysin, apigenin, 2'-hydroxyisoflavone, luteolin, 6-hydroxyluteolin, gallocatechin, quercetin -O- acetylhexoside and apigenin -7-O-[β-D-glucuronide (1→2) -O-β-D-glucuronide], were used as marker components to discriminate newly harvested PCRC and aging PCRC. In addition, the antioxidant potency composite index (APC) indicated that the PCRC stored for three or four years had stronger antioxidant activity than the PCRC stored for other periods. By means of molecular docking, it was reviewed that the amount of antiviral components against SARS-CoV-2 in freshly harvested PCRC was significantly higher than that in aging PCRC. The results in this study supplied scientific data for quality control, evaluation, and rational utilization of PCRC and basic information for further analysis of the metabolic regulation of the active components of the PCRC.

Detection of sivelestat and its metabolite in small volumes of plasma from Chinese ALI/ARDS patients with SIRS via high-throughput UPLC-MS/MS: A pharmacokinetic study.

In this study, we developed a sensitive and efficient analytical approach combining a 96-well plate-based protein precipitation strategy with ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS/MS) in order to assess the pharmacokinetic (PK) properties of sivelestat and its metabolite XW-IMP-A in samples of plasma from ALI/ARDS patients with SIRS. The samples were separated via gradient elution with a C18 column (Phenomenex Kinetex, C18, 2.6 µm, 100 Å, 50 × 2.1 mm) using 0.1 % formic acid aqueous solution (A) and acetonitrile-methanol (1:1, V:V) (B) as a mobile phase at a 0.6 mL/min flow rate. UPLC-MS/MS spectra were generated in positive ion mode, and multiple reaction monitoring (MRM) was used to detect the following transitions: m/z 435.1 → 360.0 for sivelestat, m/z 469.0 → 394.0 for sivelestat-IS, m/z 351.0 → 276.0 for XW-IMP-A, and m/z 384.9 → 310.0 for XW-IMP-A-IS. This assay was run for 2.5 min in total, and achieved lowest limit of quantitation values of 2.0 ng/mL and 0.5 ng/mL for sivelestat and XW-IMP-A, respectively, while remaining highly linear from 2-500 ng/mL for sivelestat (r2 ≥ 0.9900) and from 0.5-125 ng/mL for XW-IMP-A (r2 ≥ 0.9900). These validated data were consistent with US Food and Drug Administration (FDA) and European Medicines Agency (EMA) acceptance criteria. In addition, this method was successfully applied to the steady-state PK evaluation of ALI/ARDS patients with SIRS.

Qualitative and Quantitative Analysis of Ukrainian Iris Species: A Fresh Look on Their Antioxidant Content and Biological Activities.

The major groups of antioxidant compounds (isoflavonoids, xanthones, hydroxycinnamic acids) in the rhizome methanol extracts of four Ukrainian Iris sp. (Iris pallida, Iris hungarica, Iris sibirica, and Iris variegata) were qualitatively and quantitatively analyzed using HPLC-DAD and UPLC-MS/MS. Gallic acid, caffeic acid, mangiferin, tectoridin, irigenin, iristectorigenin B, irisolidone, 5,6-dihydroxy-7,8,3',5'-tetramethoxyisoflavone, irisolidone-7-O-ß-d-glucopyranoside, germanaism B, and nigricin were recognized by comparing their UV/MS spectra, chromatographic retention time (tR) with those of standard reference compounds. I. hungarica and I. variegata showed the highest total amount of phenolic compounds. Germanaism B was the most abundant component in the rhizomes of I. variegata (7.089 ± 0.032 mg/g) and I. hungarica (6.285 ± 0.030 mg/g). The compound analyses showed good calibration curve linearity (r2 > 0.999) and low detection and quantifications limit. These results validated the method for its use in the simultaneous quantitative evaluation of phenolic compounds in the studied Iris sp. I. hungarica and I. variegata rhizomes exhibited antioxidant activity, as demonstrated by the HPLC-ABTS system and NRF2 expression assay and anti-inflammatory activity on respiratory burst in human neutrophils. Moreover, the extracts showed anti-allergic and cytotoxic effects against cancer cells. Anti-coronavirus 229E and lipid formation activities were also evaluated. In summary, potent antioxidant marker compounds were identified in the examined Iris sp.

Vitamin C supplementation is necessary for patients with coronavirus disease: An ultra-high-performance liquid chromatography-tandem mass spectrometry finding.

To administer vitamin C (VC) with precision to patients with the coronavirus disease (COVID-19), we developed an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to assess plasma VC concentrations. 31 patients with COVID-19 and 51 healthy volunteers were enrolled. VC stability was evaluated in blood, plasma, and precipitant-containing stabilizers. A proportion of 7.7 % of VC was degraded in blood at room temperature (RT) (approximately 20-25 °C) at 1.5 h post administration with respect to the proportion degraded at 0.5 h, but without statistical difference. VC was stable in plasma for 0.75 h at RT, 2 h at 4 °C, 5 days at -40 °C, and 4 h in precipitant-containing stabilizer (2 % oxalic acid) at RT. The mean plasma concentration of VC in patients with COVID-19 was 2.00 mg/L (0.5-4.90) (n = 8), which was almost 5-fold lower than that in healthy volunteers (9.23 mg/L (3.09. 35.30)) (n = 51). After high-dose VC treatment, the mean VC concentration increased to 13.46 mg/L (3.93. 34.70) (n = 36), higher than that in healthy volunteers, and was within the normal range (6-20 mg/L). In summary, we developed a simple UPLC-MS/MS method to quantify VC in plasma, and determined the duration for which the sample remained stable. VC levels in patients with COVID-19 were considerably low, and supplementation at 100 mg/kg/day is considered highly essential.