Metabolomics analysis of (Geo)propolis from Brazilian stingless bees by FIA and UHPLC-HRMS (Orbitrap).

Native stingless bees (Meliponini) from Brazil make (geo)propolis which is largely used in folk medicine, specially by indigenous and quilombos communities and beekeepers´ families but are progressively being recognized for their pharmacological activities. In this study, the ethanolic extracts of (geo)propolis (EEGs) from Melipona marginata, M. quadrifasciata, M. scutellaris, and Tetragonisca angustula were analysed by Flow injection analysis (FIA) and Ultra-high performance liquid chromatography (UHPLC) in a high resolution Orbitrap mass analyser (HRMS) to investigate and compare their chemical profile. Untargeted metabolomic approach based on UHPLC-HRMS experiments, and bioinformatic tools, allowed to annotate 59 compounds from diverse classes such as: flavonoids, phenolic compounds, sugars, terpenoids, and lipids. In addition, using multivariate tools and Flow injection- high resolution mass spectrometry (FIA-HRMS), it was possible to classify samples and identify marker ions related to the bee species or genus and to the geographical origin as a proof of concept.

An integrated strategy for in-depth profiling of N-acylethanolamines in biological samples by UHPLC-HRMS.

BACKGROUND: N-acylethanolamines (NAEs) are a class of naturally occurring bioactive lipids that play crucial roles in various physiological processes, particularly exhibiting neuroprotective and anti-inflammatory properties. However, the comprehensive profiling of endogenous NAEs in complex biological matrices is challenging due to their low abundance, structural similarity and the limited availability of commercial standards. Here, we propose an integrated strategy for comprehensive profiling of NAEs that combines chemical derivatization and a three-dimensional (3D) prediction model based on quantitative structure-retention time relationship (QSRR) using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS). RESULTS: After acetyl chloride (ACC) derivatization, the detection sensitivity of NAEs was significantly improved. We developed a QSRR prediction model to construct an in-house database for 141 NAEs, encompassing information on RT, MS1 (m/z), and MS/MS spectra. Propargylamine-labeled fatty acids were synthesized as RT calibrants across various analytical conditions to enhance the robustness of the RT prediction model. NAEs in biological samples were then in-depth profiled using parallel reaction monitoring (PRM) acquisition. This integrated strategy identified and annotated a total of 50 NAEs across serum, hippocampus and cortex tissues from a 5xFAD mouse model of Alzheimer's disease (AD). Notably, the levels of polyunsaturated NAEs, particularly NAE 20:5 and NAE 22:6, were significantly decreased in 5xFAD mice compared to WT mice, as confirmed by accurate quantitation using ACC-d0/d3 derivatization. SIGNIFICANCE: Our integrated strategy exhibits great potential for the in-depth profiling of NAEs in complex biological samples, facilitating the elucidation of NAE functions in diverse physiological and pathological processes.

Rennets differing in chymosin-to-pepsin ratio shape the metabolomic and sensory profile of Grana Padano PDO cheese during ripening.

Utilizing different chymosin and pepsin ratios in cheesemaking may represent a potential strategy to shape the sensory profile of hard cheeses. This study investigated the impact of rennet with varying chymosin and pepsin ratios on the chemical profile and sensory attributes of Grana Padano PDO cheese at different ripening times (10 to 20 months). The research involved the analysis of hard cheese manufactured with distinct calf chymosin percentages (99 %, 95 %, and 83 %), exploiting sensory analyses and untargeted metabolomics to identify marker compounds correlating with specific sensory traits. The results demonstrated that varying the rennet composition significantly affected sensory profile; in particular, the rennet made by 83 % chymosin and 17 % pepsin generated a more complex sensory profile starting from 12 months. AMOPLS and ASCA analysis on untargeted metabolomics signatures revealed that ripening time was the only significant factor when compared with rennet type and the interaction ripening x rennet. Finally, at more advanced ripening times, 3-methylbutanoic acid and homoethone were significantly up-accumulated in cheese samples manufactured with higher pepsin percentages, likely explaining sensory outcomes. This study provides valuable insights into using rennet to tailor the sensory qualities of hard cheeses, underscoring the importance of enzyme selection in cheese manufacturing to drive innovation in the dairy industry.

Metabolomic Profiling and Biological Investigation of the Marine Sponge-Derived Fungus Aspergillus sp. SYPUF29 in Response to NO Condition.

Marine-derived fungi are assuming an increasingly central role in the search for natural leading compounds with unique chemical structures and diverse pharmacological properties. However, some gene clusters are not expressed under laboratory conditions. In this study, we have found that a marine-derived fungus Aspergillus sp. SYPUF29 would survive well by adding an exogenous nitric oxide donor (sodium nitroprusside, SNP) and nitric oxide synthetase inhibitor (L-NG-nitroarginine methyl ester, L-NAME) in culture conditions. Moreover, using the LC-MS/MS, we initially assessed and characterized the difference in metabolites of Aspergillus sp. SYPUF29 with or without an additional source of nitrogen. We have found that the metabolic pathway of Arginine and proline metabolism pathways was highly enriched, which was conducive to the accumulation of alkaloids and nitrogen-containing compounds after adding an additional source of nitrogen in the cultivated condition. Additionally, the in vitro anti-neuroinflammatory study showed that the extracts after SNP and L-NAME were administrated can potently inhibit LPS-induced NO-releasing of BV2 cells with lower IC50 value than without nitric oxide. Further Western blotting assays have demonstrated that the mechanism of these extracts was associated with the TLR4 signaling pathway. Additionally, the chemical investigation was conducted and led to nine compounds (SF1-SF9) from AS1; and six of them belonged to alkaloids and nitrogen-containing compounds (SF1-SF6), of which SF1, SF2, and SF8 exhibited stronger activities than the positive control, and showed potential to develop the inhibitors of neuroinflammation.

Tea Prepared from Dried Cannabis: What Do We Drink?

Besides many other uses, dried Cannabis may be used for "tea" preparation. This study focused on a comprehensive characterization of an aqueous infusion prepared according to a common practice from three fairly different Cannabis cultivars. The transfer of 42 phytocannabinoids and 12 major bioactive compounds (flavonoids) into the infusion was investigated using UHPLC-HRMS/MS. Phytocannabinoid acids were transferred generally in a higher extent compared to their counterparts; in the case of Δ9-THC, it was only in the range of 0.4-1.9% of content in the Cannabis used. A dramatic increase of phytocannabinoids, mainly of the neutral species, occurred when cream was added during steeping, and the transfer of Δ9-THC into "tea" achieved a range of 53-64%. Under such conditions, drinking a 250 mL cup of such tea by a 70 kg person might lead to multiple exceedance of the Acute Reference Dose (ARfD), 1 µg/kg b.w., even in the case when using hemp with a Δ9-THC content below 1% in dry weight for preparation.

Untargeted metabolomics analyses to identify a new sweet compound released during post-fermentation maceration of wine.

The sensory quality of a wine is mainly based on its aroma and flavor. Sweetness contributes in the gustatory balance of red wines. The investigation of compounds involved in this flavor was based on empirical observations, such as the increase in wine sweetness during yeast autolysis, concomitant to post-fermentation maceration in red winemaking. An untargeted metabolomics approach using UHPLC-HRMS has been developed to discover a new sweet molecule released during this stage. Among several markers highlighted, one compound was selected to be isolated by various separative techniques. It was unambiguously identified by NMR as N6-succinyladenosine and is reported for the first time in wine at an average concentration of 3.16 mg/L in 85 red wines. Furthermore, sensory analysis has highlighted its sweetness. In addition to discovering a new sweet compound in wine, this study proposes new tools for studying taste-active compounds in natural matrices.

Study of Leachable Compounds in Hospital Pharmacy-Compounded Prefilled Syringes, Infusion Bags and Vials.

Hospital pharmacy compoundings are crucial for maintaining patient care. They are time- and cost-effective in hospital pharmacy settings because they prevent waste, preparation errors, dosage errors, microbial contamination and breakage due to handling. Unfortunately, the drawbacks of hospital pharmacy compounding include the selection of inappropriate medical devices (MDs) for long-term storage, which could directly impact patients. In this study, three important hospital pharmaceutical compoundings, vancomycin in prefilled syringes (PFSs) made of polypropylene (PP) material, paediatric parenteral nutrition (PN) in ethylene vinyl acetate (EVA) bags and diluted insulin in cyclic olefin copolymer (COC) vials, were selected for leachate study and risk assessment. These compounds were studied via a semiquantitative screening approach by means of an ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) with postcolumn infusion and an in-house built database. 17 leachable compounds for the PFS, 25 for the PN, and 10 for the vial were identified, and their concentrations were estimated for toxicological assessments. In conclusion, all MDs used in hospital pharmacy compoundings were observed suitable thanks to risk assessments. However, suitable MDs recommended for long-term storage would remain with polymers like COC, for higher safety when exposed to frail and vulnerable patients like neonates and infants.

Simultaneous determination of free DL-amino acids in human hair with a novel DBD-M-Pro derivatization by UHPLC-HRMS: An application in diabetes patients.

Human hair is a non-invasive biological sample that is easy to collect and store and can reflect long-term body health. However, the correlation between DL-amino acids and metabolic diseases in hair samples has not been studied. Therefore, we propose a novel UHPLC-HRMS method for analyzing seven free chiral amino acids (DL-Thr, DL-Glu, DL-Ala, DL-Val, DL-Pro, DL-Leu, and DL-Phe) simultaneously in hair samples by derivatization of chiral probe 4-(N,N-dmethylaminosulfonyl)-2,1,3-benzoxadiazole-trans-2-methyl-L-proline (DBD-M-Pro) labeled with targeted amino functional groups. Gradient elution was carried out using an ACQUITYTM BEH C18 (100×2.1 mm,1.7 µm) column with a mobile phase of 0.15 % formic acid (FA) in 10 mM ammonium acetate (CH3-COONH4) and 0.2 % FA in acetonitrile. The labelled DL-amino acid diastereoisomers could be completely separated, with a resolution (Rs) of 1.59-11.44. These amino acids show a strong linear correlation within the range of 3.1-99.2 pmol (R2 ≥ 0.9990). Intraday and interday precision was 1.87 %-14.87 %. The average recovery was 96.12 %-105.33 %. The limit of detection (LOD) ranged from 0.29 to 2.11 pmol. We then employed the method to determine the concentration of free chiral amino acids in hair samples from 30 healthy volunteers (HVs) and 30 diabetes patients (DPs). Male diabetes patients had significantly higher levels of L-Thr, L-Val, L-Leu (p < 0.05), and D-Ala (p < 0.01) in their hair samples than male healthy volunteers and female diabetes patients had significantly higher levels of D-Ala (p < 0.05) in their hair samples than female healthy volunteers. This is the first study to confirm the feasibility of using free DL-amino acids in human hair as potential biomarkers for diabetes.

Prevalence of perfluoroalkyl substances in paired batches of precooked and canned bovine meat and their implication on consumer safety.

Meat is a highly nutritious food but there is a lot of significant evidence of negative health outcomes related to its excessive consumption, especially for processed one. Among the variety of emerging contaminants of concern for human health, a key role is played by poly- and per-fluoroalkyl substances (PFASs), which show adverse effects in humans who are exposed to them through diet. In the present study, for the first time, 70 paired batches of pre-cooked and canned bovine meat were analysed by Liquid Chromatography coupled to High Resolution Mass Spectrometry to evaluate the presence and concentration of 18 PFASs. These data were used to assess Italian consumers' health risks by performing the PFAS intake evaluation. PFBA and PFOS were detected in the precooked and canned meat samples, with PFBA mean concentration of 0.22 ± 0.36 ng g-1, and

Oil/Water Biphasic Solvent System for the Eco-Extraction and Cosmetic Formulation of Bixa orellana L.

Annatto, obtained from the seeds of achiote (Bixa orellana L.), is a widely used orange pigment rich in bixin and other apocarotenoids. This work reports the optimisation of a green extraction method of pigments and antioxidant compounds from achiote as well as its integration in a one-step green extraction-cosmetic formulation process. A biphasic solvent system of water and oil was used to recover simultaneously polar polyphenols, and less polar compounds, such as δ-tocotrienol and bixin. The optimisation of the ultrasound assisted extraction is presented, as well as a comparison of different vegetable oils used as extraction solvents. The composition, physicochemical properties and antioxidant activity of the oils were studied and their extraction performance was compared. Refined sunflower oil proved to be a better solvent than virgin olive, jojoba, coconut and grapeseed oils. Both aqueous and oil phases displayed an interesting antioxidant capacity. The oil phase contained 0.9% of bixin, as well as minor apocarotenoids and δ-tocotrienol. Twelve compounds, mainly phenolics, were identified by UHPLC-DAD-HRMS/MS in the aqueous phase. Twenty-one volatile compounds were identified in the volatile fraction by SPME-GC-MS. Lastly, a one-step green process is proposed to combine the extraction and the cosmetic formulation of the bioactive compounds.

Identification of N-piperidinyl etonitazene in alternative keratinous matrices from a decomposing cadaver.

This short report describes research on N-piperidinyl etonitazene, also known as etonitazepipne, in keratinous matrices (hair and nails) after death related to a suspected opioid overdose. Etonitazepipne belongs to the family of benzimidazole opioids, a class of new synthetic opioids that has penetrated the illicit drug market. Analysis in the case under study showed the presence of etonitazepipne in both hair and nails, confirming that the substance accumulates in the body with repeated intake.

Salvadora persica leaves: phytochemical profile and in vitro-inhibitory activity on inflammatory mediators implicated in periodontal disease.

CONTEXT: Virtually all parts of Salvadora persica L. (Salvadoraceae) are used in traditional medicine. The twigs and leaves are used for oral health, but leaves are far less investigated. OBJECTIVE: This study assesses the oral health-promoting potential of S. persica leaves with emphasis on anti-inflammatory and antiproliferative effects and provides an in depth-characterization of their metabolite profile. MATERIALS AND METHODS: Hot-water and methanolic S. persica leaf extracts (1, 10, and 100 µg/mL) and their major constituents (5, 10, and 50 µM), were subjected to cellular assays on IL-8 and TNFα release in LPS-stimulated human neutrophils, NO-release in LPS/IFNγ stimulated mouse macrophages, and proliferation of HNO97 human tongue carcinoma cells. Metabolite profiling was performed by UHPLC-HRMS analysis. Major constituents were isolated and structurally elucidated. RESULTS AND DISCUSSION: Both extracts showed pronounced anti-inflammatory activity in LPS-stimulated neutrophils. Major identified compound classes were flavonoid glycosides, the glucosinolate glucotropaeolin, phenyl- and benzylglycoside sulfates, and megastigmane glycosylsulfates, the latter ones identified for the first time in S. persica. Glucotropaeolin strongly inhibited the release of IL-8 and TNF-α (13.3 ± 2.0 and 22.7 ± 2.6% of the release of stimulated control cells at 50 µM), while some flavonoids and 3-(3'-O-sulfo-ß-d-glucopyranosyloxy)-7,8-dihydro-ß-ionone, a newly isolated megastigmane glycosylsulfate, were moderately active. Benzylisothiocyanate, which is likely formed from glucotropaeolin during traditional application of S. persica, showed considerable antiproliferative activity (IC50 in HNO97 cells: 10.19 ± 0.72 µM) besides strongly inhibiting IL-8 and TNFα release. CONCLUSIONS: Glucotropaeolin and benzylisothiocyanate are likely implicated in the oral health-promoting effects of S. persica leaves. The chemistry and pharmacology of the newly identified megastigmane glycosylsulfates should be further evaluated.

Chemometrics-based analysis of the phytochemical profile and antioxidant activity of Salvia species from Iran.

In recent years, the exploration of the therapeutic potential of Salvia has gained considerable attention, leading to a growing number of scientific studies emphasizing its pharmacological properties. Despite this, therapeutic applications of Salvia remain underexploited, requiring further investigation. Iran is a major center for sage diversity in Asia, boasting 60 Salvia species, 17 of which are unique to the area. This study aimed to comprehensively explore and compare the extracts of 102 Salvia samples belonging to 20 distinct Salvia species from Iran, providing a deeper understanding of their specific polyphenol content and, consequently, their antioxidant capabilities and potential therapeutic uses. All samples were analyzed to determine the contents of total phenolics, total flavonoids, total tannin, photosynthetic pigments, and ascorbic acid, along with their antioxidant activity. These data were then combined with the forty distinct chemical fingerprints identified by ultrafast high-pressure liquid chromatography coupled with high-resolution mass spectrometry. Multivariate data analysis was employed to find correlations and differences among the huge number of data obtained and to identify Salvia species with similar phytochemical and/or antioxidant properties. The results show that each Salvia species is characterized by a distinct class of polyphenols recognized for their antidiabetic, anti-inflammatory, cardioprotective and neuroprotective properties. Overall, our findings reveal the potential of some Salvia species for targeted therapeutic applications and provide a rational basis for the development of Salvia-derived nutraceuticals, ultimately improving the prospects for the use of Salvia in medicine.

Innovative Cosmeceutical Ingredients: Harnessing Selenosugar-Linked Hydroxycinnamic Acids for Antioxidant and Wound-Healing Properties.

Selenosugars are gaining growing interest due to their antioxidant efficacy, and their ability to inhibit glycosidases, repair skin tissue or reduce endothelial dysfunction. Among selenosugars, those in which selenium replaces heterocyclic oxygen in a 5-membered sugar were our focus, and their coupling with phenolic compounds appears to be a strategy aimed at producing new compounds with enhanced antioxidant efficacy. In this context, the Mitsunobu reaction has been advantageously explored to obtain trans-p-coumaroyl-1,4-deoxy-2,3-O-isopropylidene-4-seleno-d-ribose, trans-caffeoyl-1,4-deoxy-2,3-O-isopropylidene-4-seleno-d-ribose, and trans-feruloyl-1,4-deoxy-2,3-O-isopropylidene-4-seleno-d-ribose. These compounds underwent removal of the iso-propylidene group, to provide the corresponding hydroxycinnamoyl-1,4-deoxy-4-seleno-d-ribose. All compounds were characterized by Nuclear Magnetic Resonance (NMR) spectroscopy and High-Resolution Mass Spectrometry (HRMS). This latter technique was pivotal for ensuing cellular metabolomics analyses. In fact, after evaluating the anti-radical efficacy through 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods, which underline the massive role of the phenolic moiety in establishing efficacy, the compounds, whose cytotoxicity was first screened in two highly oxidative-stress-sensitive cells, were tested for their wound healing properties towards human HaCaT keratinocytes cells. Caffeoyl- and feruloyl selenosugars exerted a dose-dependent repair activity, while, as highlighted by the metabolomic approach, they were poorly taken up within the cells.

Thermally induced changes in the profiles of phytocannabinoids and other bioactive compounds in Cannabis sativa L. inflorescences.

Phytocannabinoids occurring in Cannabis Sativa L. are unique secondary metabolites possessing interesting pharmacological activities. In this study, the dynamics of thermally induced (60 and 120 °C) phytocannabinoid reactions in four cannabis varieties were investigated. Using UHPLC-HRMS/MS, 40 phytocannabinoids were involved in target analysis, and an additional 281 compounds with cannabinoid-like structures and 258 non-cannabinoid bioactive compounds were subjected to suspect screening. As expected, the key reaction was the decarboxylation of acidic phytocannabinoids. Nevertheless, the rate constants differed among cannabis varieties, documenting the matrix-dependence of this process. Besides neutral counterparts of acidic species, ́new bioactive compounds such as hydroxyquinones were found in heated samples. In addition, changes in other bioactive compounds with both cannabinoid-like and non-cannabinoid structures were documented during cannabis heating at 120 °C. The data document the complexity of heat-induced processes and provide a further understanding of changes in bioactivities occurring under such conditions.

Effects of photochemical aging on the molecular composition of organic aerosols derived from agricultural biomass burning in whole combustion process.

Biomass burning organic aerosols (BBOA) are key components of atmospheric particulate matter, yet the effects of aging process on their chemical composition and related properties remain poorly understood. In this study, fresh smoke emissions from the combustion of three types of agricultural biomass residues (rice, maize, and wheat straws) were photochemically aged in an oxidation flow reactor. The changes in BBOA composition were characterized by offline analysis using ultrahigh performance liquid chromatography coupled with Orbitrap mass spectrometry. The BBOA molecular composition varied dramatically with biomass type and aging process. Fresh and aged BBOA were predominated by CHO and nitrogen-containing CHON, CHN, and CHONS species, while with very few CHOS and other non­oxygen species. The signal peak area variations revealed that individual molecular species underwent dynamic changes, with 77-81 % of fresh species decreased or even disappeared and 33-46 % of aged species being newly formed. A notable increase was observed in the number and peak area of CxHyO≥6 compounds in aged BBOA, suggesting that photochemical process served as an important source of highly oxygenated species. Heterocyclic CxHyN2 compounds mostly dominated in fresh CHN species, whereas CxHyN1 were more abundant in aged ones. Fragmentation and homologs oxidation by addition of oxygen-containing functional groups were important pathways for the BBOA aging. The changes in BBOA composition with aging would have large impacts on particle optical properties and toxicity. This study highlights the significance of photochemical aging process in altering chemical composition and related properties of BBOA.

Differences in Metabolite Profiles of Dihydroberberine and Micellar Berberine in Caco-2 Cells and Humans-A Pilot Study.

We investigated the pharmacokinetic pathway of berberine and its metabolites in vitro, in Caco-2 cells, and in human participants following the administration of dihydroberberine (DHB) and micellar berberine (LipoMicel®, LMB) formulations. A pilot trial involving nine healthy volunteers was conducted over a 24 h period; blood samples were collected and subjected to Ultra High-Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) analyses to quantify the concentrations of berberine and its metabolites. Pharmacokinetic correlations indicated that berberrubine and thalifendine follow distinct metabolic pathways. Additionally, jatrorrhizine sulfate appeared to undergo metabolism differently compared to the other sulfated metabolites. Moreover, berberrubine glucuronide likely has a unique metabolic pathway distinct from other glucuronides. The human trial revealed significantly higher blood concentrations of berberine metabolites in participants of the DHB treatment group compared to the LMB treatment group-except for berberrubine glucuronide, which was only detected in the LMB treatment group. Similarly, results from in vitro investigations showed significant differences in berberine metabolite profiles between DHB and LMB. Dihydroberberine, dihydroxy-berberrubine/thalifendine and jatrorrhizine sulfate were detected in LMB-treated cells, but not in DHB-treated cells; thalifendine and jatrorrhizine-glucuronide were detected in DHB-treated cells only. While DHB treatment provided higher blood concentrations of berberine and most berberine metabolites, both in vitro (Caco-2 cells) and in vivo human studies showed that treatment with LMB resulted in a higher proportion of unmetabolized berberine compared to DHB. These findings suggest potential clinical implications that merit further investigation in future large-scale trials.

Tannic acids and proanthocyanidins in tea inhibit SARS-CoV-2 variants infection.

The COVID-19 pandemic has caused hundreds million cases and millions death as well as continues to infect human life in the world since late of 2019. The breakthrough infection caused from mutation of SARS-CoV-2 is rising even the vaccinated population has been increasing. Currently, the severe threat posed by SARS-CoV-2 has been alleviated worldwide, and the situation has transitioned to coexisting with the virus. The dietary food with antiviral activities may improve to prevent virus infection for living with COVID-19 pandemic. Teas containing enriched phenolic ingredients such as tannins have been reported to be antitumor agents as well as be good inhibitors for coronavirus. This study developed a highly sensitive and selective ultra-high performance liquid chromatography-high resolution mass spectrometric method for quantification of tannic acids, a hydrolysable tannin, and proanthocyanidins, a condense tannin, in teas with different levels of fermentation. The in vitro pseudoviral particles (Vpp) infection assay was used to evaluate the inhibition activities of various teas. The results of current research demonstrate that the tannins in teas are effective inhibitors against infection of SARS-CoV-2 and its variants.

Prospects for the Protective Mechanisms and Compounds of Bufei Huoxue Capsule against COVID-19 Convalescence: Evaluation of Integrating UHPLC-HRMS Analysis and Network Pharmacology Strategy.

INTRODUCTION: Most COVID-19 survivors are troubled with chronic persistent symptoms, which have currently no definitive treatments. Bufei Huoxue (BFHX) capsule exerts clinical benefit, while the material basis and molecular mechanism remain unclear. AIM: The study aimed to elucidate the protective mechanisms of BFHX capsules against COVID-19 convalescence. UHPLC-HRMS and various databases were employed to explore potential compounds and targets. PPI, MCODE, transcription factor (TF), and miRNA analyses were conducted to receive hub targets and corresponding upstream regulators. METHOD: Molecular docking was applied to verify the binding activity of compound and target. Further, GO, KEGG, WIKI, and Reactome analyses were performed, and compound-targetsymptom and gene-disease networks were constructed. A total of 127 compounds and 313 targets were acquired. A sum of 10 hub targets were screened and showed good binding affinities with critical compounds. RESULT: MLLT1, CBFB, and EZH2 were identified as key TFs, and hsa-mir-146a-5p, hsa-mir- 26b-5p, and hsa-mir-24-3p were predicted to be important miRNAs. BFHX capsule may alleviate the symptoms by targeting TNF, IL-6, IFNG, and TGF-ß1. Besides, BFHX capsule may exert a therapeutic effect on respiratory disease (especially pulmonary fibrosis and lung infection) and multi-system damage during COVID-19 convalescence by regulating cytokine-cytokine receptor interaction, as well as TGF-ß, TNF, and Toll-like receptor signaling pathways. CONCLUSION: In summary, BFHX capsule may exert a therapeutic effect on multi-system damages during COVID-19 convalescence through multiple compounds (such as albiflorin, isopsoralen, and neobavaisoflavone), multiple targets (such as TNF, IL-6, and EGF) and multiple pathways (TGF-ß, TNF, and Toll-like receptor signaling pathways).

Mechanism of action of Sambucus williamsii Hance var. miquelii in the treatment of osteoporosis analyzed by UHPLC-HRMS/MS combined network pharmacology and experimental validation.

Sambucus williamsii Hance var. miquelii(SWH) is a precious wild Chinese herb whose fruit, rhizome, leaves and root bark can be used as medicine. Sambucus Linn has pharmacological effects such as anti-osteoporosis, promoting fracture healing, anti-viral and anti-inflammatory. In this study, the main chemical components of the alcoholic extracts from SWH were rapidly identified by ultra-high performance liquid chromatography-quadrupole orbit trap high-resolution mass spectrometry (UHPLC- HRMS MS), and a total of 42 compounds were characterized from the alcoholic extracts of SWH. The results of network pharmacological validation showed that kaempferol, quercetin, luteolin, isorhamnetin and morroniside were the main active components, and KEGG enrichment demonstrated that SWH mainly affected the signaling pathways such as PI3K-Akt, TNF and FoxO by modulating the related targets such as AKT1, PIK3R1, EGFR, RELA SRC and PTGS2. The molecular docking results showed binding solid activity between the main active components of SWH and the targets. The network pharmacology was validated by establishing an animal model of osteoporosis (OP) in rats by gavage administration of vitamin A acid. The results of the pharmacological experiments showed that SWH could improve the degree of bone loss in the femur of osteoporotic rats, increase the number of trabeculae and decrease trabeculae porosity, up-regulate the Ca and P content in the serum of OP rats, down-regulate the scope of ALP and BGP in the serum, and promote the calcification of the bone matrix, and then exert the anti-OP efficacy. In this study, network pharmacology and pharmacological experiments verified the pharmacological mechanism of SWH in anti-OP rats. This provides a theoretical basis for the research and development of anti-OP drugs and a reference for the application of other traditional Chinese medicines in treating OP diseases.