Snake antivenoms remain the only effective treatment for envenomation; however, current manufacturing practices often result in formulations containing substantial amounts of non-specific serum proteins and heterologous immunoglobulins, which contribute to adverse reactions and limit therapeutic performance. Here, we developed and evaluated a venom-based affinity chromatography platform in which Crotalus durissus terrificus toxins were covalently immobilized on CNBr-activated Sepharose 4B to enable selective isolation of F(ab′)₂ fragments (BF) from crotalic antivenom (CAv). Ten chromatographic runs were conducted across two experimental sets to assess method repeatability and intermediate precision. Chromatographic profiles were highly consistent across all assays, with intra-run and inter-run relative standard deviations (RSD) ≤ 15%, confirming the robustness of the purification method. Quantitative proteomics demonstrated substantial enrichment of BF and a marked depletion of non-specific serum proteins such as albumin, fibrinogen, fibronectins, and serotransferrins. ELISA assays revealed an 8-fold increase in BF by the enriched fraction relative to the CAv. In vitro functional assays demonstrated that BF exhibited greater inhibition of enzymatic activity than CAv across the tested time points. Together, these results demonstrate the high selectivity and reliability of the venom-based affinity matrix and may support its integration as an incremental bioprocessing step to enhance the purity, quality, and safety of antivenoms. This platform demonstrates a proof of concept for advancing next-generation crotalic antivenom production.
Coronary heart disease combined with depression (dual-heart disorder) exhibits high morbidity and poor prognosis, lacking safe unified therapeutic agents. Shenqi Jieyu Decoction (SQJY) relieves depressive and cardiovascular symptoms in percutaneous coronary intervention (PCI) patients, yet its underlying multiple potential mechanisms remain unclear. In this study, an ApoE−/− mouse model of dual-heart disorder was induced via high-fat diet combined with chronic unpredictable mild stress (CUMS). Behavioral testing, aortic pathological staining, hippocampal neurochemistry, and ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS)-based untargeted metabolomics of serum and cecal contents were integrated with 16S rRNA sequencing, establishing an integrated multi-omics workflow to examine the potential modulatory effects of SQJY and to explore its possible mechanism. Behavioral and pathological assays indicated that SQJY alleviated atherosclerosis and depression-like phenotypes, elevated hippocampal 5-hydroxytryptamine (5-HT), dopamine (DA) and brain-derived neurotrophic factor (BDNF), and suppressed neuroinflammatory cytokines. 16S sequencing suggested SQJY remodeled dysregulated gut microbiota toward a more balanced profile. Metabolomics identified disturbed sphingolipid and glycerophospholipid metabolism as important metabolic signatures that H-SQJY group corrected more extensively in cecal content (68 of 265 metabolites) than in serum (8 of 103). Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2)-based prediction of microbial functions was integrated, and Spearman correlation analysis showed significant associations between predicted microbial functions, differential gut microbes and lipid metabolites. Collectively, SQJY showed concurrent potential cardioprotective and antidepressant effects, possibly through restoring gut microbial homeostasis and modulating lipid metabolic disorders. These correlative and predicted findings provide preliminary evidence for the potential application of SQJY in dual-heart disorder.
Following the COVID-19 pandemic, mRNA vaccines have been widely deployed, with lipid nanoparticles (LNPs) serving as the prevailing delivery vehicles for mRNA vaccines. Separation and quantitative determination of mRNA and LNP lipid components have therefore attracted considerable attention. Reported mRNA assays suffer from several limitations, including interferences originating from metallic instrument components and complex formulation matrices, insufficient resolution, and prolonged elution durations. Existing analytical approaches for LNP lipid quantification usually involve labor-intensive sample pretreatment and fail to eliminate matrix interferences. Even reported methods capable of simultaneous measurement of nucleic acids and lipids still exhibit deficiencies in resolution and sensitivity. Herein, an integrated analytical platform combining Bio-RP-HPLC-UV and UHPLC-ELSD was established. A biocompatible inert liquid-chromatography system was adopted for mRNA quantification to mitigate adsorption and carry-over effects commonly observed in conventional stainless-steel systems, while chromatographic conditions were optimized to shorten analysis time. A 2/6-port valve-switching splitting device was installed upstream of the UHPLC-ELSD detector to prevent rapid detector contamination induced by direct injection of complex mRNA-LNP formulation matrices and substantially reduce instrument-maintenance frequency. Mobile-phase compositions were further optimized to improve resolution and sensitivity for LNP lipid detection. The validated workflow exhibited favorable precision and excellent linearity (R > 0.9990). The recovery of the mRNA quantification method ranged from 87.89% to 100.31%, and the mean recoveries of the four LNP lipids at three concentration levels varied between 99.52% and 102.46%, guaranteeing assay accuracy. Furthermore, this integrated platform was applied to characterize mRNA-LNP samples before and after size-exclusion-chromatography (SEC) purification. Correlation analysis was performed among mRNA contents, drug-to-lipid ratios, and particle-size data acquired by high-throughput dynamic light scattering (DLS). Results from method-validation experiments and testing of three batches of samples demonstrated that the proposed platform enables simultaneous quantification of mRNA and lipids in mRNA-LNP formulations and multi-attribute parameter correlation verification throughout the SEC purification procedure. Satisfactory inter-batch consistency was achieved across three production batches. This work provides technical support for constructing a facile and practicable quality-evaluation system for mRNA-LNP vaccine formulations.
Humulus lupulus L. is a phytochemically rich medicinal plant with antioxidant and pharmacological potential; however, the direct hemolytic effects of extracts obtained using different solvents remain poorly characterized. This study integrated experimental and computational approaches to evaluate the phytochemical composition, antioxidant potential, elemental profile, and hemolytic activity of aqueous, methanolic, ethanolic, and acetone extracts of H. lupulus L. Total phenolic and flavonoid contents, DPPH and ABTS radical-scavenging activities, and IC50 values were determined. Chemical and elemental profiles were characterized using gas chromatography–mass spectrometry (GC–MS) and inductively coupled plasma mass spectrometry, respectively. Hemolytic activity was assessed in human erythrocytes, and selected tentatively identified constituents were further investigated by molecular docking and density functional theory. The extraction solvent markedly influenced the chemical composition and biological responses. Methanol yielded the highest phenolic content and strongest ABTS activity (IC50 = 54.25 μg/mL), whereas ethanol showed the highest flavonoid content and the lowest DPPH IC50 value among the extracts (161.0 μg/mL). Despite its pronounced antioxidant activity, the methanol extract consistently produced the greatest hemolytic effect, while aqueous and ethanolic extracts exhibited substantially greater erythrocyte compatibility. GC–MS revealed distinct solvent-dependent chemical profiles, whereas potassium, calcium, and magnesium were the predominant elements. Docking identified 2,3-dihydroxypropyl hexadecanoate as the most consistently interacting constituent, particularly toward hemoglobin, erythrocyte Band 3, and TRPV4. Density functional theory revealed substantial differences in electronic reactivity, with Xanthosine exhibiting the lowest HOMO-LUMO energy gap. Overall, antioxidant potency and erythrocyte compatibility did not necessarily align, identifying extraction solvent as a critical determinant of the bioactivity-hemocompatibility balance of H. lupulus extracts.
Background Allergic asthma is a common allergen-driven phenotype of asthma with a high prevalence. Current first-line therapies are limited by poor treatment adherence, frequent relapse after discontinuation, and heterogeneous therapeutic responses. Sangmei Zhike Granules (SMZK), a traditional Chinese medicine formula formulated to dispel wind, diffuse the Lung, and nourish yin, have demonstrated therapeutic efficacy against allergic asthma; however, their underlying mechanisms remain unclear. Objective This study aimed to identify the lung-distributed constituents of SMZK and elucidate their mechanisms of action against allergic asthma. Methods Ultra-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) was used to characterize the chemical constituents of SMZK and identify SMZK-derived prototype compounds in rat lung tissue. Network pharmacology analysis was subsequently performed based on the screened prototype compounds to predict potential targets and signaling pathways. An ovalbumin (OVA)-induced rat model of allergic asthma was established, and the therapeutic effects of SMZK were evaluated using pulmonary function testing, histopathological examination, and enzyme-linked immunosorbent assay (ELISA). The predicted signaling pathway was further validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and ELISA. Results A total of 812 chemical constituents were identified in SMZK, and 41 SMZK-derived prototype compounds were detected in rat lung tissue. Network pharmacology analysis of 26 prototype compounds that met the screening criteria identified IL6, MAPK8, ESR1, SRC, STAT3, TNF, MAPK3, MAPK1, and RELA as key targets, with the PI3K-Akt and Toll-like receptor signaling pathways emerging as major candidate pathways. SMZK improved pulmonary function, alleviated pulmonary inflammatory cell infiltration, and attenuated type 2 airway inflammation. Mechanistically, SMZK downregulated the expression of TLR4 and PI3Kγ and inhibited NF-κB p65 activation, thereby suppressing the TLR4/PI3Kγ/NF-κB signaling axis and ameliorating type 2 airway inflammation. Conclusion The lung-distributed constituents of SMZK may attenuate type 2 airway inflammation in allergic asthma primarily by suppressing the TLR4/PI3Kγ/NF-κB signaling axis.
