The advancement of carbonic anhydrase (CA) activity and its inhibitor assays is crucial for drug development. Screening effective components from complex traditional Chinese medicine (TCM) systems as inhibitors is an important step in drug development. However, developing a simple and rapid method for this purpose remains challenging. Herein, a fluorescence (FL) sensing probe based on carbon quantum dots (CDs) integrated with a target affinity identification model for sensitive and specific CA detection and CA inhibitor screening was proposed. A FL sensing probe based on CDs was designed for the rapid detection of potential inhibitors in TCM samples on CA at first. Then the target affinity strategy was employed to accurately capture, isolate, and identify the active ingredient with inhibitory effects in the TCM sample. The developed method integrates two analytical models for screening CA inhibitors, significantly improving the accuracy and reproducibility of screening. The method was validated with positive, negative control and applied to screen CA inhibitors from 80 different TCM samples. The results revealed that ursolic acid and oleanolic acid from Chaenomeles speciosa (Sweet) Nakai, as well as cis-cinnamic acid and trans-cinnamic acid from Cinnamomum cassia Presl, are active ingredients that inhibit CA. Furthermore, isothermal titration calorimetry (ITC), molecular docking and dynamic simulations were employed to validate their interaction effects. Thus, this innovative detection method has great potential for rapidly screening CA inhibitor drugs. We believe that this strategy will stimulate further exploration and serve as a versatile and practical tool for screening enzyme inhibitors in TCM samples.

Osteoporosis is a prevalent skeletal disorder characterized by imbalanced bone remodeling, leading to excessive bone loss. Cannabinoid receptor 2 (CB2) has emerged as a promising therapeutic target in bone metabolism due to its peripheral expression and dual regulation of osteoblast and osteoclast activity. Desmethoxyyangonin (DMY) has demonstrated CB2 agonist activity, yet its therapeutic efficacy and molecular mechanisms in bone remain incompletely understood. Here, we investigated the anti-osteoporotic effects of DMY and its mechanistic insight on CB2 signaling. An ovariectomized (OVX) mouse model was used to assess the in vivo effects of DMY on trabecular bone microarchitecture and biomechanical properties. The impact of DMY on osteoblast and osteoclast differentiation was examined in vitro using MC3T3-E1 cells and bone marrow-derived macrophages (BMMs), respectively. Molecular docking and molecular dynamics simulations were conducted to investigate the potential interactions between DMY and CB2. Untargeted metabolomics was performed to explore metabolic alterations associated with DMY treatment. Western blot studies evaluated key osteogenic and signaling markers. DMY treatment significantly restored bone mineral density and improved trabecular microarchitecture. In vitro, DMY promoted osteoblast differentiation and mineralization while concurrently suppressing osteoclast formation and function. Pharmacological blockade of CB2 with its antagonist (SR144528) abrogated DMY's effects on both cell types, indicating CB2-mediated action. Molecular docking and dynamics simulations predicted a stable interaction between DMY and CB2. Untargeted metabolomics revealed DMY-induced shifts in metabolic pathways related to bone remodeling. Mechanistically, DMY upregulated osteogenic markers and activated PI3K/Akt and Wnt/β-catenin signaling cascades. Collectively, these findings demonstrate that DMY exerts bone-protective effects via CB2-dependent modulation of bone formation and resorption, offering a dual-target strategy for osteoporosis intervention.

Protein aggregation is an important pathological feature of cardiovascular disease. Therein, thrombin-mediated fibrin aggregation is one of the mechanisms of thrombosis, accurate monitoring of which is significant for the research of thrombosis. In this study, the optical properties and theoretical calculations confirmed that the AIE probe could specifically illuminate fibrin aggregates without interference, and its signal response was positively correlated with thrombin activity. Therefore, the biosensing technique can realize in situ monitoring of the coagulation process and rapid identification of active substances in complex systems. Furthermore, to detect anticoagulant active monomers, a biosensing targeted affinity screening (BioSTAS) technology was established by combining the above biosensing technique with the affinity chromatography technique. The rapid identification of active substances was achieved through biosensing, and then active monomers were captured by affinity chromatography. As a result, two agents with anticoagulant activity, rhein and oleanolic acid, were discovered via the screening of more than 30 kinds of natural products and commercial preparations. This study not only provides a new idea for the application of AIE probes in the dynamic monitoring of protein aggregation but also establishes an innovative strategy for screening active agents from a complex system through the integration of biosensing and affinity chromatography technologies.

Network pharmacology is instrumental in understanding how TCM works by targeting specific pathways. This study examines different mechanisms using three network pharmacology approaches, focusing on the TCM "Bai Mi Decoction" (BMD). The neuroprotective effects of BMD were evaluated in a middle cerebral artery occlusion-reperfusion (MCAO/R) rat model. The composition of BMD extract (BMDE) and its components in brain (BMDB) were analyzed using UHPLC-QTOF-MS/MS, alongside documented constituents from the database (BMDD). A network pharmacological analysis was conducted to explore the similarities and differences in BMD's neuroprotective mechanisms. BMD showed a strong neuroprotective effect in MCAO/R rats, as indicated by lower neurological deficit scores, smaller cerebellar infarct sizes, and improved histopathological changes. Analysis identified 45, 11, and 22 components in BMDE, BMDB, and BMDD, respectively, with 29, 26, and 23 potential therapeutic targets. However, most database-listed compounds were not found in actual samples. Functional enrichment and pathway network analysis showed that BMDE and BMDB shared the most targets. MAPK1 was the only common target across all groups, targeted by crocetin, the sole shared compound. BMD proved highly effective in MCAO/R rats, with compounds found in BMD extracts or in vivo better reflecting the actual pharmacological mechanisms than database-derived ones.

Sophora japonica L. pollen (SJP) is an emerging cause of urban seasonal allergies such as asthma and rhinitis. This study systematically evaluated the allergenicity of SJP using both in vivo and in vitro experiments. In vivo, a mouse model was employed in which SJP induced typical allergic symptoms, including sneezing and rubbing behavior, elevated serum IgE and IL-4 levels, and reduced IFN-γ levels. In vitro, IgE immunoblotting and ELISA using sera from allergic patients identified five potential allergenic protein bands separated by SDS-PAGE. LC-MS analysis further revealed a novel potential allergen, a 35 kDa protein belonging to the cytochrome f family, designated as Sop j 1. Structural comparison by CD spectroscopy and AlphaFold, combined with matching molecular weight and IgE reactivity profiles, confirmed that this cytochrome f protein is indeed Sop j 1. Recombinant Sop j 1 was produced and structurally validated. Western blot and inhibition ELISA demonstrated its specific binding to IgE in patient sera and its ability to inhibit IgE binding to native SJP. These findings indicate that Sop j 1 is a key allergenic protein in SJP with high IgE sensitization rates in the studied population, which will facilitate the development of precise diagnostics and treatments.

The discovery of active ingredients from natural products is an important source for drug development, but their complex matrices still hinder the establishment of efficient discovery strategies. Therefore, in order to achieve efficient and accurate analysis of active components in complex systems, this study proposes an efficient screening method assisted by biosensing technology. First, an iminopyrazine-based Fe-covalent organic framework (Fe-COF) was prepared, it was demonstrated that the prepared mimetic enzymes exhibited excellent peroxidase-like activity through structural characterization, activity evaluation, and density functional theory (DFT) calculations. Then, a biosensing analysis method for detecting xanthine oxidase activity was established using the prepared mimetic enzymes. This method was applied to rapidly screen xanthine oxidase inhibitors from complex natural product extracts. The results showed that acorn extracts were quickly screened for significant inhibition of xanthine oxidase activity with the assistance of biosensing methods. However, the efficient identification and quantification of bioactive compounds within complex matrices still pose a major challenge. In this study, computer-aided drug discovery approaches were employed to identify purpurogallin as a xanthine oxidase inhibitor from acorn extract, greatly improving screening efficiency and saving screening costs. Further research has shown that purpurogallin has good inhibitory activity on xanthine oxidase (IC50 = 177.67 μM). Furthermore, traditional UV spectrophotometry and SPR method were used to evaluate the binding and inhibitory abilities of the screened compounds, and the ability of the screened compounds to reduce uric acid was evaluated at the cellular level. This work demonstrates that the use of computer-aided analysis strategies assisted by biosensing technology can efficiently and accurately discover active monomers, providing a new approach for the analysis of active compounds in complex systems.

Current Alzheimer's drugs exhibit limited effectiveness, highlighting the necessity for multi-target treatments. This study developed an innovative and efficient screening platform combining hydrogen peroxide test strip-based colorimetric sensing with affinity chromatography for rapid identification of acetylcholinesterase (AChE) inhibitors from complex herbal medicines. Applying this strategy, from Lycium barbarum leaves, we identified three potent inhibitors: chlorogenic acid, N-acetyl-N'-caffeoylputrescine (NANCP), and N-caffeoylputrescine (NCP), with IC50 ranging from 55.7 to 143.2 µm. Molecular analyses confirmed their stable binding to AChE. In a D-galactose and AlCl3-induced Alzheimer's disease (AD) mouse model, NCP treatment significantly rescued cognitive deficits in AD mice, with the spontaneous alternation rate in the Y-maze test improved by up to 50%. It markedly reduced cerebral Aβ levels (by 54%) and pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, alleviated oxidative stress, and attenuated hippocampal neuronal damage. Mechanistically, NCP modulated glycerophospholipid metabolism, reshaped gut microbiota, and targeted the proteasome-autophagy pathway, revealing a multi-faceted synergistic mechanism. The research offers a new screening tool for AChE inhibitors and highlights a promising natural multi-target candidate, NCP, for AD therapy.

The environmental residues of penicillin antibiotics and their induced spread of resistance genes pose a serious challenge to global public health and ecological safety. Conventional wastewater treatment technologies face challenges in meeting current demands due to their suboptimal efficiency and the potential for secondary contamination. In this study, a covalent organic framework (COF)-based enzyme immobilization strategy was designed to construct a composite biocatalyst (β-Lac@Tp-Pa) through dynamic Schiff base condensation, which covalently anchored β-lactamase onto the imine COF (Tp-Pa) backbone, developing an enzyme core-COF shell structure that integrates enzymatic functionality with framework stability. The preparation conditions for β-Lac@Tp-Pa were systematically optimized. Under the optimal conditions, an enzyme activity recovery rate of 36.8% was achieved, while the enzyme leaching rate during recycling was maintained below 10%. In conjunction with a recirculating flow reaction device, approximately 99% removal of 50-200 mg L-1 penicillin in filtered lake water was achieved. Additionally, it demonstrated a retention of approximately 60% of its initial activity after twenty cycles. This study demonstrates the potential application of enzymes encapsulated in covalent organic frameworks in the field of wastewater treatment, providing a valuable reference for research in this area.

Discovery of active ingredients from complex natural products remains a critical challenging task in drug development, primarily due to the lack of rapid and precise analytical tools for bioactive compound screening. To address this challenge, we developed a biosensing-assisted computer-aided screening strategy. A covalent organic framework (COF) derived photocatalytic artificial oxidase was synthesized with robust catalytic capability toward TMB under natural light excitation. Leveraging arbutin as the chromogenic substrate, a sensitive biosensing assay was developed for α-glucosidase (α-Glu) activity detection with a linear range of 2-30 μg mL-1, enabling precise screening of active inhibitors within complex natural extract. This approach facilitated the successful identification of bioactive extracts from 25 natural product candidates. Subsequently, combined with computer-aided virtual screening, two active monomers (Honokiol and Magnolol) were rapidly resolved and characterized, with IC50 values of 175 ± 5.62 μM and 991.9 ± 36.5 μM against α-Glu, respectively. The screened active monomers exhibited significant inhibitory activity against α-Glu. Compared to conventional methods, the biosensing-assisted strategy effectively constrains the analytical scope and minimizes false-positive results, whereas the computational approach obviously accelerates the identification and analysis efficiency of active monomers. This study provides a new strategy for efficiently and accurately discovering bioactive compounds in complex systems, with promising prospects for high-throughput drug screening.

Discovery of active ingredient from complex natural products is crucial for drug discovery. However, the discovery of bioactive compounds from complex natural products is frequently limited by challenges in both precise identification and efficient analysis. This study developed a biosensing-assisted computer-aided screening strategy for the rapid and accurate discovery of bioactive components in complex natural product systems. A covalent organic framework (COF) derived photocatalytic artificial oxidase was synthesized with robust catalytic capability toward TMB under natural light excitation. Leveraging arbutin as the chromogenic substrate, a sensitive biosensing assay was developed for α-glucosidase (α-Glu) activity detection, enabling precise screening of active inhibitors within complex natural extract. This approach facilitated the successful identification of bioactive extracts from 25 natural product candidates. Subsequently, combined with computer-aided virtual screening, two active monomers (Honokiol and Magnolol) were rapidly resolved and characterized. The screened active monomers exhibited significant inhibitory activity against α-Glu. The biosensing-assisted strategy effectively constrains the analytical scope and minimizes false-positive outcomes, whereas the computational approach obviously accelerates the identification and analysis efficiency of active monomers. This study provides a new paradigm for efficiently and accurately discovering bioactive compounds in complex systems.

Regulation and screening of biomacromolecule (e.g., enzyme) activity is one of the core tasks in disease treatment and drug discovery. Natural products are an important source of enzyme inhibitors, but their complex composition makes it difficult to develop effective candidate drug molecules. As a typical biomacromolecular target, chymotrypsin (CHT) exhibits aberrant activity closely associated with diseases such as wound healing, chronic pancreatitis, and emphysema. Therefore, efficient screening of enzyme inhibitors from natural products holds significant importance for clinical diagnosis, treatment, and drug development. This study establishes an integrated screening platform for enzyme inhibitors by combining a Cu2+-casein-mediated fluorescence-enhanced biosensing system with targeted ligand capture technology, enabling highly sensitive screening and precise identification of chymotrypsin inhibitors from natural products. In this platform, Cu2+-casein functions as a biomacromolecular substrate while also acting as a peroxidase-mimetic enzyme, significantly enhancing the fluorescence signal amplification effect of o-phenylenediamine oxidation. When chymotrypsin hydrolyzes casein, the structure of casein is disrupted, reducing its copper ion-binding capacity. Which leads to decreased catalytic activity and subsequent changes in fluorescence intensity, allowing quantitative detection of chymotrypsin activity with a detection limit as low as 13.6 ng mL-1. In the targeted fishing module, immobilized chymotrypsin (CHT@NH2-SiO2) is employed as an affinity medium, and combined with HPLC and LC-MS analysis, enables precise identification of active components in complex samples. This integrated strategy mitigates certain limitations of individual techniques, providing a feasible approach for efficient, high-throughput screening of inhibitors targeting biomacromolecules from complex systems.

Hyaluronidase (HAase) is a potent urinary biomarker for bladder cancer, yet its clinical detection and the discovery of related inhibitors are often hindered by complex procedures and susceptibility to interference. To address these challenges, this study developed an integrated strategy that combines a dual-phase biosensor with immobilized HAase-based ligand fishing. The HAase-responsive biosensor enables both high-throughput screening of inhibitors from herbal extracts and direct, pretreatment-free analysis of clinical urine samples, effectively distinguishing bladder cancer patients from healthy individuals. Meanwhile, the complementary ligand fishing module allows precise capture and identification of bioactive compounds from complex mixtures, leading to the discovery of cinnamaldehyde as a candidate inhibitor (IC50 = 396.5 mu g/mL). In conclusion, this platform provides a versatile strategy that not only facilitates the rapid discovery of HAase-targeting agents from natural products but also offers a simple and interference-resistant approach for non-invasive monitoring of bladder cancer.

Hyaluronidase (HAase), an enzyme responsible for hyaluronic acid (HA) degradation, compromises the structural integrity of HA by cleaving anti-inflammatory high-molecular-weight HA into pro-inflammatory lower molecular weight forms. Consequently, developing simple and efficient strategies for screening HAase inhibitors is of critical importance. Herein, a novel biosensing-based targeted affinity screening method was established for the identification of HAase inhibitors from complex herbal medicines. In the biosensing system, carbon dots served as fluorescent probes, while HA-functionalized gold nanoparticles acted as quenchers, and the addition of HAase induced a marked fluorescence recovery. Under optimized conditions, HAase exhibited a good linear response over the concentration range of 5.3 to 343.8 U mL- 1. This method enables rapid and efficient detection of HAase inhibitory activity in complex herbal extracts. Subsequently, active components were selectively captured, separated, and identified using immobilized HAase affinity chromatography. Following validation with both negative and positive control drugs, the screening model successfully identified Rhodiola rosea L. among 43 herbal medicines as a potent HAase. Gallic acid (GA) was identified as the active constituent, exhibiting an IC50 value of 111.7 & micro;M. Results from molecular docking and molecular dynamics simulations demonstrated a strong binding affinity between GA and HAase. Additionally, cellular experiments confirmed the anti-inflammatory activity of GA, as evidenced by its significant inhibition of LPS-induced inflammatory markers IL-4, IL-5, IL-6, and TNF-a. Collectively, the integrated biosensing and affinity-based screening strategy established in this study provides a precise and efficient platform for the discovery of bioactive compounds from complex natural products.

Lactose can cause health problems in patients with lactose intolerance. (3-Galactosidase is commonly used to catalyze the hydrolysis of lactose, thereby producing low-lactose or lactose-free products. However, most existing methods employ free enzymes, which significantly increases the economic cost. In this study, the polyurethane sponge (PUS) was selected as the carrier, and the cross-linked enzyme aggregates (CLEAs) were loaded onto the carrier after the sponge was modified. The reaction products were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier infrared transform spectroscopy, and simultaneous thermal analysis, respectively. The loading amount of (3-galactosidase under the optimal conditions was 63 U/g support . The prepared PUS@(3-galactosidase-CLEAs was successfully used for the hydrolysis of lactose in milk and could hydrolyze 228-304 mu g of lactose in 30 min with good stability (RSD = 4.4%) and reusability. The method proposed in this study effectively combined the advantages of sponge and cross-linking enzyme aggregates, successfully achieving the decomposition of lactose in milk and rapid separation of the product. It is worth mentioning that this study provided a more effective and convenient industrial option for the production of lactose-free milk.

Hyaluronidase (HAase) degrades hyaluronic acid (HA), a key glycosaminoglycan of the extracellular matrix involved in tissue hydration, cell motility, and inflammation. Excessive HAase activity is associated with diseases, including cancer metastasis and inflammation, making HAase inhibitors vital for treatment strategies. Meanwhile, efficiently analyzing active ingredients in complex herbal medicines is key for drug discovery, but developing a simple and accurate method remains challenging. Here, an integrated approach combining paperbased sensing and column-free affinity chromatography to efficiently screen potential HAase inhibitors from herbal sources was established. The paper-based sensing module uses HA's rheological properties to quickly assess the inhibitory effects of herbal extracts on HAase. Herbals with inhibitory activity are then analyzed using affinity chromatography to capture, separate, and identify bioactive components interacting with HAase. The feasibility of both biosensing and affinity chromatography was validated using negative and positive control drugs, and Galla chinensis was successfully identified from 40 herbal samples as a potent HAase inhibitor, with tannic acid (TA) being the active compound (IC50 = 51.22 mu M). Molecular docking and dynamics simulations revealed a strong binding affinity between TA and HAase. TA's anti-inflammatory effects were further confirmed by its significant inhibition of LPS-induced IL-4, IL-5, IL-6, and TNF-alpha in cell experiments. In summary, this integrated strategy presents a promising and efficient approach for the discovery and development of novel HAase inhibitors, as well as other enzyme inhibitors derived from natural medicinal sources.

(3-Lactamases ((3-LS) are a significant threat to public health due to their role in conferring antibiotic resistance against nearly all clinically used (3-lactam antibiotics. Consequently, the search for (3-lactamase inhibitors ((3-LSI), which can reversibly bind to and inactivate (3-LS, has intensified. Here, a sensitive and rapid cellulose-based colorimetric sensor integrated with affinity identification model was designed for high-throughput screening of (3-LSI. The innovative concept of a nitrocefin-based colorimetric paper sensor strategy was effective in rapidly detecting the inhibitory effects of herbals on (3-LS. Then, potential (3-LSI present in the screened herbals were captured, separated, and identified utilizing affinity identification model. The efficacy of the developed method was validated with both negative and positive drugs and was subsequently applied to the screening of (3-LSI in 7 different herbal samples. Notably, Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou exhibited significant (3-LS inhibitory activity. Further affinity separation and LC-MS analyses identified 2 bioactive compounds, spinosin, and magnoflorine, as the agents responsible for this inhibitory effect, with IC50 values of 143.53 mu M and 294.66 mu M, respectively. The inhibitory effects and mechanisms were validated through antibacterial activity testing, molecular docking, and dynamic simulation. Notably, spinosin combined with aztreonam showed greater antibacterial effectiveness than avibactam. This study not only establishes a platform for the effective preliminary screening of (3-LSI as a potential antimicrobial resistance-reversing agent for future clinical applications, but also inspires the further development of other cellulose-based colorimetric sensors for the rapid and highthroughput discovery of various enzyme inhibitors in complex natural medicines.

Ethanol metabolism relies on acetaldehyde dehydrogenase (ALDH) to convert toxic acetaldehyde into harmless acetic acid. Some food-derived ALDH inhibitors can disrupt this process, raising health risks from alcohol consumption. Thus, identifying these inhibitors quickly and accurately in foods is crucial. Here we developed a novel analytical technique combining biosensing with affinity chromatography to detect ALDH inhibitors. The new concept of ALDH-Acetaldehyde-(3-Nicotinamide adenine dinucleotide hydrate (NAD+) based colorimetric sensing protocol exhibited good linearity within an ALDH activity range of 1.465-4.395 U/mL. The optimized detection procedure uses 0.1 % acetaldehyde, 10 mM NAD+, a 3:3 WST-8 to methoxy-PMS ratio, and two 30-min reactions at 37 degrees C. The efficacy of the method was validated with both negative and positive drugs and then applied to screen ALDH inhibitors in eleven foods, identifying soybean [Glycine max (L.) merr.] as a significant inhibitory. Within soybean, daidzin was found to have an IC50 of 194.09 mu M. Molecular docking studies reveal that daidzin effectively binds to ALDH's active site. The detection method developed here is simple, highly sensitive, and reproducible, providing an efficient approach for identifying and screening new enzyme inhibitors in foods.

The sol-gel method demonstrates significant potential for fabricating protein molecularly imprinted polymers (MIPs). In order to maintain good protein structure and provide more recognition functional groups during the preparation of protein MIPs. In this study, an exceptionally mild sol-gel protocol for protein MIPs synthesis was developed, in which no catalyst or organic solvent was employed and abundant recognition groups were introduced through in-situ derivatization. This method has been applied in the preparation of MIPs using lysozyme (lys) as a template. The developed MIPs exhibit high adsorption capacity and selectivity toward lys. The generation of the imprinting effect was successfully demonstrated by observing the surface morphology and evaluating the physical/chemical properties of the synthesized MIPs. In subsequent adsorption experiments, MIPs also showed excellent adsorption performance (389.85 mg/g) and quickly reached the adsorption equilibrium within 60 min. In addition, the selectivity of MIPs for lys was improved by the sol-gel imprinting process, and good reusability was observed after five adsorption-desorption cycles. The method was also successfully applied to isolate lys from egg white samples. This work demonstrates the great potential of the sol-gel strategy in the field of molecular imprinting.