RSC Advances
·
2026
·Vol. 16
·期 6
·5220-5227
·
IF 4.6
Qian Qin,Jinwei Zhang,Guo Feng,Yinhao Liao,Youli Chen,Caiyao Han,Yan Lei,Kexin Ma,Wei Li,Yibao Jin,Jinxi Wang,Ping Wang,Bing Wang,Xie-an Yu
Fermentation is a significant and specialized processing technology that primarily serves to enhance efficacy and reduce toxicity. Yaomu (YM) was fermented in a cellar using bovine bile as the fermentation matrix. However, the alterations in its active ingredients before and after processing remain poorly understood. An aggregation-induced emission fluorescence sensor (NITPA-S) was developed that employed S-2238 (S) to encapsulate the NITPA-Py-603 (NITPA) probe with the help of glutaraldehyde, creating an “ON” state. Thrombin hydrolyzed the sensor, releasing the probe in the “OFF” state, while thrombin inhibitors reversed this effect. We combined an HPLC fully automatic partial fraction collector and UHPLC-Q-TOF/MS for the separation, screening and identification of YM active compounds, which were used as indicators of anticoagulant activity to explore the changes in the content of YM before and after processing by using UHPLC-MS/MS and the sensor. A total of 211 compounds were collected and identified. Notably, karakoline, talatisamine, neoline, chasmanine and schaftoside exhibited the highest inhibition rates in YM. Furthermore, these compounds generally increased in concentration after processing ( p < 0.05). The anticoagulant inhibition rates of the mixture of these five compounds were 86.48% and 83.44% when compared with those of unfermented YM and YM, respectively. Consequently, the fluorescent sensor could completely screen out the anticoagulant active ingredients, providing a novel analytical approach for exploring and screening changes in the active components before and after TCM processing.
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Journal of Pharmaceutical Analysis
·
2026
·
IF 11.2
Lingling Yang,Shuxian Zhang,Xiaoxuan Fan,Keshuai Liu,Rui Mai,Wangyan Zhao,Qiang Han,Yongjian Ai,Qionglin Liang,Xueqin Ma,Guoning Chen
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.
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ANALYTICAL LETTERS
·
2022
·
IF 2.267
Ru Li,Xuan Zou,Pan Luan,Xiaokun Liu,Ning Wang,Qian Wang,Huashi Guan,Zhe Xu
Thrombosis is the main cause of many cardiovascular diseases. The conventional pharmaceuticals used for preventing thrombosis can induce significant adverse side effects, especially bleeding risk. Hence, the development of more effective and safe antithrombotic agents is strongly demanded to improve clinical treatment. In order to rapidly screen and identify novel antithrombotic substances from natural marine products, a multi-target screening strategy is reported to quantify the antithrombotic activity with high-performance liquid chromatography – mass spectrometry (HPLC-MS). Five key thrombosis-related targets, including thrombin, coagulation factors Xa, XIa and XIIa and platelet phosphodiesterase 3, were selected to establish the multi-target antithrombotic activity screening assay. The Michaelis-Menten constants (Km) were 8.16 µM, 58.85 µM, 120.03 µM, 35.24 µM and 79.67 µM, respectively. Five known inhibitors, namely argatroban, rivaroxaban, benzamidine, 3-carboxamide-coumarinand, and milrinone, were used for validation. The reproducibility of the screening strategy was verified by calculating the overall average Z′ value, which was 0.88. Subsequently, this method was assessed by screening a small marine library of 78 compounds. Five coumarin compounds were selected, and their antithrombotic activities were further validated in standard assessments of enzyme inhibition and on the coagulometer. According to the validation, the multi-target assay based on dried blood spots and HPLC-MS was suitable for screening antithrombotic substances with considerable reliability.
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ANALYTICA CHIMICA ACTA
·
2017
·
IF 4.95
Zhe Xu,Ruonan Liu,Huashi Guan
An accurate, rapid, and cost-effective methodology for enzyme assay is highly demanded to screen the effect of compounds on target at the molecular level. Thrombin (EC 3.4.21.5) and factor Xa (FXa, EC 3.4.21.6) have been identified as the critical targets for the development of potential drugs with anticoagulant activity. In this study, a rapid, sensitive and accurate assay based on UHPLC-MS/MS method has been developed for inhibitor screening against thrombin and factor Xa simultaneously. For thrombin and factor Xa, the Michaelis-Menten constants ( K m ) were calculated to be 6.14 and 57.27 μM, respectively. The inhibition constants ( K i ) for two known inhibitors, argatroban and rivaroxaban , were determined to be 16.23 and 0.41 nM, respectively. The assay was further validated through the determination of a high Z′ factor value of 0.89. Finally, the developed assay was applied to screen a chemical library against two enzymes . Three hit compounds belonging to a class of sulfated polysaccharides were identified and their targets of inhibition action were further evaluated. The results indicated that the dual-target assay by UHPLC-MS/MS analysis could be used as a reliable method for screening anticoagulant agents.
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BIOSENSORS & BIOELECTRONICS
·
2024
·
IF 10.7
Xiaoxuan Fan,Shuxian Zhang,Keshuai Liu,Xiaofei Wang,Hui Yuan,Zhiping Lv,Lijuan Ma,Xueqin Ma,Xia Zhang,Guoning Chen
Efficient analysis of active ingredient in complex natural products is crucial for drug discovery, but developing a simple method for this is challenging. The discovery of drugs against bacterial resistance is urgent because drug-resistant bacteria produce β-lactamases, which inactivate antibiotics and increase infection risks, particularly the AmpC β-lactamase. Here, an integrated analytical model based on colorimetric sensing and magnetic nanoparticles (MNPs) affinity chromatography was developed for screening AmpC β-lactamase inhibitors. A paper-based colorimetric microdevice was designed for rapid identification of inhibitors in complex samples. Magnetic nanoparticles affinity was then utilized to capture, isolate and identify the active ingredient by immobilized enzyme technology. This comprehensive analytical model greatly improves the efficiency and accuracy of inhibitor screening compared to other screenin g methods. Finally, the method was applied to screen AmpC β-lactamase activity inhibitors from five natural medicines. As a result, epicatechin gallate, a potential active component of AmpC β-lactamase, was successfully identified from Rhodiola rosea L . After thorough integration with in vitro antibacterial assays, the activity of the screened compounds was further substantiated. Their binding was further explored by molecular docking and molecular dynamics simulations. Consequently, potential AmpC β-lactamase inhibitors are rapidly and precisely captured from complex natural products by this method, which will offer novel insights into the screening of enzyme inhibitors within natural products.
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Shuxian Zhang,Xiaofei Wang,Jili Han,Xiaoxuan Fan,Keshuai Liu,Lingling Yang,Hao Yang,Xiaofei Chen,Xueqin Ma,Guoning Chen
Biological aggregates play a crucial role in the pathogenesis of thrombotic diseases, especially thrombin-induced biological aggregates. Therefore, the efficient discovery of thrombin inhibitors is of great significance for the prevention and treatment of thrombotic diseases. In this study, the aggregation precursor protein fluorescent probe was successfully prepared for monitoring the production of biological aggregates induced by thrombin. In this program, the aggregation degree of biomolecules can be quickly monitored through a fluorescence sensing technology. To facilitate the modulation of the biological aggregation process, the application of this advanced fluorescence sensing technology was utilized for the screening of thrombin inhibitors, which are pivotal regulatory molecules in biological aggregation. In addition, by combining the target fishing technique, an integrated model for rapid screening of potential inhibitors in complex extracts was further established. This model not only swiftly detects the presence of inhibitory components within complex systems but also precisely captures and identifies active monomers. After positive drug validation of the screening model, three active monomers, namely, ginkgetin, isoginkgetin, and bilobetin, were accurately screened from 30 natural products. These results highlighted the immense potential of the proposed approach for screening active ingredients from a wide range of natural products.
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Nature Communications
·
2024
·Vol. 15
·期 1
·1-19
·
IF 14.7
Lv Keyu,Chen Shuai,Xu Xulin,Chiu Joyce,Wang Haoqing J.,Han Yunyun,Yang Xiaodan,Bowley Sheryl R.,Wang Hao,Tang Zhaoming,Tang Ning,Yang Aizhen,Yang Shuofei,Wang Jinyu,Jin Si,Wu Yi,Schmaier Alvin H.,Ju Lining A.,Hogg Philip J.,Fang Chao
The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency ( Hrg −/− ) leads to delayed onset but enhanced formation of thrombus compared to WT . However, in the combined FXII deficiency ( F12 −/− ) and HRG deficiency (by siRNA or Hrg −/− ), there is further thrombosis reduction compared to F12 −/− alone, confirming HRG’s procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa.
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