Analytical and Bioanalytical Chemistry

, Volume 411, Issue 10, pp 1989–2000 | Cite as

Ligand fishing with cellular membrane-coated cellulose filter paper: a new method for screening of potential active compounds from natural products

  • Liang Xu
  • Cheng Tang
  • Xin Li
  • Xiaofan Li
  • Huiping Yang
  • Ruizhi Mao
  • Jiahui He
  • Wanqing Li
  • Jiyang Liu
  • Yalong Li
  • Shuobo Shi
  • Xuefeng XiaoEmail author
  • Xianhua WangEmail author
Paper in Forefront


Ligand fishing is a widely used approach for screening active compounds from natural products. Recently, cell membrane (CM) as affinity ligand has been applied in ligand fishing, including cell membrane chromatography (CMC) and CM-coated magnetic bead. However, these methods possess many weaknesses, including complicated preparation processes and time-consuming operation. In this study, cheap and easily available cellulose filter paper (CFP) was selected as carrier of CM and used to fabricate a novel CM-coated CFP (CMCFP) for the first time. The type of CFP was optimized according to the amount of immobilized protein, and the immobilization of CM onto CFP by the insertion and self-fusion process was verified by confocal imaging. The CMCFP exhibited good selectivity and stability and was used for fishing potentially active compounds from extracts of Angelica dahurica. Three potentially active compounds, including bergapten, pabulenol, and imperatorin, were fished out and identified. The traditional Chinese medicine systems pharmacology database and analysis platform was used to build an active compound-target protein network, and accordingly, the gamma-aminobutyric acid receptor subunit alpha-1 (GABRA1) was deduced as potential target of CM for the active compounds of Angelica dahurica. Molecular docking was performed to evaluate the interaction between active compounds and GABRA1, and bergapten was speculated as a new potentially active compound. Compared with other methods, the fishing assay based on CMCFP was more effective, simpler, and cheaper.


Ligand fishing Cellular membrane-coated cellulose filter paper Active compounds Network pharmacology 



We would like to thank Prof. H. Duan of Tianjin Medical University and Prof. L. He and Prof. S. Wang of Xi’an Jiaotong University for their valuable help on this work.

Funding information

This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 21605114, 81402889, 81303191), 131 innovative talents training project in Tianjin, Tianjin Municipal Administration of Traditional Chinese Medicine (Grant No. 2017077), and Tianjin Institute of Higher Vocational Education (Grant No. VII308).

Compliance with ethical standards

The procedure about rabbit was approved by Animal Ethics Committee, Tianjin Medical University, Tianjin, China. All the experiments on rabbits were performed in compliance with the guide of care and use of laboratory animals.

Conflict of interest

The authors declare that they no conflict of interest.

Supplementary material

216_2019_1662_MOESM1_ESM.pdf (3.1 mb)
ESM 1 (PDF 3137 kb)


  1. 1.
    Tu Y. The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine. Nat Med. 2011;17:1217–20.CrossRefGoogle Scholar
  2. 2.
    Wang Y, Fan X, Qu H, Gao X, Cheng Y. Strategies and techniques for multi-component drug design from medicinal herbs and traditional Chinese medicine. Curr Top Med Chem. 2012;12(12):1356–62.CrossRefGoogle Scholar
  3. 3.
    Wang Z, Li X, Chen M, Liu F, Han C, Kong L, et al. A strategy for screening of α-glucosidase inhibitors from Morus alba root bark based on the ligand fishing combined with high-performance liquid chromatography mass spectrometer and molecular docking. Talanta. 2018;180:337–45.CrossRefGoogle Scholar
  4. 4.
    Cieśla Ł, Moaddel R. Comparison of analytical techniques for the identification of bioactive compounds from natural products. Nat Prod Rep. 2016;33(10):1131–45.CrossRefGoogle Scholar
  5. 5.
    Hage DS, Anguizola JA, Bi C, Li R, Matsuda R, Papastavros E, et al. Pharmaceutical and biomedical applications of affinity chromatography: recent trends and developments. J Pharm Biomed Anal. 2012;69:93–105.CrossRefGoogle Scholar
  6. 6.
    Zhuo R, Liu H, Liu N, Wang Y. Ligand fishing: a remarkable strategy for discovering bioactive compounds from complex mixture of natural products. Molecules. 2016;21(11):1516–31.CrossRefGoogle Scholar
  7. 7.
    Ding X, Cao Y, Yuan Y, Gong Z, Liu Y, Zhao L, et al. Development of APTES-decorated HepG2 cancer stem cell membrane chromatography for screening active components from Salvia miltiorrhiza. Anal Chem. 2016;88(24):12081–9.CrossRefGoogle Scholar
  8. 8.
    Li F, Zhang Y, Qiu D, Kang J. Screening of epidermal growth factor receptor inhibitors in natural products by capillary electrophoresis combined with high performance liquid chromatography–tandem mass spectrometry. J Chromatogr A. 2015;1400:117–23.CrossRefGoogle Scholar
  9. 9.
    Schejbal J, Řemínek R, Zeman L, Mádr A, Glatz Z. On-line coupling of immobilized cytochrome P450 microreactor and capillary electrophoresis: a promising tool for drug development. J Chromatogr A. 2016;1437:234–40.CrossRefGoogle Scholar
  10. 10.
    Yin Z, Zhao W, Tian M, Zhang Q, Guo L, Yang L. A capillary electrophoresis-based immobilized enzyme reactor using graphene oxide as a support via layer by layer electrostatic assembly. Analyst. 2014;139(8):1973–9.CrossRefGoogle Scholar
  11. 11.
    Moaddel R, Marszałł MP, Bighi F, Yang Q, Duan X, Wainer IW. Automated ligand fishing using human serum albumin-coated magnetic beads. Anal Chem. 2007;79(14):5414–7.CrossRefGoogle Scholar
  12. 12.
    Song HP, Chen J, Hong JY, Hao H, Qi LW, Lu J, et al. A strategy for screening of high-quality enzyme inhibitors from herbal medicines based on ultrafiltration LC-MS and in silico molecular docking. Chem Commun. 2015;51(8):1494–7.CrossRefGoogle Scholar
  13. 13.
    Hu Y, Fu A, Miao Z, Zhang X, Wang T, Kang A, et al. Fluorescent ligand fishing combination with in-situ imaging and characterizing to screen Hsp 90 inhibitors from Curcuma longa L. based on InP/ZnS quantum dots embedded mesoporous nanoparticles. Talanta. 2018;178:258–67.CrossRefGoogle Scholar
  14. 14.
    Wang H, Zhao X, Wang S, Tao S, Ai N, Wang Y. Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures. J Chromatogr A. 2015;1392:20–7.CrossRefGoogle Scholar
  15. 15.
    Hou G, Niu J, Song F, Liu Z, Liu S. Studies on the interactions between ginsenosides and liposome by equilibrium dialysis combined with ultrahigh performance liquid chromatography-tandem mass spectrometry. J Chromatogr B. 2013;923-924:1–7.CrossRefGoogle Scholar
  16. 16.
    Chen L, Wang X, Liu Y, Di X. Dual-target screening of bioactive components from traditional Chinese medicines by hollow fiber-based ligand fishing combined with liquid chromatography–mass spectrometry. J Pharm Biomed Anal. 2017;143:269–76.CrossRefGoogle Scholar
  17. 17.
    de Almeida FG, Vanzolini KL, Cass QB. Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing. J Pharm Biomed Anal. 2017;132:159–64.CrossRefGoogle Scholar
  18. 18.
    Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov. 2006;5:993–6.CrossRefGoogle Scholar
  19. 19.
    Hou X, Wang S, Zhang T, Ma J, Zhang J, Zhang Y, et al. Recent advances in cell membrane chromatography for traditional Chinese medicines analysis. J Pharm Biomed Anal. 2014;101:141–50.CrossRefGoogle Scholar
  20. 20.
    Xu L, Xu B, Zhao ZY, Yang HP, Tang C, Dong LY, et al. Preparation and characterization of micro-cell membrane chromatographic column with N-hydroxysuccinimide group-modified silica-based porous layer open tubular capillary. J Chromatogr A. 2017;1516:125–30.CrossRefGoogle Scholar
  21. 21.
    Dong ZB, Li SP, Hong M, Zhu Q. Hypothesis of potential active components in Angelica sinensis by using biomembrane extraction and high performance liquid chromatography. J Pharm Biomed Anal. 2005;38(4):664–9.CrossRefGoogle Scholar
  22. 22.
    Tang C, Mao R, Liu F, Yu Y, Xu L, Zhang Y. Ligand fishing with cellular membrane-coated magnetic beads: a new method for the screening of potentially active compounds from natural products. Chromatographia. 2017;80(10):1517–25.CrossRefGoogle Scholar
  23. 23.
    Ma J, Wang C, Wei Y. Polyethyleneimine-facilitated high-capacity boronate affinity membrane and its application for the adsorption and enrichment of cis-diol-containing molecules. RSC Adv. 2016;6(49):43648–55.CrossRefGoogle Scholar
  24. 24.
    Yuan LM, Ma W, Xu M, Zhao HL, Li YY, Wang RL, et al. Optical resolution and mechanism using enantioselective cellulose, sodium alginate and hydroxypropyl-β-cyclodextrin membranes. Chirality. 2017;29(6):315–24.CrossRefGoogle Scholar
  25. 25.
    Wang X, Xu L, Mao R, Zhao X, Xu B, Tang C, et al. An insertion/self-fusion mechanism for cell membrane immobilization on porous silica beads to fabricate biomimic carriers. Biomater Sci. 2017;5(7):1334–41.CrossRefGoogle Scholar
  26. 26.
    Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science. 1972;175(4023):720–31.CrossRefGoogle Scholar
  27. 27.
    Ru J, Li P, Wang J, Zhou W, Li B, Huang C, et al. TCMSP: a database of systems pharmacology for drug discovery from herbal medicines. Aust J Chem. 2014;6(1):13–8.Google Scholar
  28. 28.
    Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–504.CrossRefGoogle Scholar
  29. 29.
    Hsin K-Y, Matsuoka Y, Asai Y, Kamiyoshi K, Watanabe T, Kawaoka Y, et al. systemsDock: a web server for network pharmacology-based prediction and analysis. Nucleic Acids Res. 2016;44(W1):W507–13.CrossRefGoogle Scholar
  30. 30.
    Hsin KY, Ghosh S, Kitano H. Combining machine learning systems and multiple docking simulation packages to improve docking prediction reliability for network pharmacology. PLoS One. 2014;8(12):e83922.CrossRefGoogle Scholar
  31. 31.
    Lee KS, Tsien RW. Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells. Nature. 1983;302:790–4.CrossRefGoogle Scholar
  32. 32.
    He L, Wang S, Geng X. Coating and fusing cell membranes onto a silica surface and their chromatographic characteristics. Chromatographia. 2001;54(1):71–6.CrossRefGoogle Scholar
  33. 33.
    Du H, He J, Wang S, He L. Investigation of calcium antagonist–L-type calcium channel interactions by a vascular smooth muscle cell membrane chromatography method. Anal Bioanal Chem. 2010;397(5):1947–53.CrossRefGoogle Scholar
  34. 34.
    Wubshet SG, Brighente IMC, Moaddel R, Staerk D. Magnetic ligand fishing as a targeting tool for HPLC-HRMS-SPE-NMR: α-glucosidase inhibitory ligands and alkylresorcinol glycosides from eugenia catharinae. J Nat Prod. 2015;78(11):2657–65.CrossRefGoogle Scholar
  35. 35.
    Pochet L, Heus F, Jonker N, Lingeman H, Smit AB, Niessen WMA, et al. Online magnetic bead based dynamic protein affinity selection coupled to LC–MS for the screening of acetylcholine binding protein ligands. J Chromatogr B. 2011;879(20):1781–8.CrossRefGoogle Scholar
  36. 36.
    Xie Y, Chen Y, Lin M, Wen J, Fan G, Wu Y. High-performance liquid chromatographic method for the determination and pharmacokinetic study of oxypeucedanin hydrate and byak-angelicin after oral administration of Angelica dahurica extracts in mongrel dog plasma. J Pharm Biomed Anal. 2007;44(1):166–72.CrossRefGoogle Scholar
  37. 37.
    Li B, Zhang X, Wang J, Zhang L, Gao B, Shi S, et al. Simultaneous characterisation of fifty coumarins from the roots of angelica dahurica by off-line two-dimensional high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry. Phytochem Anal. 2014;25(3):229–40.CrossRefGoogle Scholar
  38. 38.
    He JY, Zhang W, He LC, Cao YX. Imperatorin induces vasodilatation possibly via inhibiting voltage dependent calcium channel and receptor-mediated Ca2+ influx and release. Eur J Pharmacol. 2007;573(1):170–5.CrossRefGoogle Scholar
  39. 39.
    Schofield PR, Pritchett DB, Sontheimer H, Kettenmann H, Seeburg PH. Sequence and expression of human GABAA receptor α1 and β1 subunits. FEBS Lett. 1989;244(2):361–4.CrossRefGoogle Scholar
  40. 40.
    Horiuchi Y, Nakayama J, Ishiguro H, Ohtsuki T, Detera-Wadleigh SD, Toyota T, et al. Possible association between a haplotype of the GABA-A receptor alpha 1 subunit gene (GABRA1) and mood disorders. Biol Psychiatry. 2004;55(1):40–5.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Liang Xu
    • 1
    • 2
  • Cheng Tang
    • 1
  • Xin Li
    • 1
  • Xiaofan Li
    • 1
  • Huiping Yang
    • 1
  • Ruizhi Mao
    • 1
    • 3
  • Jiahui He
    • 4
    • 5
  • Wanqing Li
    • 1
  • Jiyang Liu
    • 2
  • Yalong Li
    • 2
  • Shuobo Shi
    • 6
  • Xuefeng Xiao
    • 4
    Email author
  • Xianhua Wang
    • 1
    Email author
  1. 1.Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnosis, School of PharmacyTianjin Medical UniversityTianjinChina
  2. 2.Tianjin Medical CollegeTianjinChina
  3. 3.People’s Hospital of TongliangquChongqingChina
  4. 4.School of Chinese Materia MedicaTianjin University of Traditional Chinese MedicineTianjinChina
  5. 5.Acchrom Technologies Co., Lid.BeijingChina
  6. 6.Beijing Advanced Innovation Center for Soft Matter Science and EngineeringBeijing University of Chemical TechnologyBeijingChina

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