Skip to main content
SpringerLink
Log in

An Anti-microbial Terpenoid Fraction from Gymnema sylvestre Induces Flip-flop of Fluorescent-Phospholipid Analogs in Model Membrane

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Therapeutic potential of Gymnema sylvestre on diverse cell types is predominantly due to a variety of terpenoids and their derivatives. However, their bioavailability becomes limited due to poor solubility and lower lipophilic properties, provoking the search for novel membranotropic terpenoids and their mechanism of action. A terpenoid fraction purified from Gymnema sylvestre exhibited broad spectrum antimicrobial activity against both Gram positive and Gram negative bacteria with IC50 ˂ 0.1 mg/ml. Evaluation of its membranotropic effect in vitro on reconstituted model membrane revealed that the fraction induced flip-flop of fluorescent phospholipid analogs across the lipid bilayer. The terpenoid-induced lipid flipping was biphasic with a fast linear phase (rate constant (k1) = 3 to 5 S−1) and a second slow exponential phase (rate constant (k2) = (4 to 9) × 10−3 S−1). The lipid-flippase activity of the terpenoid fraction showed concentration and incubation-dependent cooperativity, indicating their lipophilic nature and membrane-destabilizing activity that facilitated lipid translocation. For the first time, our study reveals the flippase activity of a terpenoid fraction of Gymnema sylvestre that could be further explored for their membrane-mediated pharmacological properties.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

CETFGS:

CHCl3-extracted terpenoid fraction of Gymnema sylvestre

DMSO:

Dimethyl sulfoxide

Egg-PC:

Egg-phosphatidylcholine

EA:

Ethyl acetate

FTIR:

Fourier-transform infrared spectroscopy

GA:

Gymnemic acid

GUV:

Giant unilamellar vesicle

HEPES:

N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid

IC50 :

Inhibitory concentration for 50% growth reduction

LSS:

Low salt solution

NAO:

10 N-nonyl acridine orange

NBD-PE:

N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt

PE:

Phosphatidylethanolamine

PL:

Phospholipid

References

  1. Park, J. I., Bae, H. R., Kim, C. G., Stonik, V. A., & Kwak, J. Y. (2014). Relationships between chemical structures and functions of triterpene glycosides isolated from sea cucumbers. Frontiers in Chemistry, 2, 77.

    Google Scholar 

  2. Inoue, Y., Shiraishi, A., Hada, T., Hirose, K., Hamashima, H., & Shimada, J. (2004). The antibacterial effects of terpene alcohols on Staphylococcus aureus and their mode of action. FEMS Microbiology Letters, 237(2), 325–331.

    CAS  Google Scholar 

  3. Dupont, C., Chen, Y., Xu, Z., Roquet-Banères, F., Blaise, M., Witt, A. K., Dubar, F., Biot, C., Guérardel, Y., Maurer, F. P., & Chng, S. S. (2019). A piperidinol-containing molecule is active against Mycobacterium tuberculosis by inhibiting the mycolic acid flippase activity of MmpL3. Journal of Biological Chemistry, 294(46), 17512–17523.

    Article  CAS  Google Scholar 

  4. Ingólfsson, H. I., Thakur, P., Herold, K. F., Hobart, E. A., Ramsey, N. B., Periole, X., De Jong, D. H., Zwama, M., Yilmaz, D., Hall, K., & Maretzky, T. (2014). Phytochemicals perturb membranes and promiscuously alter protein function. ACS Chemical Biology, 9(8), 1788–1798.

    Article  Google Scholar 

  5. Gauthier, C., Legault, J., Girard-Lalancette, K., Mshvildadze, V., & Pichette, A. (2009). Haemolytic activity, cytotoxicity and membrane cell permeabilization of semi-synthetic and natural lupaneand oleanane-type saponins. Bioorganic & Medicinal Chemistry, 17(5), 2002–2008.

    Article  CAS  Google Scholar 

  6. Lorent, J., Le Duff, C. S., Quetin-Leclercq, J., & Mingeot-Leclercq, M. P. (2013). Induction of highly curved structures in relation to membrane permeabilization and budding by the triterpenoid saponins, α-and δ-Hederin. Journal of Biological Chemistry, 288(20), 14000–14017.

    Article  CAS  Google Scholar 

  7. Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88(2), 308–316.

    Article  CAS  Google Scholar 

  8. Tiwari, P., Mishra, B. N., & Sangwan, N. S. (2014). Phytochemical and pharmacological properties of Gymnema sylvestre: An important medicinal plant. BioMed Research International, 2014, 830285.

    Google Scholar 

  9. Persaud, S. J., Al-Majed, H., Raman, A., & Jones, P. M. (1999). Gymnema sylvestre stimulates insulin release in vitro by increased membrane permeability. Journal of Endocrinology, 163(2), 207–212.

    Article  CAS  Google Scholar 

  10. Di Fabio, G., Romanucci, V., Zarrelli, M., Giordano, M., & Zarrelli, A. (2013). C-4 gem-dimethylated oleanes of Gymnema sylvestre and their pharmacological activities. Molecules, 18(12), 14892–14919.

    Article  Google Scholar 

  11. Chen, J., Jiang, Q. D., Chai, Y. P., Zhang, H., Peng, P., & Yang, X. X. (2016). Natural terpenes as penetration enhancers for transdermal drug delivery. Molecules, 21(12), 1709–1730.

    Article  Google Scholar 

  12. Singh, S., Fatima, Z., Ahmad, K., & Hameed, S. (2020). Repurposing of respiratory drug theophylline against Candida albicans: Mechanistic insights unveil alterations in membrane properties and metabolic fitness. Journal of Applied Microbiology, 1–37. (ahead of print). https://doi.org/10.1111/jam.14669.

  13. Blicharski, T., & Oniszczuk, A. (2017). Extraction methods for the isolation of isoflavonoids from plant material. Open Chemistry Journal, 15(1), 34–45.

    Article  CAS  Google Scholar 

  14. Ejikeme, C., Ezeonu, C. S., & Eboatu, A. N. (2014). Determination of physical and phytochemical constituents of some tropical timbers indigenous to nigerdelta area of Nigeria. European Scientific Journal, 10(48), 247–270.

    Google Scholar 

  15. Pathan, R. A., & Bhandari, U. (2011). Gymnemic acid-phospholipid complex: preparation and characterization. Journal of Dispersion Science and Technology, 32(8), 1165–1172.

    Article  CAS  Google Scholar 

  16. Razmavar, S., Abdulla, M. A., Ismail, S. B., & Hassandarvish, P. (2014). Antibacterial activity of leaf extracts of Baeckea frutescens against methicillin-resistant Staphylococcus aureus. BioMed Research International, 2014, 521287.

    Article  Google Scholar 

  17. Ansari, M. A., Fatima, Z., & Hameed, S. (2016). Anticandidal effect and mechanisms of monoterpenoid, perillyl alcohol against Candida albicans. PLoS One, 11(9), e0162465.

    Article  Google Scholar 

  18. Pereno, V., Carugo, D., Bau, L., Sezgin, E., de la Serna, J. B., Eggeling, C., & Stride, E. (2017). Electroformation of giant unilamellar vesicles on stainless steel, electrodes. ACS Omega, 2(3), 994–1002.

    Article  CAS  Google Scholar 

  19. Fernandez, M. I. G., Ceccarelli, D., & Muscatello, U. (2004). Use of the fluorescent dye 10-N-nonyl acridine orange in quantitative and location assays of cardiolipin: a study on different experimental models. Analytical Biochemistry, 328(2), 174–180.

    Article  Google Scholar 

  20. Sahu, S. K., & Gummadi, S. N. (2008). Flippase activity in proteoliposomes reconstituted with Spinacea oleracea endoplasmic reticulum membrane proteins: evidence of biogenic membrane flippase in plants. Biochemistry, 47(39), 10481–10490.

    Article  CAS  Google Scholar 

  21. Arora, D. S., & Sood, H. (2017). In vitro antimicrobial potential of extracts and phytoconstituents from Gymnema sylvestre R. Br. leaves and their biosafety evaluation. AMB Express, 7(1), 115.

    Article  Google Scholar 

  22. Chodisetti, B., Rao, K., & Giri, A. (2013). Phytochemical analysis of Gymnema sylvestre and evaluation of its antimicrobial activity. Natural Product Research, 27(6), 583–587.

    Article  CAS  Google Scholar 

  23. Liu, W., Liu, J., Yin, D., & Zhao, X. (2015). Influence of ecological factors on the production of active substances in the anti-cancer plant Sinopodophyllum hexandrum (Royle) TS Ying. PLoS One, 10(4), 17–35.

    Google Scholar 

  24. Nagarajan, S., Nagarajan, R., Braunhut, S. J., Bruno, F., McIntosh, D., Samuelson, L., & Kumar, J. (2008). Biocatalytically oligomerized epicatechin with potent and specific anti-proliferative activity for human breast cancer cells. Molecules, 13(11), 2704–2716.

    Article  CAS  Google Scholar 

  25. Rajarajeshwari, T., Shivashri, C., & Rajasekar, P. (2014). Synthesis and characterization of biocompatible gymnemic acid-gold nanoparticles: a study on glucose uptake stimulatory effect in 3T3-L1 adipocytes. RSC Advances, 4(108), 63285–63295.

    Article  CAS  Google Scholar 

  26. Syed Ali Fathima, M., & Johnson, M. (2018). Spectroscopic studies on pouzolzia wight II benn. International Journal of Pharmacy and Biological Sciences, 10(3), 124–132.

    CAS  Google Scholar 

  27. Turina, A. D. V., Nolan, M. V., Zygadlo, J. A., & Perillo, M. A. (2006). Natural terpenes: self-assembly and membrane partitioning. Biophysical Chemistry, 122(2), 101–113.

    Article  CAS  Google Scholar 

  28. Kumar, S., Scheidt, H. A., Kaur, N., Kang, T. S., Gahlay, G. K., Huster, D., & Mithu, V. S. (2019). Effect of the alkyl chain length of amphiphilic ionic liquids on the structure and dynamics of model lipid membranes. Langmuir, 35(37), 12215–12223.

    Article  CAS  Google Scholar 

  29. Epand, R. M., & Epand, R. F. (2011). Bacterial membrane lipids in the action of antimicrobial agents. Journal of Peptide Science, 17(5), 298–305.

    Article  CAS  Google Scholar 

  30. Sanyal, S., & Menon, A. K. (2009). Specific transbilayer translocation of dolichol-linked oligosaccharides by an endoplasmic reticulum flippase. Proceedings of the National Academy of Sciences of the United States of America, 106(3), 767–772.

    Article  CAS  Google Scholar 

  31. Ploier, B., Caro, L. N., Morizumi, T., Pandey, K., Pearring, J. N., Goren, M. A., Finnemann, S. C., Graumann, J., Arshavsky, V. Y., Dittman, J. S., & Ernst, O. P. (2016). Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants. Nature Communications, 7(1), 1–11.

    Article  Google Scholar 

  32. Goren, M. A., Morizumi, T., Menon, I., Joseph, J. S., Dittman, J. S., Cherezov, V., Stevens, R. C., Ernst, O. P., & Menon, A. K. (2014). Constitutive phospholipid scramblase activity of a G protein-coupled receptor. Nature Communications, 5(1), 5115.

    Article  CAS  Google Scholar 

  33. Taylor, G., Nguyen, M. A., Koner, S., Freeman, E., Collier, C. P., & Sarles, S. A. (2019). Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop. Biochimica et Biophysica Acta - Biomembranes, 1861(1), 335–343.

    Article  CAS  Google Scholar 

  34. Allhusen, J. S., & Conboy, J. C. (2017). The ins and outs of lipid flip-flop. Accounts of Chemical Research, 50(1), 58–65.

    Article  CAS  Google Scholar 

  35. Caetano, W., Gelamo, E. L., Tabak, M., & Itri, R. (2002). Chlorpromazine and sodium dodecyl sulfate mixed micelles investigated by small angle X-ray scattering. Journal of Colloid and Interface Science, 248(1), 149–157.

    Article  CAS  Google Scholar 

  36. Sánchez, M. E., del V Turina, A., Garcıa, D. A., Nolan, M. V., & Perillo, M. A. (2004). Surface activity of thymol: implications for an eventual pharmacological activity. Colloids and Surfaces B: Biointerfaces, 34(2), 77–86.

    Article  Google Scholar 

  37. Lee, B. C., Menon, A. K., & Accardi, A. (2016). The nhTMEM16 scramblase is also a nonselective ion channel. Biophysical Journal, 111(9), 1919–1924.

    Article  CAS  Google Scholar 

  38. Suzuki, J., & Nagata, S. (2014). Phospholipid scrambling on the plasma membrane. Methods in Enzymology, 544, 381–393.

    Article  CAS  Google Scholar 

  39. Fatima, Z., & Hameed, S. (2019). Lipidomic insight of anticandidal perillyl alcohol and sesamol induced Candida membrane disruption. Infectious Disorders Drug Targets, (ahead of print). https://doi.org/10.2174/1871526519666191023125020.

  40. Togashi, N., Inoue, Y., Hamashima, H., & Takano, A. (2008). Effects of two terpene alcohols on the antibacterial activity and the mode of action of farnesol against Staphylococcus aureus. Molecules, 13(12), 3069–3076.

    Article  CAS  Google Scholar 

Download references

Funding

This work is supported by the INSPIRE fellowship to Mr. Himadri Gourav Behuria from DST, Govt. of India.

Author information

Authors and Affiliations

  1. Department of Biotechnology, North Orissa University, Mayurbhanj, Baripada, Odisha, 757003, India

    Himadri Gourav Behuria & Santosh Kumar Sahu

Authors
  1. Himadri Gourav Behuria
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Santosh Kumar Sahu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Santosh Kumar Sahu.

Ethics declarations

Competing Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Behuria, H.G., Sahu, S.K. An Anti-microbial Terpenoid Fraction from Gymnema sylvestre Induces Flip-flop of Fluorescent-Phospholipid Analogs in Model Membrane. Appl Biochem Biotechnol 192, 1331–1345 (2020). https://doi.org/10.1007/s12010-020-03399-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-020-03399-3

Keywords

Search

Navigation