Diterpene alcohol fraction of Cyperus rotundus Linn essential oil regulates Bcl-2 and Bax expression inducing apoptosis on HeLa in vitro and in silico

Susianti Susianti; Yanwirasti Yanwirasti; Eryati Darwin; Jamsari Jamsari; Arif Setiawansyah

doi:10.7324/JAPS.2024.188231

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Abstract

Apoptosis, or programmed cell death, is a crucial mechanism in preventing cancer growth, and one of the targets is reducing the expression of the anti-apoptotic protein Bcl-2. This study aimed to evaluate the potential of fractions from nutsedge (Cyperus rotundus) essential oil in inducing apoptosis and inhibiting the progression of cervical cancer. The researchers investigated the apoptosis-inducing activity of these fractions against the HeLa cervical cancer cell line using in vitro and in silico approaches. The cytotoxic effects were assessed through an MTT assay on HeLa cells cultured in 96-well plates. Additionally, flow cytometry with Annexin/PI staining was employed to analyze the induction of apoptosis by the fractions. The immunocytochemical staining of cells was also implemented to assess Bax and Bcl-2 expressions. The biologically active compound in fractions was screened using a molecular docking approach to Bcl-2 co-crystalized structure and their pharmacokinetics and toxicity profile were assessed. The cytotoxic activity differed significantly from each fraction. The highest cytotoxicity was observed in fraction 1 (IC50: 8.307 + 0.186 mcg/ml), and the lowest cytotoxicity was observed in fraction 4 (IC50:>500 mcg/ml). Fraction 1 decreased the Bcl-2 expression and increased the Bax expression. Molecular docking screening revealed that 5-(7a-isopropenyl-4,5-dimethyl-octahydro-inden-4-yl)-3-methyl-pent-2-en-1-ol was predicted as the main contributor to apoptosis-inducing activity of fraction 1. The supplementation of fraction 1 induces cell apoptosis on HeLa cells, indicating the potential of this fraction of nutsedge essential oil for developing an anti-cervical cancer agent.

Keyword: Diterpene alcohol Cyperus rotundus Apoptosis HeLa Bcl-2 Bax

Citation:

Susianti S, Yanwirasti Y, Darwin E, Jamsari J, Setiawansyah A. Diterpene alcohol fraction of Cyperus rotundus Linn essential oil regulates Bcl-2 and Bax expression inducing apoptosis on HeLa in vitro and in silico. J Appl Pharm Sci. 2024. Online First. http://doi.org/10.7324/JAPS.2024.188231

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Reference

1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of incidence and mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49. https://doi.org/10.3322/caac.21660
2. Singh N, Kumar A. Insights into Ovarian Cancer: chemo-diversity, dose depended toxicities and survival responses. Med Oncol. 2023;40(4):111. Available from: https://doi.org/10.1007/s12032-023-01976-0
3. Jasrotia R, Dhanjal DS, Bhardwaj S, Sharma P, Chopra C, Singh R, et al. Nanotechnology based vaccines: cervical cancer management and perspectives. J Drug Deliv Sci Technol. 2022;71:103351.Available from: https://www.sciencedirect.com/science/article/pii/S1773224722002611 https://doi.org/10.1016/j.jddst.2022.103351
4. Newman DJ, Cragg GM. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J Nat Prod. 2020 Mar 27;83(3):770-803. Available from: https://doi.org/10.1021/acs.jnatprod.9b01285
5. Farnsworth NR. Screening plants for new medicine. In: Staff S, Wilson E, editors. Biodiversity. Washington, DC: National Academy of Sciences Press; 1988. p. 83-97.
6. Cragg GM, Newman DJ. Plants as a source of anti-cancer agents. J Ethnopharmacol. 2005;100(1):72-9. https://doi.org/10.1016/j.jep.2005.05.011
7. Kamala A, Middha SK, Karigar CS. Plants in traditional medicine with special reference to Cyperus rotundus L.: a eeview. 3 Biotech. 2018;8(3):309. https://doi.org/10.1007/s13205-018-1328-6
8. Essaidi I, Koubaier HBH, Snoussi A, Casabianca H, Chaabouni MM, Bouzouita N. Chemical composition of Cyperus rotundus L. tubers essential oil from the South of Tunisia, antioxidant potentiality and antibacterial activity against foodborne pathogens. J Essent Oil-Bearing Plants. 2014;17(3):522-32. https://doi.org/10.1080/0972060X.2014.895182
9. Rajamanickam M, Rajamanickam A. Analgesic and anti-inflammatory activity of the extracts from Cyperus rotundus Linn Rhizomes. J Appl Pharm Sci. 2016;6(9):197-203. https://doi.org/10.7324/JAPS.2016.60929
10. Singh N, Pandey BR, Verma P, Bhalla M, Gilca M. Phyto-pharmacotherapeutics of Cyperus rotundus Linn. (Motha): an overview. Indian J Nat Prod Resour. 2012;3(4):467-76.
11. Sivapalan SR. Medicinal uses and pharmacological activities of Cyperus rotundus Linn-a review. Int J Sci Res Public. 2013;3(1):2250-3153.
12. Susianti S, Yanwirasti Y, Darwin E, Jamsari. The cytotoxic effects of purpel nutsedge (Cyperus rotundus L.) tuber essential oil on the hela cervical cancer cell line. Paksitan J Biotech. 2016;15(2):1-23.
13. Utami N, Susianti S, Bakri S, Kurniawan B, Setiawansyah A. Cytotoxic activity of Cyperus rotundus L. Rhizome collected from three ecological zones in Lampung-Indonesia Against HeLa Cervical Cancer Cell. J Appl Pharm Sci. 2023;13(10):141-8. 14. Simorangkir D, Masfria M, Harahap U, Satria D. Activity anticancer N-hexane fraction of Cyperus rotundus L. Rhizome to breast cancer MCF-7 cell l;ine. Open Access Maced J Med Sci. 2019;7(22):3904-6. https://doi.org/10.3889/oamjms.2019.530
15. Mannarreddy P, Denis M, Munireddy D, Pandurangan R, Thangavelu KP, Venkatesan K. Cytotoxic effect of Cyperus rotundus Rhizome extract on human cancer cell lines. Biomed Pharmacother. 2017;95:1375-87. https://doi.org/10.1016/j.biopha.2017.09.051
16. Memariani T, Hosseini T, Kamali H, Mohammadi A, Ghorbani M, Shakeri A, et al. Evaluation of the cytotoxic effects of Cyperus longus extract, fractions and its essential oil on the PC3 and MCF7 cancer cell lines. Oncol Lett. 2016;11(2):1353-60. https://doi.org/10.3892/ol.2015.4050
17. Hu QP, Cao XM, Hao DL, Zhang LL. Chemical composition, antioxidant, DNA damage protective, cytotoxic and antibacterial activities of Cyperus rotundus Rhizomes essential oil against foodborne pathogens. Sci Rep. 2017;7(December 2016):1-9. https://doi.org/10.1038/srep45231
18. Wang Q, Yi C, Duan W, Duan Y, Lou J, Zeng G, et al. Two new sesquiterpenoids isolated from Cyperus rotundus L. Nat Prod Commun. 2021;16(2):1934578X2199168. https://doi.org/10.1177/1934578X21991687
19. Jeong SJ, Miyamoto T, Inagaki M, Kim YC, Higuchi R. Rotundines A−C, Three novel Sesquiterpene Alkaloids from Cyperus rotundus. J Nat Prod. 2000;63(5):673-5. https://doi.org/10.1021/np990588r
20. Samariya K, Sarin R. Isolation and identification of flavonoids from Cyperus rotundus Linn. in vivo and in vitro. J Drug Deliv Therap. 2013;3(2):109-13. https://doi.org/10.22270/jddt.v3i2.460
21. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63. https://doi.org/10.1016/0022-1759(83)90303-4
22. Touré BB, Miller-Moslin K, Yusuff N, Perez L, Doré M, Joud C, et al. The role of the acidity of N-Heteroaryl sulfonamides as inhibitors of Bcl-2 family protein-protein interactions. ACS Med Chem Lett. 2013;4(2):186-90. https://doi.org/10.1021/ml300321d
23. Pires DE, Blundell TL, Ascher DB. pkCSM: predicting small-molecule pharmacokinetic properties using graph-based signatures (Theory-How to Enterpret pkCSM Result). pKCSM. 2015;58(9):4066-72. Available from: http://biosig.unimelb.edu.au/pkcsm/theory https://doi.org/10.1021/acs.jmedchem.5b00104
24. Setiawansyah A, Arsul MI, Sukrasno S, Damayanti S, Insanu M, Fidrianny I. Anti-hyperuricemic potential of caryophyllene from Syzygium aromaticum essential oil: SiO2-AgNO3-based column chromatography purification, antioxidant, and Xanthine Oxidase inhibitory activities. Adv Trad Med. 2024;24:475-87. https://doi.org/10.1007/s13596-023-00710-5
25. Sayed HM, Mohamed MH, Farag SF, Mohamed GA, Proksch P. A new steroid glycoside and furochromones from Cyperus rotundus L. Nat Prod Res. 2007 Apr;21(4):343-50. https://doi.org/10.1080/14786410701193056
26. Kilani S, Ledauphin J, Bouhlel I, Ben Sghaier M, Boubaker J, Skandrani I, et al. Comparative study of Cyperus rotundus essential oil by a modified GC/MS analysis method. Evaluation of its antioxidant, cytotoxic, and apoptotic effects. Chem Biodivers. 2008;5(5):729-42. https://doi.org/10.1002/cbdv.200890069
27. Susianti S. Selektivitas Ekstrak Umbi Rumput Teki (Cyperus rotundus L.) Terhadap Sel HeLa dan SiHa Serta Pengaruhnya Terhadap Apoptosis. Yogyakarta, Indonesia: Universitas Gadjah Mada; 2009.
28. Gautam N, Mantha AK, Mittal S. Essential oils and their constituents as anticancer agents: a mechanistic view. BioMed Res Int. 2014;24:154106-29. https://doi.org/10.1155/2014/154106
29. Mohamed Abdoul-Latif F, Ainane A, Houmed Aboubaker I, Mohamed J, Ainane T. Exploring the potent anticancer activity of essential oils and their bioactive compounds: mechanisms and prospects for future cancer therapy. Pharmaceuticals. 2023;16(8):1086-112. https://doi.org/10.3390/ph16081086
30. Huang SY, Grinter SZ, Zou X. Scoring functions and their evaluation methods for protein-ligand docking recent advances and future directions. Phys Chem Chem Phys. 2010;12(40):12899-908. https://doi.org/10.1039/c0cp00151a
31. Mukherjee S, Balius TE, Rizzo RC. Docking validation resources: protein family and ligand flexibility experiments. J Chem Inf Model. 2010;50(11):1986-2000. https://doi.org/10.1021/ci1001982
32. Setiawansyah A, Reynaldi MA, Tjahjono DH, Sukrasno S. Molecular docking-based virtual screening of antidiabetic agents from Songga (Strychnos lucida R.Br.): an Indonesian Native Plant. Curr Res Biosci Biotech. 2022;3(2):208-14. https://doi.org/10.5614/crbb.2022.3.2/82KYTCPW
33. Reynaldi MA, Setiawansyah A. Potensi anti-Kanker Payudara Tanaman Songga (Strychnos lucida R. Br): Tinjauan Interaksi Molekuler Terhadap Reseptor Estrogen-α In Silico. Sasambo J Pharm. 2022;3(1):30-5. https://doi.org/10.29303/sjp.v3i1.149
34. Setiawansyah A, Arsul MI, Adliani N, Wismayani L. HMG-CoA reductase inhibitory activity potential of Iota-, Kappa-, and Lambda-carrageenan: a molecular docking approach. Ad-Dawaa' J Pharm Sci. 2022;5(2):94-102. https://doi.org/10.24252/djps.v5i2.32721
35. Pérez MAC, Sanz MB, Torres LR, Avalos RG, González MP, Díaz HG. A topological sub-structural approach for predicting human intestinal absorption of drugs. Eur J Med Chem. 2004;39(11):905-16. https://doi.org/10.1016/j.ejmech.2004.06.012
36. Issa NT, Wathieu H, Ojo A, Byers SW, Dakshanamurthy S. Drug metabolism in preclinical drug development: a survey of the discovery process, toxicology, and computational tools. Curr Drug Metab. 2017;18(6):556-65. https://doi.org/10.2174/1389200218666170316093301
37. Durán-Iturbide NA, Díaz-Eufracio BI, Medina-Franco JL. In Silico ADME/Tox profiling of natural products: a focus on BIOFACQUIM. ACS Omega. 2020;5(26):16076-84. https://doi.org/10.1021/acsomega.0c01581
38. Doogue MP, Polasek TM. Drug dosing in renal disease. Clin Biochem Rev. 2011;32(2):69-73.
39. Paine SW, Ménochet K, Denton R, McGinnity DF, Riley RJ. Prediction of human renal clearance from preclinical species for a diverse set of drugs that exhibit both active secretion and net reabsorption. Drug Metab Dispos. 2011 Jun;39(6):1008-13. https://doi.org/10.1124/dmd.110.037267
40. Hacker K, Maas R, Kornhuber J, Fromm MF, Zolk O. Substrate-dependent inhibition of the human organic cation transporter OCT2: a comparison of metformin with experimental substrates. PLoS One. 2015;10(9):e0136451. https://doi.org/10.1371/journal.pone.0136451

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1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of incidence and mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49. https://doi.org/10.3322/caac.21660

2. Singh N, Kumar A. Insights into Ovarian Cancer: chemo-diversity, dose depended toxicities and survival responses. Med Oncol. 2023;40(4):111. Available from: https://doi.org/10.1007/s12032-023-01976-0

3. Jasrotia R, Dhanjal DS, Bhardwaj S, Sharma P, Chopra C, Singh R, et al. Nanotechnology based vaccines: cervical cancer management and perspectives. J Drug Deliv Sci Technol. 2022;71:103351.Available from: https://www.sciencedirect.com/science/article/pii/S1773224722002611 https://doi.org/10.1016/j.jddst.2022.103351

4. Newman DJ, Cragg GM. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J Nat Prod. 2020 Mar 27;83(3):770-803. Available from: https://doi.org/10.1021/acs.jnatprod.9b01285

5. Farnsworth NR. Screening plants for new medicine. In: Staff S, Wilson E, editors. Biodiversity. Washington, DC: National Academy of Sciences Press; 1988. p. 83-97.

6. Cragg GM, Newman DJ. Plants as a source of anti-cancer agents. J Ethnopharmacol. 2005;100(1):72-9. https://doi.org/10.1016/j.jep.2005.05.011

7. Kamala A, Middha SK, Karigar CS. Plants in traditional medicine with special reference to Cyperus rotundus L.: a eeview. 3 Biotech. 2018;8(3):309. https://doi.org/10.1007/s13205-018-1328-6

8. Essaidi I, Koubaier HBH, Snoussi A, Casabianca H, Chaabouni MM, Bouzouita N. Chemical composition of Cyperus rotundus L. tubers essential oil from the South of Tunisia, antioxidant potentiality and antibacterial activity against foodborne pathogens. J Essent Oil-Bearing Plants. 2014;17(3):522-32. https://doi.org/10.1080/0972060X.2014.895182

9. Rajamanickam M, Rajamanickam A. Analgesic and anti-inflammatory activity of the extracts from Cyperus rotundus Linn Rhizomes. J Appl Pharm Sci. 2016;6(9):197-203. https://doi.org/10.7324/JAPS.2016.60929

10. Singh N, Pandey BR, Verma P, Bhalla M, Gilca M. Phyto-pharmacotherapeutics of Cyperus rotundus Linn. (Motha): an overview. Indian J Nat Prod Resour. 2012;3(4):467-76.

11. Sivapalan SR. Medicinal uses and pharmacological activities of Cyperus rotundus Linn-a review. Int J Sci Res Public. 2013;3(1):2250-3153.

12. Susianti S, Yanwirasti Y, Darwin E, Jamsari. The cytotoxic effects of purpel nutsedge (Cyperus rotundus L.) tuber essential oil on the hela cervical cancer cell line. Paksitan J Biotech. 2016;15(2):1-23.

13. Utami N, Susianti S, Bakri S, Kurniawan B, Setiawansyah A. Cytotoxic activity of Cyperus rotundus L. Rhizome collected from three ecological zones in Lampung-Indonesia Against HeLa Cervical Cancer Cell. J Appl Pharm Sci. 2023;13(10):141-8. 14. Simorangkir D, Masfria M, Harahap U, Satria D. Activity anticancer N-hexane fraction of Cyperus rotundus L. Rhizome to breast cancer MCF-7 cell l;ine. Open Access Maced J Med Sci. 2019;7(22):3904-6. https://doi.org/10.3889/oamjms.2019.530

15. Mannarreddy P, Denis M, Munireddy D, Pandurangan R, Thangavelu KP, Venkatesan K. Cytotoxic effect of Cyperus rotundus Rhizome extract on human cancer cell lines. Biomed Pharmacother. 2017;95:1375-87. https://doi.org/10.1016/j.biopha.2017.09.051

16. Memariani T, Hosseini T, Kamali H, Mohammadi A, Ghorbani M, Shakeri A, et al. Evaluation of the cytotoxic effects of Cyperus longus extract, fractions and its essential oil on the PC3 and MCF7 cancer cell lines. Oncol Lett. 2016;11(2):1353-60. https://doi.org/10.3892/ol.2015.4050

17. Hu QP, Cao XM, Hao DL, Zhang LL. Chemical composition, antioxidant, DNA damage protective, cytotoxic and antibacterial activities of Cyperus rotundus Rhizomes essential oil against foodborne pathogens. Sci Rep. 2017;7(December 2016):1-9. https://doi.org/10.1038/srep45231

18. Wang Q, Yi C, Duan W, Duan Y, Lou J, Zeng G, et al. Two new sesquiterpenoids isolated from Cyperus rotundus L. Nat Prod Commun. 2021;16(2):1934578X2199168. https://doi.org/10.1177/1934578X21991687

19. Jeong SJ, Miyamoto T, Inagaki M, Kim YC, Higuchi R. Rotundines A−C, Three novel Sesquiterpene Alkaloids from Cyperus rotundus. J Nat Prod. 2000;63(5):673-5. https://doi.org/10.1021/np990588r

20. Samariya K, Sarin R. Isolation and identification of flavonoids from Cyperus rotundus Linn. in vivo and in vitro. J Drug Deliv Therap. 2013;3(2):109-13. https://doi.org/10.22270/jddt.v3i2.460

21. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63. https://doi.org/10.1016/0022-1759(83)90303-4

22. Touré BB, Miller-Moslin K, Yusuff N, Perez L, Doré M, Joud C, et al. The role of the acidity of N-Heteroaryl sulfonamides as inhibitors of Bcl-2 family protein-protein interactions. ACS Med Chem Lett. 2013;4(2):186-90. https://doi.org/10.1021/ml300321d

23. Pires DE, Blundell TL, Ascher DB. pkCSM: predicting small-molecule pharmacokinetic properties using graph-based signatures (Theory-How to Enterpret pkCSM Result). pKCSM. 2015;58(9):4066-72. Available from: http://biosig.unimelb.edu.au/pkcsm/theory https://doi.org/10.1021/acs.jmedchem.5b00104

24. Setiawansyah A, Arsul MI, Sukrasno S, Damayanti S, Insanu M, Fidrianny I. Anti-hyperuricemic potential of caryophyllene from Syzygium aromaticum essential oil: SiO2-AgNO3-based column chromatography purification, antioxidant, and Xanthine Oxidase inhibitory activities. Adv Trad Med. 2024;24:475-87. https://doi.org/10.1007/s13596-023-00710-5

25. Sayed HM, Mohamed MH, Farag SF, Mohamed GA, Proksch P. A new steroid glycoside and furochromones from Cyperus rotundus L. Nat Prod Res. 2007 Apr;21(4):343-50. https://doi.org/10.1080/14786410701193056

26. Kilani S, Ledauphin J, Bouhlel I, Ben Sghaier M, Boubaker J, Skandrani I, et al. Comparative study of Cyperus rotundus essential oil by a modified GC/MS analysis method. Evaluation of its antioxidant, cytotoxic, and apoptotic effects. Chem Biodivers. 2008;5(5):729-42. https://doi.org/10.1002/cbdv.200890069

27. Susianti S. Selektivitas Ekstrak Umbi Rumput Teki (Cyperus rotundus L.) Terhadap Sel HeLa dan SiHa Serta Pengaruhnya Terhadap Apoptosis. Yogyakarta, Indonesia: Universitas Gadjah Mada; 2009.

28. Gautam N, Mantha AK, Mittal S. Essential oils and their constituents as anticancer agents: a mechanistic view. BioMed Res Int. 2014;24:154106-29. https://doi.org/10.1155/2014/154106

29. Mohamed Abdoul-Latif F, Ainane A, Houmed Aboubaker I, Mohamed J, Ainane T. Exploring the potent anticancer activity of essential oils and their bioactive compounds: mechanisms and prospects for future cancer therapy. Pharmaceuticals. 2023;16(8):1086-112. https://doi.org/10.3390/ph16081086

30. Huang SY, Grinter SZ, Zou X. Scoring functions and their evaluation methods for protein-ligand docking recent advances and future directions. Phys Chem Chem Phys. 2010;12(40):12899-908. https://doi.org/10.1039/c0cp00151a

31. Mukherjee S, Balius TE, Rizzo RC. Docking validation resources: protein family and ligand flexibility experiments. J Chem Inf Model. 2010;50(11):1986-2000. https://doi.org/10.1021/ci1001982

32. Setiawansyah A, Reynaldi MA, Tjahjono DH, Sukrasno S. Molecular docking-based virtual screening of antidiabetic agents from Songga (Strychnos lucida R.Br.): an Indonesian Native Plant. Curr Res Biosci Biotech. 2022;3(2):208-14. https://doi.org/10.5614/crbb.2022.3.2/82KYTCPW

33. Reynaldi MA, Setiawansyah A. Potensi anti-Kanker Payudara Tanaman Songga (Strychnos lucida R. Br): Tinjauan Interaksi Molekuler Terhadap Reseptor Estrogen-α In Silico. Sasambo J Pharm. 2022;3(1):30-5. https://doi.org/10.29303/sjp.v3i1.149

34. Setiawansyah A, Arsul MI, Adliani N, Wismayani L. HMG-CoA reductase inhibitory activity potential of Iota-, Kappa-, and Lambda-carrageenan: a molecular docking approach. Ad-Dawaa' J Pharm Sci. 2022;5(2):94-102. https://doi.org/10.24252/djps.v5i2.32721

35. Pérez MAC, Sanz MB, Torres LR, Avalos RG, González MP, Díaz HG. A topological sub-structural approach for predicting human intestinal absorption of drugs. Eur J Med Chem. 2004;39(11):905-16. https://doi.org/10.1016/j.ejmech.2004.06.012

36. Issa NT, Wathieu H, Ojo A, Byers SW, Dakshanamurthy S. Drug metabolism in preclinical drug development: a survey of the discovery process, toxicology, and computational tools. Curr Drug Metab. 2017;18(6):556-65. https://doi.org/10.2174/1389200218666170316093301

37. Durán-Iturbide NA, Díaz-Eufracio BI, Medina-Franco JL. In Silico ADME/Tox profiling of natural products: a focus on BIOFACQUIM. ACS Omega. 2020;5(26):16076-84. https://doi.org/10.1021/acsomega.0c01581

38. Doogue MP, Polasek TM. Drug dosing in renal disease. Clin Biochem Rev. 2011;32(2):69-73.

39. Paine SW, Ménochet K, Denton R, McGinnity DF, Riley RJ. Prediction of human renal clearance from preclinical species for a diverse set of drugs that exhibit both active secretion and net reabsorption. Drug Metab Dispos. 2011 Jun;39(6):1008-13. https://doi.org/10.1124/dmd.110.037267

40. Hacker K, Maas R, Kornhuber J, Fromm MF, Zolk O. Substrate-dependent inhibition of the human organic cation transporter OCT2: a comparison of metformin with experimental substrates. PLoS One. 2015;10(9):e0136451. https://doi.org/10.1371/journal.pone.0136451