Open Access
Research (Published online: 11-09-2018)
6. The neuroprotective effect of Ocimum sanctum Linn. ethanolic extract on human embryonic kidney-293 cells as in vitro model of neurodegenerative disease
Puspa Hening, Made Bagus Auriva Mataram, Nastiti Wijayanti, Dwi Liliek Kusindarta and Hevi Wihadmadyatami
Veterinary World, 11(9): 1237-1243

Puspa Hening: Research Center of Biotechnology, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.
Made Bagus Auriva Mataram: Department of Anatomy, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.
Nastiti Wijayanti: Department of Animal Physiology, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.
Dwi Liliek Kusindarta: Department of Anatomy, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.
Hevi Wihadmadyatami: Department of Anatomy, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.

doi: 10.14202/vetworld.2018.1237-1243

Share this article on [Facebook] [LinkedIn]

Article history: Received: 13-05-2018, Accepted: 24-07-2018, Published online: 11-09-2018

Corresponding author: Hevi Wihadmadyatami


Citation: Hening P, Mataram MBA, Wijayanti N, Kusindarta DL, Wihadmadyatami H (2018) The neuroprotective effect of Ocimum sanctum Linn. ethanolic extract on human embryonic kidney-293 cells as in vitro model of neurodegenerative disease, Veterinary World 11(9): 1237-1243.

Aim: This study aimed to analyze the neuroprotective effect of Ocimum sanctum Linn. ethanolic extract (OSE) on human embryonic kidney-293 (HEK-293) cells as the in vitro model of neurodegenerative diseases.

Materials and Methods: In this research, HEK-293 cells divided into five groups consisting of normal and healthy cells (NT), cells treated with Camptothecin 500 μM as the negative control, cells treated with trimethyltin 10 μM (TMT), cells treated with OSE 75 μg/ml, and cells pre-treated with OSE 75 μg/ml then induced by TMT 10 μM (OSE+TMT). MTT assay and phase contrast microscopy were applied to observe the cell viability quantitatively and morphological after Ocimum sanctum Linn extract treatment. Finally, the reverse transcription polymerase chain reaction was employed to study the expression of choline acetyltransferase (ChAT).

Results: The MTT assay and phase contrast microscopy showed that OSE pre-treatment significantly increased the viability of TMT-induced apoptotic cells and maintained cell viability of the normal HEK-293 cells. Expression of ChAT markedly reduced on TMT treatment group, but OSE administration stabilized ChAT expression in TMT-induced HEK-293 cells.

Conclusion: This present study proved that OSE administration has neuroprotective effect by increased HEK-293 cells viability and maintain ChAT expression.

Keywords: choline acetyltransferase, human embryonic kidney-293, neurodegenerative diseases, Ocimum sanctum Linn. ethanolic extract.


1. Crankshaw C.L. (2012) Neurodegenerative diseases. Biofiles Sigma-Aldrich, 7(2): 1-22.

2. Mattson, M.P. (2000) Apoptosis in neurodegenerative disorders. Nat. Rev. Mol. Cell Biol., 1: 120-129. [Crossref] [PubMed]

3. Olanow, C.W., Kordower, J.H., Lang, A.E., Obeso, J.A. (2009) Dopaminergic transplantation for Parkinson's disease: Current status and future prospects. Ann. Neurol., 66(5): 591-596. [Crossref] [PubMed]

4. Winblad, B., Palmer, K., Kivipelto, M., Jelic, V., Fratiglioni, L., Wahlund, L.O., Nordberg, A., Backman, L., Albert, M., Almkvist, O., Arai, H., Basun, H., Blennow, K., de Leon, M., DeCarli, C., Erkinjuntti, T., Giacobini, E., Graff, C., Hardy, J., Jack, C., Jorm, A., Ritchie, K., van Duijn, C., Visser, P., Petersen, R.C. (2004) Mild cognitive impairment - beyond controversies, towards a consensus: Report of the International Working Group on Mild Cognitive Impairment. J. Intern. Med., 256(3): 240-246. [Crossref] [PubMed]

5. Modgil, S., Lahiri, D.K., Sharma, V.L., Anand, A. (2014) Role of early life exposure and environment on neurodegeneration: Implications on brain disorders. Transl. Neurodegener., 3(1): 9. [Crossref] [PubMed] [PMC]

6. Resende, R.R., Adhikari, A. (2009) Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation. Cell Commun. Signal., 7(1): 20. [Crossref]

7. Hasselmo, M.E. (2006) The role of acetylcholine in learning and memory. Curr. Opin. Neurobiol., 16(6): 710-715. [Crossref] [PubMed] [PMC]

8. Kasa, P., Rakonczay, Z., Gulya, K. (1997) The cholinergic system in Alzheimer's disease. Prog. Neurobiol,. 52(6): 511-535. [Crossref]

9. Terry Jr. A.V., Buccafusco, J.J. (2003) The cholinergic hypothesis of age and Alzheimer's disease-related cognitive deficits: Recent challenges and their implications for novel drug development. J. Pharm. Exp. Ther., 306(3): 821-827. [Crossref] [PubMed]

10. Nunes-Tavares, N., Santos, L.E., Stutz, B., Brito-Moreira, J., Klein, W.L., Ferreira, S.T, de Mello, F.G. (2012) Inhibition of choline acetyltransferase as a mechanism for cholinergic dysfunction induced by amyloid-β peptide oligomers. J. Biol. Chem., 287(23): 19377-19385. [Crossref] [PubMed] [PMC]

11. Oda, Y. (1999) Choline acetyltransferase: The structure, distribution and pathologic changes in the central nervous system. Pathol. Int., 49: 921-937. [Crossref]

12. Kadian, R., Parle, M. (2012) Therapeutic potential and phytopharmacology of tulsi. Int. J. Pharm. Life Sci., 3(7): 1858-1867.

13. Gupta, S.K., Prakash, J., Srivastava, S. (2002) Validation of traditional claim of Tulsi, Ocimum sanctum Linn. As a medicinal plant. Indian J. Exp. Biol., 40(7): 765-773. [PubMed]

14. Ramesh, B., Satakopan, V.N. (2010) Antioxidant activities of hydroalcoholic extract of Ocimum sanctum against cadmium-induced toxicity in rats. Indian J. Clin. Biochem., 25(3): 307-310. [Crossref] [PubMed] [PMC]

15. Elmi, A., Ventrella, D., Barone, F., Filippini, G., Benvenuti, S., Pisi, A., Scozzoli, M, Bacci, M.L. (2017) Thymbra capitata (L.) Cav. and Rosmarinus officinalis (L.) essential oils: In vitro effects and toxicity on swine spermatozoa. Molecules., 22: 2162. [Crossref]

16. Zaker, A., Asili, J., Abrishamchi, P., Tayarani-najaran, Z., Hadi, S. (2017) Cytotoxic and apoptotic effects of root extract and tanshinones isolated from Perovskia abrotanoides Kar. Iran. J. Basic Med. Sci., 2020(20): 1377-1384.

17. Gunes-Bayir, A., Kocyigit, A., Guler, E.M. (2018) In vitro effects of two major phenolic compounds from the family Lamiaceae plants on the human gastric carcinoma cells. Toxicol. Ind. Health., 20(10): 1-15. [Crossref]

18. Venuprasad, M.P., Kumar, H., Razack, S., Amruta, N., Khanum, F. (2017) Chemical composition of Ocimum sanctum by LC-ESI - MS / MS analysis and its protective effects against smoke-induced lung and neuronal tissue damage in rats. Biomed. Pharmacother., 91: 1-12. [Crossref]

19. Joshi, H., Parle, M. (2006) Evaluation of nootropic potential of Ocimum sanctum Linn. In Mice. Indian J. Exp. Biol., 44(2): 133-136. [PubMed]

20. Kusindarta, D.L., Wihadmadyatami, H., Haryanto, A. (2018) The analysis of hippocampus neuronal density (CA1 and CA3) after Ocimum sanctum ethanolic extract treatment on the young adulthood and middle age rat model. Vet. World., 11: 135-140. [Crossref]

21. Kusindarta, D.L., Wihadmadyatami, H., Haryanto, A. (2016) Ocimum sanctum Linn. Stimulate the expression of choline acetyltransferase on the human cerebral microvascular endothelial cells. Vet. World., 9(12): 1348-1354. [Crossref] [PubMed] [PMC]

22. Venuprasad, M.P., Kandikattu, H.K., Khanum, F. (2013) Neuroprotective effects of hydroalcoholic extract of Ocimum sanctum against H2O2 induced neuronal cell damage in SH-SY5Y cells via its antioxidative defence mechanism. Neurochem. Res., 38: 2190-2200. [Crossref]

23. Shaw, G., Morse, S., Ararat, M., Graham, F.L. (2002) Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol., 16(8): 869-871. [Crossref]

24. Thomas, P., Smart, T.G. (2005) HEK293 cell line: A vehicle for the expression of recombinant proteins. J. Pharmacol. Toxicol. Methods., 51(3): 187-200. [Crossref] [PubMed]

25. Geloso, M.C., Corvino, V., Michetti, F. (2011) Trimethyltin-induced hippocampal degeneration as a tool to investigate neurodegenerative processes. Neurochem. Int., 58(7): 729-738. [Crossref] [PubMed]

26. Kusindarta, D.L., Wihadmadyatami, H., Jadi, A.R., Karnati, S., Lochnit, G., Hening, P., Haryanto, A., Auriva, M.B., Purwaningrum, M. (2018) Ethanolic extract Ocimum sanctum. Enhances cognitive ability from young adulthood to middle-aged mediated by increasing choline acetyltransferase activity in rat model. Res. Vet. Sci., 118: 431-438. [Crossref] [PubMed]

27. Schirmer, S. (2011) Expression Profile of Components of the Acetylcholine - System in Rat Testicular Tissue and Function In Non-Germ Cell Populations. Dissertation. Justus-Liebig-Universitat Giessen, Giessen.

28. Imai, K., Kamio, N., Cueno, M.E., Saito, Y., Inoue, H., Saito, I., Ochiai, K. (2014) Role of the histone H3 lysine 9 methyltransferase Suv39 h1 in maintaining Epstein-Barr virus latency in B95-8 cells. FEBS J., 281(9): 2148-2158. [Crossref] [PubMed]

29. Choi, B.S., Sapkota, K., Kim, S., Lee, H.J., Choi, H.S., Kim, S.J. (2010) Antioxidant activity and protective effects of Tripterygium regelii extract on hydrogen peroxide-induced injury in human dopaminergic cells, SH-SY5Y. Neurochem. Res., 35(8): 1269-1280. [Crossref] [PubMed]

30. Uma, D. P. (2001) Radioprotective, anticarcinogenic and antioxidant properties of the Indian holy basil, Ocimum sanctum (Tulasi). Indian J. Exp. Biol., 39(3): 185-190.

31. Jagetia, C.G. (2007) Radioprotective potential of plants and herbs against the effects of ionizing radiation. J. Clin. Biochem. Nutr.,40(2): 74-81. [Crossref] [PubMed] [PMC]

32. Adhvaryu, M.R., Reddy, N., Parabia, M.H. (2008) Anti-tumor activity of four Ayurvedic herbs in Dalton lymphoma ascites bearing mice and their short-term in vitro cytotoxicity on DLA-cell-line. Afr. J. Tradit. Complement Altern. Med., 5(4): 409-418. [Crossref]

33. Bader, S., Klein, J., Diener, M. (2014) Choline acetyltransferase and organic cation transporters are responsible for synthesis and propionate-induced release of acetylcholine in colon epithelium. Eur. J. Pharmacol., 733: 23-33. [Crossref] [PubMed]

34. Lips, K.S., Luhrmann, A., Tschernig, T., Stoeger, T., Alessandrini, F., Grau, V., Haberberger, R.V., Koepsell, H., Pabst, R., Kummer, W. (2007) Down-regulation of the non-neuronal acetylcholine synthesis and release machinery in acute allergic airway inflammation of rat and mouse. Life Sci., 80(24-25): 2263-2269. [Crossref] [PubMed]

35. Hussmann, G.P., Yasuda, R.P., Xiao, Y., Wolfe, B.B., Kellar, K.J. (2011) Endogenously expressed muscarinic receptors in HEK293 cells augment up-regulation of stably expressed α4β2 nicotinic receptors. J. Biol. Chem., 286(46): 39726-39737. [Crossref] [PubMed] [PMC]

36. Kim, D.J., Kim, Y.S. (2016) Magnolol protects against trimethyltin-induced neuronal damage and glial activation in vitro and in vivo. Neurotoxicology., 53: 173-185. [Crossref] [PubMed]

37. Gunasekar, P., Li, L., Prabhakaran, K., Eybl, V., Borowitz, J.L., Isom, G.E. (2001) Mechanisms of the apoptotic and necrotic actions of trimethyltin in cerebellar granule cells. Toxicol. Sci., 64(1): 83-89. [Crossref]

38. Shin, E.J., Suh, S.K., Lim, Y.K., Jhoo, W.K., Hjelle, O.P., Ottersen, O.P, Shin, C.Y., Ko, K.H., Kim, W.K., Kim, D.S., Chun, W., Ali, S., Kim, H.C. (2005) Ascorbate attenuates trimethyltin-induced oxidative burden and neuronal degeneration in the rat hippocampus by maintaining glutathione homeostasis. Neurosci., 133(3): 715-727. [Crossref] [PubMed]

39. Martin, S., Gonzalez-Burgos, E., Carretero, M.E., Gomez-Serranillos, M.P. (2011) Neuroprotective properties of Spanish red wine and its isolated polyphenols on astrocytes. Food Chem., 128(1): 40-48. [Crossref] [PubMed]

40. Grando, S.A. (1997) Biological functions of keratinocyte cholinergic receptors. J. Invest. Dermatol. Symp. Proc., 2(1): 41-48. [Crossref]

41. Kawashima, K., Fujii, T. (2000) Extraneuronal cholinergic system in lymphocytes. Pharm. Ther., 86(1): 29-48. [Crossref]

42. Resende, R.R., Alves, A.S., Britto, L.R.G., Ulrich, H. (2008) Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells. Exp. Cell. Res., 314(7): 1429-1443. [Crossref] [PubMed]

43. Tobin, A.B., Budd, D.C. (2003) The anti-apoptotic response of the Gq/11-coupled muscarinic receptor family. Biochem. Soc. Trans., 31(6): 1182-1185. [Crossref]

44. Wessler, I., Kirkpatrick, C.J., Racke, K. (1998) Non-neuronal acetylcholine, a locally acting molecule, widely distributed in biological systems: Expression and function in humans. Pharm. Ther., 77(1): 59-79. [Crossref]