Cytotoxic Evaluation of Malaysian Kelulut Honey on Human Gingival Fibroblast Cell Line using MTT Assay

Authors

Chee Zi Yun, School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, MalaysiaFollow
Nurul Hafizah Mohd Nor, School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, MalaysiaFollow
Zurairah Berahim, School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, MalaysiaFollow
Kannan Thirumulu Ponnuraj, School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia; Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, MalaysiaFollow

Abstract

Kelulut honey or stingless bee honey is a type of honey produced by stingless bees of the Trigona species where the nest is found in living trees.

Objective: The aim of this study was to evaluate the cytotoxic potential of Malaysian Kelulut honey by employing MTT assay on a human gingival fibroblast cell line.

Methods:Human gingival fibroblast cell line was cultured in minimal essential medium alpha (α-MEM) with 10% foetal bovine serum and 1% penicillin-streptomycin solution in a 5% CO2 incubator at 37°C in a humidified atmosphere. The cells were seeded at a cell density of 5x103 cells/well in a 96-well culture plate for 24 hours. The cells were treated with seven different concentrations (200, 100, 50, 25, 12.5, 6.25, and 3.125mg/ml) of Malaysian Kelulut honey and incubated in a CO2 incubator. The negative control comprised cells treated with growth media alone. The cell viability was assessed using MTT assay at 24, 48, and 72 hours. The test plate was shaken using a microplate shaker and the absorbance of the solution was measured at 570nm using an ELISA reader with the Magellan software. Statistical analysis of the data was carried out using Kruskal-Wallis test and SPSS 24.0.0 for Windows. A p value

Results: There was no cytotoxic effect of Malaysian Kelulut honey on HGF-1 based on the MTT assay at different concentrations and at different time points tested as the cell viability was above 70%. The highest percentage of cell viability at all three different durations of treatment were observed at 3.125mg/ml, whereas the lowest cell viability was observed at 200mg/ml of Kelulut honey concentration. However, statistically significant differences were seen between some of the concentrations at various time points.

Conclusion: Since the cell viability of HGF-1 treated with Malaysian Kelulut honey was more than 70% at all concentrations ranging from 3.125mg/ml to 200mg/ml at three different time points (24, 48 and 72 hours), Malaysian Kelulut honey can be considered as non-cytotoxic on human gingival fibroblasts based on MTT assay under the present test conditions.

References

1. Kek SP, Chin NL, Yusof YA, Tan, Chua LS. Total phenolic contents and colour intensity of Malaysian honeys from the Apis spp. and Trigona spp. Bees. Agric Agric Sci Procedia. 2014;2:150–5.
2. Zainol MI, Mohd Yusoff K, Mohd Yusof MY. Antibacterial activity of selected Malaysian honey. BMC Complement Altern Med. 2013;13:129.
3. Sabir A, Tabbu CR, Agustiono P, Sosroseno W. Histological analysis of rat dental pulp tissue capped with propolis. J Oral Sci. 2005;47(3):135–8.
4. Soenen SJ, Manshian B, Montenegro JM, Amin F, Meermann B, Thiron T. Cytotoxic effects of gold nanoparticles: a multi-parametric study. ACS Nano. 2012;6(7):5767-583.
5. Damas BA, Wheater MA, Bringas JS, Hoen MM. Cytotoxicity comparison of mineral trioxide aggregates and EndoSequence bio-ceramic root repair materials. J Endod. 2011;37(3):372-5.
6. Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C. Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles. Comparison with conventional monocultures. Part Fibre Toxicol. 2011;8(1):1-16.
7. Uboldi C, Giudetti G, Broggi F, Gilliland D, Ponti J, Rossi F. Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts. Mutat Res Genet Toxicol Environ Mutagen. 2012;745(1):11-20.
8. Wang S, Yu H, Wickliffe JK. Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nanoscale TiO2. Toxicol In Vitro. 2011;25(8):2147-51.
9. Buch K, Peters T, Nawroth T, Sänger M, Schmidberger H, Langguth P. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay-A comparative study. Radiat Oncol. 2012;7(1):1-6.
10. Mendes LP, Delgado JMF, Costa ADA, Vieira MS, Benfica PL, Lima EM. Biodegradable nanoparticles designed for drug delivery: The number of nanoparticles impacts on cytotoxicity. Toxicol In Vitro. 2015;29(6):1268-74.
11. Stepanenko A, Dmitrenko V. Pitfalls of the MTT assay: direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene. 2015;574(2):193-203.
12. Madu Kelulut Syamille – Terbaik untuk anda. Viewed 19 July 2021. http://kelulutmalaysia. blogspot.com/.
13. 13. Al-Hatamleh MAI, Boer JC, Wilson KL, Plebanski M, Mohamud R, Mustafa MZ. Antioxidant-based medicinal properties of stingless bee products: recent progress and future directions. Biomolecules. 2020;10(6):923.
14. Souza BA, Roubik DW, Barth OM, Heard TA, Enríquez E, Carvalho C, et al. Composition of stingless bee honey: setting quality standards. Interciencia. 2006;31:867–75.
15. ISO 10993-12. Biological evaluation of medical devices - Part 12: Sample preparation and reference materials. 2012;1-20.
16. Kek SP, Chin NL, Yusof YA, Tan SW, Chua LS. Classification of Entomological origin of honey based on its physicochemical and antioxidant properties. Int J Food Prop. 2017;20:2723–38.
17. Vit P, Pedro SR, Roubik D. Pot-Honey: A Legacy of Stingless Bees; Springer: New York, USA. 2013;654.
18. Mohd Fadzelly AB, Shuaibu BS, Fazleen IAB, Ong JC, Zakbah M. Physicochemical and antioxidant potential of raw unprocessed honey from Malaysian stingless bees. Pak J Nutr. 2017;16: 888-94.
19. Lee I, Lee M, Jang H. The interrelationship between human gingival fibroblast differentiation and cultivating time. Tissue Eng Regen Med. 2013;10(2):60–4.
20. Supraja A, Dinesh MG, Rajasekaran S, Balaji TM, Rao SR. Effect of cyclosporin A and angiotensin II on cytosolic calcium levels in primary human gingival fibroblasts. Dent Res J. 2016;13(5):405-12.
21. Giannopoulou C, Cimasoni G. Functional characteristics of gingival and periodontal ligament fibroblasts. J Dent Res. 2011;75(3):895- 902.
22. Bartold PM, Walsh LJ, Narayanan AS. Molecular and cell biology of the gingiva. Periodontol. 2000;24:28-55.
23. Poggi P, Rodriguezy Baena R, Rizzo S, Rota MT. Moouthrinses with alcohol: cytotoxic effects on human gingival fibroblasts in vitro. J Periodontol. 2003;74(5):623-9.
24. Egusa H, Okita K, Kayashima H, Yu G, Fukuyasu S, Saeki M, et al. Gingival fibroblasts as a promising source of induced pluripotent stem cells. PLoS One. 2010;5(9):e12743.
25. Nordin A, Omar N, Sainik NQAV, Chowdhury SR, Omar E, Saim AB, et al. Low dose stingless bee honey increases viability of human dermal fibroblasts that could potentially promote wound healing. Wound Med. 2018;23:22–7.
26. Abdul Malik N, Mohamed M, Mustafa MZ, Zainuddin A. In vitro modulation of extracellular matrix genes by stingless bee honey in cellular aging of human dermal fibroblast cells. J Food Biochem. 2020;44:e13098.
27. Jacob A, Parolia A, Pau A. The effects of Malaysian propolis and Brazilian red propolis on connective tissue fibroblasts in the wound healing process. BMC Complement Altern Med. 2015;15:294.
28. Saiful Yazan L, Muhamad Zali MF, Mohd Ali R, Zainal NA, Esa N, Sapuan S, et al. Chemopreventive properties and toxicity of kelulut honey in sprague dawley rats induced with azoxymethane. BioMed Res Int. 2016;2016:4036926.
29. Pashinskiĭ VG, Gribel NV. The antitumor properties of honey. Vopr Onkol. 1990;36(6):704–9.
30. Kustiawan PM, Puthong S, Arung ET, Chanchao C. In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines. Asian Pac J Top Biomed. 2014;4(7):549–56.
31. Nafi NE, Zin NB, Pauzi N, Khadar AS, Anisava AR, Badiazaman AA, et al. Cytotoxicity, antioxidant and phytochemical screening of propolis extracts from four different Malaysian stingless bee species. Mal J Fund Appl Sci. 2019;307-12.
32. Borrelli F, Izzo AA, Di Carlo G, Maffia P, Russo A, Maiello FM, et al. Effect of a propolis extract and caffeic acid phenethyl ester on formation of aberrant crypt foci and tumors in the rat colon. Fitoterapia 2002;73(1):38–43.
33. He YJ, Liu BH, Xiang DB, Qiao ZY, Fu T, He YH. Inhibitory effect of caffeic acid phenethyl ester on the growth of SW480 colorectal tumor cells involves β-catenin associated signaling pathway down-regulation. World J Gastroenterol. 2006;12(31):4981–5.
34. Orsolić N, Terzić S, Mihaljević Z, Sver L, Basić I. Effects of local administration of propolis and its polyphenolic compounds on tumor formation and growth. Biol Pharm Bull. 2005;28(10):1928–1933.
35. ISO 10993-5. Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity. 2009;1-34.
36. Mohamad MAM, Mazlan MA, Ibrahim M, Yusof AM, Shamsuddin SAA, Hassan NFN, et al. The effect of Malaysian stingless bee, Trigona spp. honey in promoting proliferation of the undifferentiated stem cell. As Pac J Mol Biol Biotechnol. 2018;27(1):10-9

Recommended Citation

Zi Yun, C., Mohd Nor, N., Berahim, Z., & Thirumulu Ponnuraj, K. Cytotoxic Evaluation of Malaysian Kelulut Honey on Human Gingival Fibroblast Cell Line using MTT Assay. J Dent Indones. 2021;28(2): 88-93