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VOLUME 24 , ISSUE 9 ( September, 2023 ) > List of Articles


The Effect of Rosmarinus Officinalis as a Potential Root Canal Medication on the Viability of Dental Pulp Stem Cells

Nesma Mohamed Othman, Yousry Mahmoud Elhawary, Mohamed G Elbeltagy, Amany Elsaid Badr

Keywords : Calcium hydroxide, Cell viability, Dental pulp stem cells, Rosmarinic acid, Triple antibiotic paste

Citation Information : Othman NM, Elhawary YM, Elbeltagy MG, Badr AE. The Effect of Rosmarinus Officinalis as a Potential Root Canal Medication on the Viability of Dental Pulp Stem Cells. J Contemp Dent Pract 2023; 24 (9):623-631.

DOI: 10.5005/jp-journals-10024-3570

License: CC BY-NC 4.0

Published Online: 13-10-2023

Copyright Statement:  Copyright © 2023; The Author(s).


Aim: The objective of the current study was to assess and compare the impact of triple antibiotic paste (TAP) and calcium hydroxide (Ca(OH)2) with rosmarinic acid (RA) on the viability of dental pulp stem cells (DPSCs). Materials and methods: Dental pulp stem cells were isolated and characterized using flow cytometry. The cells were treated with (0.25, 0.5, 1, 2.5, and 5 mg/mL) concentrations for TAP and Ca(OH)2 and (6.25, 12.5, 25, 50, and 100 µM) concentrations for RA. Cell viability was evaluated after 3 days, with cell proliferation further analyzed over 3, 5, and 7 days utilizing the MTT assay. The optical density (OD) was quantified at 570 nm, subsequently enabling the determination of corrected OD and cell viability. ANOVA followed by the post hoc Tuckey test evaluated the statistical significance at p < 0.05. Results: Following the cell viability test, 0.25 and 0.5 mg/mL of TAP and Ca(OH)2 showed no significant difference for DPSCs compared to the control group. While dosages of 1 mg/mL, 2.5 mg/mL, and 5 mg/mL significantly reduced cell viability (p < 0.05). However, 6.25 µM and 12.5 µM concentrations of RA showed a significant increase in cell viability compared to untreated cells, 25 µM and 50 µM concentrations showed no significant difference compared to untreated cells while 100 µM concentration showed a decrease in cell viability (p < 0.05). Moreover, RA at a concentration of 12.5 µM exhibited a significant enhancement in cell proliferation rates after 5 and 7 days. Conclusion: Rosmarinic acid showed a significant increase in cell viability when used at 6.25 and 12.5 µM concentrations compared to TAP and CA(OH)2. Clinical significance: The assessment of cytotoxicity associated with bioactive compounds like RA, which processes antimicrobial and anti-inflammatory properties, holds importance. This evaluation could pave the way for novel intracanal medicaments that enhance the regenerative potential of DPSCs.

  1. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: New treatment protocol? J Endod 2004;30(4):196–200. DOI: 10.1097/00004770-200404000-00003.
  2. Galler KM, D'Souza RN, Federlin M, et al. Dentin conditioning codetermines cell fate in regenerative endodontics. J Endod 2011;37(11):1536–1541. DOI: 10.1016/j.joen.2011.08.027.
  3. Chen MY, Chen KL, Chen CA, et al. Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures. Int Endod J 2012;45(3): 294–305. DOI: 10.1111/j.1365-2591.2011.01978.x.
  4. Jeong MJ, Lim DS, Kim SO, et al. Effect of rosmarinic acid on differentiation and mineralization of MC3T3-E1 osteoblastic cells on titanium surface. Anim Cells Syst (Seoul) 2021;25(1):46–55. DOI: 10.1080/19768354.2021.1886987.
  5. Dias MI, Sousa MJ, Alves RC, et al. Exploring plant tissue culture to improve the production of phenolic compounds: A review. Industrial crops products 2016;82:9–22. DOI:
  6. Elshaer M, Chen Y, Wang XJ, et al. Resveratrol: An overview of its anti-cancer mechanisms. Life Sci 2018;207:340–349. DOI: 10.1016/j.lfs.2018.06.028.
  7. Villalva M, Jaime L, Aguado E, et al. Anti-inflammatory and antioxidant activities from the basolateral fraction of caco-2 cells exposed to a rosmarinic acid enriched extract. J Agric Food Chem 2018;66(5): 1167–1174. DOI: 10.1021/acs.jafc.7b06008.
  8. Markossian S, Grossman A, Brimacombe K, et al. Assay Guidance Manual. In: Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004. Available from:
  9. Verma P, Nosrat A, Kim J, et al. Effect of residual bacteria on the outcome of pulp regeneration in vivo. J Dent Res 2017;96(1):100–106. DOI: 10.1177/0022034516671499.
  10. Ayoub S, Cheayto A, Bassam S, et al. The effects of intracanal irrigants and medicaments on dental-derived stem cells fate in regenerative endodontics: An update. Stem Cell Rev Rep 2020;16(4):650–660. DOI: 10.1007/s12015-020-09982-9.
  11. Berkhoff JA, Chen PB, Teixeira FB, et al. Evaluation of triple antibiotic paste removal by different irrigation procedures. J Endod 2014;40(8):1172–1177. DOI: 10.1016/j.joen.2013.12.027.
  12. Oktay EA, Ersahan S, Gokyay S. Effect of intracanal medicaments used in endodontic regeneration on the push-out bond strength of a calcium-phosphate-silicate-based cement to dentin. Pak J Med Sci 2018;34(2):310–315. DOI: 10.12669/pjms.342.14630.
  13. Palombo EA. Traditional medicinal plant extracts and natural products with activity against oral bacteria: Potential application in the prevention and treatment of oral diseases. Evid Based Complement Alternat Med 2011;2011:680354. DOI: 10.1093/ecam/nep067.
  14. Rašković A, Milanović I, Pavlović N, et al. Antioxidant activity of rosemary (Rosmarinus officinalis L.) essential oil and its hepatoprotective potential. BMC Complement Altern Med 2014;14(1):1–9. DOI: 10.1186/1472-6882-14-225.
  15. Guerra-Boone L, Alvarez-Román R, Salazar-Aranda R, et al. Antimicrobial and antioxidant activities and chemical characterization of essential oils of thymus vulgaris, rosmarinus officinalis, and origanum majorana from northeastern México. Pak J Pharm Sci 2015;28(Suppl 1):363–369. PMID: 25631514.
  16. Irshaid FI, Tarawneh KA, Jacob JH, et al. Phenol content, antioxidant capacity and antibacterial activity of methanolic extracts derived from four Jordanian medicinal plants. Pak J Biol Sci 2014;17(3): 372–379. DOI: 10.3923/pjbs.2014.372.379.
  17. Gauch LMR, Silveira-Gomes F, Esteves RA, et al. Effects of Rosmarinus officinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers. Rev Soc Bras Med Trop 2014;47(3): 389–391. DOI: 10.1590/0037-8682-0137-2013.
  18. Abuzeid N, Kalsum S, Koshy RJ, et al. Antimycobacterial activity of selected medicinal plants traditionally used in Sudan to treat infectious diseases. J Ethnopharmacol 2014;157:134–139. DOI: 10.1016/j.jep.2014.09.020.
  19. Rocha J, Eduardo-Figueira M, Barateiro A, et al. Anti-inflammatory effect of rosmarinic acid and an extract of Rosmarinus officinalis in rat models of local and systemic inflammation. Basic Clin Pharmacol Toxicol 2015;116(5):398–413. DOI: 10.1111/bcpt.12335.
  20. Wang W, Li N, Luo M, et al. Antibacterial activity and anticancer activity of Rosmarinus officinalis L. essential oil compared to that of its main components. Molecules 2012;17(3):2704–2713. DOI: 10.3390/molecules17032704.
  21. Motlagh MK, Sharafi M, Zhandi M, et al. Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze–thawing process of ram sperm. Cryobiology 2014;69(2):217–222. DOI:
  22. Felicidade I, Lima J, Pesarini J, et al. Mutagenic and antimutagenic effects of aqueous extract of rosemary (Rosmarinus officinalis L.) on meristematic cells of Allium cepa. Genet Mol Res 2014;13(4):9986–9996. DOI: 10.4238/2014.November.28.3.
  23. Lin CY, Chen JH, Fu RH, et al. Induction of Pi form of glutathione S-transferase by carnosic acid is mediated through PI3K/Akt/NF-κB pathway and protects against neurotoxicity. Chem Res Toxicol 2014;27(11):1958–1966. DOI: 10.1021/tx5003063.
  24. Li XL, Liu JX, Li P, et al. Protective effect of rosmarinic acid on hypoxia/reoxygenation injury in cardiomyocytes. Zhongguo Zhong Yao Za Zhi 2014;39(10):1897–1901. PMID: 25282902.
  25. El-Demerdash FM, Abbady EA, Baghdadi HH. Oxidative stress modulation by Rosmarinus officinalis in creosote-induced hepatotoxicity. Environ Toxicol 2016;31(1):85–92. DOI: 10.1002/tox.22024.
  26. Horvathova E, Navarova J, Galova E, et al. Assessment of antioxidative, chelating, and DNA-protective effects of selected essential oil components (eugenol, carvacrol, thymol, borneol, eucalyptol) of plants and intact Rosmarinus officinalis oil. J Agric Food Chem 2014;62(28):6632–6639. DOI: 10.1021/jf501006y.
  27. Kernou ON, Azzouz Z, Madani K, et al. Application of rosmarinic acid with its derivatives in the treatment of microbial pathogens. Molecules 2023;28(10):4243. DOI: 10.3390/molecules28104243.
  28. Swamy MK, Sinniah UR, Ghasemzadeh A. Anticancer potential of rosmarinic acid and its improved production through biotechnological interventions and functional genomics. Appl Microbiol Biotechnol 2018;102(18):7775–7793. DOI: 10.1007/s00253-018-9223-y.
  29. Ghorbani A, Sadeghnia HR, Afshari AR, et al. Rosmarinic acid protects adipose tissue-derived mesenchymal stem cells in nutrient-deficient conditions. Prev Nutr Food Sci 2019;24(4):449–455. DOI: 10.3746/pnf.2019.24.4.449.
  30. Tatullo M, Marrelli M, Shakesheff KM, et al. Dental pulp stem cells: Function, isolation and applications in regenerative medicine. J Tissue Eng Regen Med 2015;9(11):1205–1216. DOI: 10.1002/term.1899.
  31. Cho YD, Kim KH, Lee YM, et al. Dental-derived cells for regenerative medicine: Stem cells, cell reprogramming, and transdifferentiation. J Periodontal Implant Sci 2022;52(6):437–454. DOI: 10.5051/jpis.2103760188.
  32. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy Position Statement. Cytotherapy 2006;8(4):315–317. DOI: 10.1080/14653240600855905.
  33. Matin MH, Jahromi MZ, Fesharaki M, et al. Cytotoxicity of triple antibiotic paste and calcium hydroxide against cultured human dental pulp fibroblasts. Journal of Dental School, Shahid Beheshti University of Medical Sciences 2015;33(3):196–204. DOI:
  34. Selis D, Pande Y, Smoczer C, et al. Cytotoxicity and genotoxicity of a new intracanal medicament, 2-hydroxyisocaproic acid – An in vitro study. J Endod 2019;45(5):578–583. DOI: 10.1016/j.joen.2019.01.012.
  35. Ji YM, Jeon SH, Park JY, et al. Dental stem cell therapy with calcium hydroxide in dental pulp capping. Tissue Eng Part A 2010;16(6): 1823–1833. DOI: 10.1089/ten.TEA.2009.0054.
  36. Ruparel NB, Teixeira FB, Ferraz CC, et al. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod 2012;38(10):1372–1375. DOI: 10.1016/j.joen.2012.06.018.
  37. Althumairy R, Hargreaves K, Teixeira F, et al. The effect of dentin conditioning with medicaments used in regenerative procedures on the survival of SCAP. J Endod 2013;39:e15. DOI:
  38. Ghasemi M, Turnbull T, Sebastian S, et al. The MTT assay: Utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis. Int J Mol Sci 2021;22(23):12827. DOI: 10.3390/ijms222312827.
  39. Chuensombat S, Khemaleelakul S, Chattipakorn S, et al. Cytotoxic effects and antibacterial efficacy of a 3-antibiotic combination: An in vitro study. J Endod 2013;39(6):813–819. DOI: 10.1016/j.joen.2012.11.041.
  40. Yadlapati M, Souza L, Dorn S, et al. Deleterious effect of triple antibiotic paste on human periodontal ligament fibroblasts. Int Endod J 2014;47(8):769–775. DOI: 10.1111/iej.12216.
  41. Aldoghachi FEH, Noor Al-Mousawi UM, Shari F. Antioxidant activity of rosmarinic acid extracted and purified from mentha piperita. Arch Razi Inst 2021;76(5):1279–1287. DOI: 10.22092/ari.2021.356072.1770.
  42. Pérez MB, Calderon NL, Croci CA. Radiation-induced enhancement of antioxidant activity in extracts of rosemary (Rosmarinus officinalis L.). Food Chem 2007;104(2):585–592. DOI:
  43. Aruoma OI, Cuppett SL. Antioxidant methodology: In vivo and in vitro concepts. The American Oil Chemists Society (AOCS); Champaign, Illnois, USA; 1997. p. 70.
  44. Petersen M, Simmonds MSJ. Rosmarinic acid. Phytochemistry 2003;62(2):121–125. DOI: 10.1016/s0031-9422(02)00513-7.
  45. Zhang J, Cui X, Zhang M, et al. The antibacterial mechanism of perilla rosmarinic acid. Biotechnol Appl Biochem 2022;69(4):1757–1764. DOI: 10.1002/bab.2248.
  46. Noguchi-Shinohara M, Ono K, Hamaguchi T, et al. Pharmacokinetics, safety and tolerability of Melissa officinalis extract which contained rosmarinic acid in healthy individuals: A randomized controlled trial. PloS one 2015;10(5):e0126422. DOI: 10.1371/journal.pone. 0126422.
  47. Andrade JMM, Maurmann N, Lopes DV, et al. Rosmarinic and chlorogenic acid, isolated from ferns, suppress stem cell damage induced by hydrogen peroxide. J Pharm Pharmacol 2022;74(11):1609–1617. DOI: 10.1093/jpp/rgac061.
  48. Lee JW, Asai M, Jeon SK, et al. Rosmarinic acid exerts an antiosteoporotic effect in the RANKL-induced mouse model of bone loss by promotion of osteoblastic differentiation and inhibition of osteoclastic differentiation. Mol Nutr Food Res 2015;59(3):386–400. DOI: 10.1002/mnfr.201400164.
  49. Cicogna F, Passaglia E, Benedettini M, et al. Rosmarinic and glycyrrhetinic acid-modified layered double hydroxides as functional additives for poly (lactic acid)/poly (butylene succinate) blends. Molecules 2023;28(1):347. DOI: 10.3390/molecules28010347.
  50. Gomaa MA, Elhawary YM, Badr AE. Glycyrrhizin enhances the proliferation of diabetic bone marrow-derived mesenchymal stem cells: A potential therapeutic agent in endodontic surgery. J Contemp Dent Pract 2023;24(7):494–499. DOI: 10.5005/jp-journals-10024-3536.
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