Assessing the Effect of Indigo Naturalis on Periodontal Ligament Fibroblast Viability: A Pilot In Vitro Study

Year 2026, Volume: 30 Issue: 2, 427 - 433, 15.03.2026

Aslı Aktan , Umut Ulaş Tosun , Sema Tuğçe Aydın , Dilruba Baykara , Ayşegül Tiryaki , Canan Ekinci Doğan , Turgut Taşkın , Oğuzhan Gündüz , Ömer Birkan Ağralı

https://doi.org/10.12991/jrespharm.1802902

https://izlik.org/JA34UB36GH

Abstract

This pilot investigation aimed to examine the impact of different concentrations of indigo naturalis on the cellular viability of human periodontal ligament fibroblast (hPDLF) cells. For experimental application on cells, indigo naturalis solutions were prepared at seven varying concentrations (0.025, 0.0375, 0.05, 0.075, 0.1, 0.1125 and 0.15 µg/mL) for the purpose of cellular treatment. To evaluate cell viability, cells were seeded in 96-well plates in triplicate for each concentration. Following a 24-hour treatment period with indigo naturalis, cell viability was assessed using the MTT assay. Statistical analysis of the data was performed using SPSS software, with a P-value of less than 0.05 considered statistically significant. The MTT assay revealed no significant difference in the viability of hPDLF cells between the control group and the indigo naturalis-treated groups (P>0.05). While indigo naturalis did not affect cell viability in this study, it provides important insights into the interactions between herbal compounds and hPDLF cells. This study is limited by the use of a single assay and a 24-hour observation period, which may restrict the generalisability of the findings. Further research is needed to explore the molecular mechanisms behind these interactions, investigate potential synergistic effects, and evaluate longer-term impacts on periodontal conditions.

Keywords

Cell viability , fibroblasts , indigo , periodontal ligament

Ethical Statement

As this study was conducted using cell cultures, approval from an ethics committee was not required.

Thanks

We would like to thank all the participants. The findings of this study were presented as a poster at the EuroPerio 11 Congress (14-17 May 2025). This work was supported by the Scientific and Technological Research Council of Türkiye (TÜBİTAK) under the 2224-A Program for participation in international scientific events.

References

• [1] Guo H, Bai X, Wang X, Qiang J, Sha T, Shi Y, et al. Development and regeneration of periodontal supporting tissues. Genesis. 2022;60(8-9):e23491. https://doi.org/10.1002/dvg.23491
• [2] Carmagnola D, Pellegrini G, Dellavia C, Rimondini L, Varoni E. Tissue engineering in periodontology: Biological mediators for periodontal regeneration. Int J Artif Organs. 2019;42(5):241-257. https://doi.org/10.1177/0391398819828558
• [3] Abdallah AT, Konermann A. Unraveling Divergent Transcriptomic Profiles: A Comparative Single-Cell RNA Sequencing Study of Epithelium, Gingiva, and Periodontal Ligament Tissues. Int J Mol Sci. 2024;25(11):15617. https://doi.org/10.3390/ijms25115617
• [4] Naruishi K. Biological Roles of Fibroblasts in Periodontal Diseases. Cells. 2022;11(21):3345. https://doi.org/10.3390/cells11213345
• [5] Hinz B. Matrix mechanics and regulation of the fibroblast phenotype. Periodontol 2000. 2013;63(1):14-28. https://doi.org/10.1111/prd.12030
• [6] Farkhatov Sunnatillokhan F, Akhmedov Alisher A. Periodontal Disease - Symptoms and Treatment. Journal of Social Sciences and Humanities Research Fundamentals. 2025;5(1):81-87. https://doi.org/10.55640/jsshrf-05-01-14
• [7] Graziani F, Karapetsa D, Alonso B, Herrera D. Nonsurgical and surgical treatment of periodontitis: how many options for one disease? Periodontol 2000. 2017;75(1):152-188. https://doi.org/10.1111/prd.12201
• [8] Mawardi HH, Elbadawi LS, Sonis ST. Current understanding of the relationship between periodontal and systemic diseases. Saudi Med J. 2015;36(2):150-158. https://doi.org/10.15537/smj.2015.2.9424
• [9] Pytko-Polończyk J, Stawarz-Janeczek M, Kryczyk-Poprawa A, Muszyńska B. Antioxidant-Rich Natural Raw Materials in the Prevention and Treatment of Selected Oral Cavity and Periodontal Diseases. Antioxidants (Basel). 2021;10(11):1848. https://doi.org/10.3390/antiox10111848
• [10] Dinu S, Dumitrel SI, Buzatu R, Dinu DC, Popovici R, Szuhanek C, et al. New Perspectives about Relevant Natural Compounds for Current Dentistry Research. Life (Basel). 2024;14(8):951. https://doi.org/10.3390/life14080951
• [11] Hu Y, Chen LL, Ye Z, Li LZ, Qian HZ, Wu MQ, et al. Indigo naturalis as a potential drug in the treatment of ulcerative colitis: a comprehensive review of current evidence. Pharm Biol. 2024;62(1):818-832. https://doi.org/10.1080/13880209.2024.2415652
• [12] Pan M, Pei W, Yao Y, Dong L, Chen J. Rapid and Integrated Quality Assessment of Organic-Inorganic Composite Herbs by FTIR Spectroscopy-Global Chemical Fingerprints Identification and Multiple Marker Components Quantification of Indigo Naturalis (Qing Dai). Molecules. 2018;23(11):2743. https://doi.org/10.3390/molecules23112743
• [13] Qi-Yue Y, Ting Z, Ya-Nan H, Sheng-Jie H, Xuan D, Li H, et al. From natural dye to herbal medicine: a systematic review of chemical constituents, pharmacological effects and clinical applications of indigo naturalis. Chin Med. 2020;15(1):127. https://doi.org/10.1186/s13020-020-00406-x
• [14] Gaitanis G, Magiatis P, Velegraki A, Bassukas ID. A traditional Chinese remedy points to a natural skin habitat: indirubin (indigo naturalis) for psoriasis and the Malassezia metabolome. Br J Dermatol. 2018;179(3):800. https://doi.org/10.1111/bjd.16807
• [15] Naganuma M. Treatment with indigo naturalis for inflammatory bowel disease and other immune diseases. Immunol Med. 2019;42(1):16-21. https://doi.org/10.1080/25785826.2019.1599158
• [16] Yu H, Li TN, Ran Q, Huang QW, Wang J. Strobilanthes cusia (Nees) Kuntze, a multifunctional traditional Chinese medicinal plant, and its herbal medicines: A comprehensive review. J Ethnopharmacol. 2021;265:113325. https://doi.org/10.1016/j.jep.2020.113325
• [17] Martínez-García M, Hernández-Lemus E. The Molecular Comorbidity Network of Periodontal Disease. Int J Mol Sci. 2024;25(18):10161. https://doi.org/10.3390/ijms251810161
• [18] Boyapati R, Pendyala R, Lanke RB, Anumala D. Herbal Treatments Used as an Alternative in the Treatment of Periodontal Diseases. Cumhuriyet Dental Journal. 2023;26(2):204-210. https://doi.org/10.7126/cumudj.1126037
• [19] Lg P. Periodontal Disease and Phytotherapy. Journal of Oral Hygiene & Health. 2015;3(1):1000172. https://doi.org/10.4172/2332-0702.1000172
• [20] Huang Y, Tang Y, Zhang R, Wu X, Yan L, Chen X, et al. Role of periodontal ligament fibroblasts in periodontitis: pathological mechanisms and therapeutic potential. J Transl Med. 2024;22(1):1136. https://doi.org/10.1186/s12967-024-05944-8
• [21] Buranaamnuay K. The MTT assay application to measure the viability of spermatozoa: A variety of the assay protocols. Open Vet J. 2021;11(2):251-269. https://doi.org/10.5455/OVJ.2021.v11.i2.9
• [22] 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
• [23] Kim E, Jeon IS, Kim JW, Kim J, Jung HS, Lee SJ. An MTT-based method for quantification of periodontal ligament cell viability. Oral Dis. 2007;13(5):495-499. https://doi.org/10.1111/j.1601-0825.2006.01328.x
• [24] Alavi SH, Rezvani G, Esfahani MN, Nobakht Lahrood F. Periodontal Ligament Fibroblast Cell Viability Following Treatment with Different Concentrations of Green Tea, Aloe Vera and a Mixture of their Extracts. Front Dent. 2022;19:40. https://doi.org/10.18502/fid.v19i40.11901
• [25] Mandroli PS, Prabhakar AR, Bhat K, Krishnamurthy S, Bogar C. An in vitro evaluation of cytotoxicity of curcumin against human periodontal ligament fibroblasts. Ayu. 2019;40(3):192-195. https://doi.org/10.4103/ayu.AYU_294_18
• [26] Al-Haj Ali SN, Al-Jundi SH, Ditto DJ. In vitro toxicity of grey MTA in comparison to white MTA on human periodontal ligament fibroblasts. Eur Arch Paediatr Dent. 2014;15(6):429-433. https://doi.org/10.1007/s40368-014-0134-z
• [27] Singh S, Kini S, Pai S, H RR, Purayil TP. Survival of human periodontal ligament fibroblast cells in Cornisol and HBSS for transportation of avulsed teeth: a comparative ex vivo study. Acta Odontol Scand. 2021;79(2):112-117. https://doi.org/10.1080/00016357.2020.1795248
• [28] Cabral MC, Costa MA, Fernandes MH. In vitro models of periodontal cells: a comparative study of long-term gingival, periodontal ligament and alveolar bone cell cultures in the presence of beta-glycerophosphate and dexamethasone. J Mater Sci Mater Med. 2007;18(6):1079-1088. https://doi.org/10.1007/s10856-007-0134-1
• [29] Cheng HM, Kuo YZ, Chang CY, Chang CH, Fang WY, Chang CN, et al. The anti-TH17 polarization effect of Indigo naturalis and tryptanthrin by differentially inhibiting cytokine expression. J Ethnopharmacol. 2020;255:112760. https://doi.org/10.1016/j.jep.2020.112760
• [30] Chang HN, Huang ST, Yeh YC, Wang HS, Wang TH, Wu YH, et al. Indigo naturalis and its component tryptanthrin exert anti-angiogenic effect by arresting cell cycle and inhibiting Akt and FAK signaling in human vascular endothelial cells. J Ethnopharmacol. 2015;174:474-481. https://doi.org/10.1016/j.jep.2015.08.050
• [31] Lin YK, Leu YL, Yang SH, Chen HW, Wang CT, Pang JH. Anti-psoriatic effects of indigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component. J Dermatol Sci. 2009;54(3):168-174. https://doi.org/10.1016/j.jdermsci.2009.02.007
• [32] ISO 10993-5:2009. Biological evaluation of medical devices—part 5: tests for in vitro cytotoxicity. International Organization for Standardization (2009).