Shahrekord University of Medical Sciences Journal of Herbmed Pharmacology 2345-5004 15 3 2026 07 01 In vitro antiviral activity of thymoquinone and its synergistic effect with acyclovir against herpes simplex virus type 1 (HSV-1) 365 371 10.34172/jhp.53593 EN Maryam Shirian https://orcid.org/0009-0009-9001-5516 Zainab Jalil Awad Al Jabree https://orcid.org/0009-0000-3229-4347 Pegah Khosravian https://orcid.org/0000-0002-4802-8534 Masoud Hafizi https://orcid.org/0000-0001-8003-9041 Fatemeh Javadi-Farsani https://orcid.org/0000-0002-3269-9704 Mohammad-Taghi Moradi https://orcid.org/0000-0002-3103-3740 Journal Article 10.34172/jhp.53593 2025 09 11 2025 12 25 Introduction: Drug resistance has heightened the demand for natural antiviral agents. Thymoquinone (TQ), a major bioactive component of black seed oil, has demonstrated antiviral activity in numerous studies. However, its potential efficacy against herpes simplex virus type 1 (HSV-1) has not been extensively explored. This research aimed to assess the in vitro antiviral effect of TQ and its synergistic relationship with acyclovir against HSV-1. Methods: In this experimental study, the cytotoxic effects of TQ and acyclovir on Vero cells were evaluated using the MTT test. The antiviral activity of TQ and acyclovir with sub-cytotoxic doses and combinations with TQ (CC10) on HSV-1 virus replication was investigated using both MTT and 50% tissue culture infectious dose (TCID50) assays. Compusyn software was used to analyze the combination index (CI) of TQ and acyclovir. Results: The 50% cytotoxic concentration (CC50) values of acyclovir and TQ were calculated to be 537.7 and 57.5 μM, respectively. The 50% inhibitory concentration (IC50) values of acyclovir, TQ, and acyclovir together with 10 μM TQ (CC10) were obtained to be 0.25, 13.38, and 0.04 μM, respectively. Based on the TCID50 findings, both drugs, whether used separately or in combination, significantly diminished viral titers. Synergistic effects were identified at most concentrations, with the strongest synergy observed at 0.3 μM TQ in combination with 0.03 μM acyclovir. Conclusion: TQ possesses potent antiviral activity against HSV-1 virus and has synergistic effects with acyclovir to inhibit virus replication. Further studies need to be carried out to assess its utility and safety profile.