ENHANCED AND SYNERGISTIC INHIBITION OF PRO-INFLAMMATORY CYTOKINES (IL-1Β, IL-17A, AND IL-6) BY A CURCUMIN- THYMOQUINONE COMPLEX (CURQNONE®), COMPARED TO CURCUMIN MONOTHERAPY IN LPS-STIMULATED RAW 264.7 MURINE MACROPHAGES

S. Mehkri, K. G. Dinesh, G. Ashok and Krathish Bopanna*

Background: Curcumin and thymoquinone (TQ) are phytochemicals with well-established anti- inflammatory and antioxidant properties.Curcumin inhibits NF-κB and MAPK signaling, and reduces transcriptional priming of inflammatory mediators, but its limited bioavailability restricts therapeutic effectiveness. TQ, derived from Nigella sativa, complements curcumin by inhibiting NLRP3 inflammasome activation and enhancing antioxidant responses through NRf2 signaling. A proprietary curcumin and TQ (CTQ) complex (CurQnone®) has been hypothesized to achieve broader suppression of inflammatory pathways than curcumin alone. This study compared the efficacy of CTQ with curcumin monotherapy in LPS-stimulated RAW 264.7 macrophages. Methods: RAW 264.7 macrophages were pre-treated with CTQ or curcumin (7.8 and 15.62 μg/mL), followed by stimulation with LPS (1 μg/mL). Cell culture supernatants were collected and analyzed for IL-6, IL-17A, and IL-1β levels using ELISA, with fold changes calculated relative to untreated controls. In parallel, transcriptional expression of iNOS and COX-2 was assessed by RT-PCR to evaluate the extent of inflammatory gene priming. Results: Both CTQ and curcumin significantly attenuated LPS-induced cytokine production and iNOS/COX-2 expression. CTQ consistently demonstrated stronger inhibition than curcumin alone, with fold reductions as follows: IL-1β (8.3-fold vs. 5.4-fold), IL-6 (5.4-fold vs. 2.4-fold), and IL-17A (9.4-fold vs. 8.6-fold). These effects were evident at both tested concentrations, suggesting a dose-responsive trend. In addition, CTQalso resulted in stronger downregulation of iNOS and COX-2 transcripts, indicating more effective blockade of transcriptional priming and pro-inflammatory mediator synthesis. Conclusion: CTQ exerted broader and more potent anti-inflammatory effect than curcumin alone in LPS- stimulated macrophages, targeting both transcriptional priming and cytokine maturation pathways. Its pronounced IL-6/IL-17 axis highlights potential application in chronic inflammatory and autoimmune conditions. These findings support CTQ as a synergistic, dose- sparing nutraceutical candidate warranting further preclinical and clinical evaluation.