Bioguided Isolation of (E)-Ethyl-12-cyclohexyl-4,5-dihydroxydodec-2-enoate from the Aerial Parts of Heliotropium indicum and Evaluation of Its Mechanism of Action Using the Formalin Test.

Background/Objectives: Heliotropium indicum (H. indicum) is a medicinal plant traditionally used for conditions associated with inflammation, but its active antinociceptive constituents remain poorly defined. This study evaluated the antinociceptive activity of the aerial parts of H. indicum through a bioassay-guided approach and explored the mechanism of action of the active compound isolated in the formalin test. Methods: Three extracts of H. indicum (hexane, dichloromethane, and methanol) were evaluated in male Swiss albino CD-1 mice using the formalin test. The most active extract was fractionated, and its major fractions were screened for antinociceptive activity. Based on the active fraction and previous phytochemical data, (E)-ethyl-12-cyclohexyl-4,5-dihydroxydodec-2-enoate (ECDE) was selected for further pharmacological evaluation in the same model. Antagonist pretreatments were used to investigate the involvement of opioid, serotonergic, gamma-aminobutyric acid (GABAA), and Nitric Oxide (NO)-soluble Guanylyl Cyclase (sGC) pathways. Results: The three extracts reduced nociceptive behavior, mainly during phase II of the formalin test, whereas the dichloromethane extract showed the broadest activity profile and was selected for fractionation. The six fractions significantly reduced phase II nociception, and fraction F5 was selected for purification. ECDE produced a clear dose-dependent antinociceptive effect in phase II, with minimal effect on phase I, and its efficacy was compared with that of ketorolac, a standard antinociceptive drug. Dose-response analysis estimated a DE50 of 0.76 mg/kg for ECDE. Pretreatment with N-nitro-L-arginine methyl ester (L-NAME) and [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) significantly attenuated the effect of ECDE, whereas naloxone, methiothepin, and bicuculline did not. Conclusions: ECDE was identified in H. indicum as one of the compounds contributing to this effect. Its activity appears to be directed mainly toward inflammatory nociception and to depend, at least in part, on the NO-sGC pathway.