ObjectivesThe study aimed to identify nutmeg compounds that indirectly interact with the endocannabinoid system through inhibition of the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) enzymes. Methods Thirteen compounds were screened for FAAH and MAGL inhibition. Compounds demonstrating significant FAAH inhibition were evaluated to determine the halfmaximal inhibitory concentration (IC 50 ). The most potent compound was investigated in the elevated plus maze (EPM) rodent anxiety model. Key findings Three compounds, licarin A (9), 5 0 -methoxylicarin A (8) and malabaricone C (6) were most active in inhibiting FAAH with IC 50 of 7.02 lM AE 2.02, 4.57 lM AE 0.66 and 38.29 lM AE 6.18, respectively. None of the purified compounds showed significant MAGL inhibition. Because of its relative high potency and selectivity, compound 8 was further evaluated in the EPM animal model of anxiety. The compound showed significant increase in number of open arm entries (P < 0.05) when administered at 120 mg/kg dose. No effect was observed on the locomotor activity.Conclusions Results collected introduce active nutmeg compounds as potential leads for further development. Of the three compounds, 8 possesses highest potency and FAAH selectivity as well as anxiolytic activity. Furthermore, in vivo testing in appropriate behavioural animal paradigms is warranted.FAAH inhibitors from nutmeg Abir T. El-Alfy et al.

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The endocannabinoid system (ECS) is a complex neuromodulatory network involved in the regulation of numerous physiological functions. These effects have been substantiated by the use of known exogenous cannabinoid, Δ9‐THC, the active component of marijuana. Thus, the ECS has emerged as an appealing pharmacologic target in drug discovery. Anecdotal reports of Myristica fragrans, commonly known as nutmeg, being used as a substitute for marijuana, suggest that one or more of its components may interact with the ECS. Previous studies in our laboratory demonstrated that administration of nutmeg extracts produced behavioral tetrad effects similar to Δ9‐THC. However, the mechanism of such action requires further exploration. Receptor binding assays revealed that nutmeg extracts did not have direct CB1 or CB2 receptor binding activity. Additional mechanistic studies showed that indirect interaction through the inhibition of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), key endocannabinoid degradation enzymes, was observed. The objective of the current study was to isolate the active components of nutmeg extract responsible for the FAAH inhibition activity and to evaluate them in an animal model of anxiety. Using bioassay guided fractionation and various chromatographic techniques, three compounds were isolated, spectroscopically characterized, and identified (MF 109‐5, MF30‐7, and MF117‐3). Concentration and time inhibition studies were conducted. The IC50 values calculated for the three compounds were 4.57 ± 0.66, 7.50 ± 2.02, and 38.29 ± 6.18 mM, respectively. The compounds were further evaluated in the elevated plus maze animal model of anxiety in comparison to diazepam as the positive control. The three nutmeg compounds were tested at doses ranging from 5 to 120 mg/kg, i.p. injection. While diazepam demonstrated a significant dose‐response increase in open arm entry and permanence time, indicative of anxiolytic action, none of the isolated compounds had a significant effect. In conclusion, the study resulted in the identification of three nutmeg compounds that significantly inhibit FAAH enzyme and hence shed light on potential mechanism of cannabis‐like action of nutmeg. While none of the compounds showed anxiolytic activity in the elevated plus maze model, additional pharmacokinetic studies are needed to optimize the dosing regimen required for significant increase in endocannabinoids that might be needed for elaboration of the anxiolytic effect.Support or Funding InformationResearch reported in this publication was supported by the National Institute on Drug Abuse of the National Institutes of Health under Award Number R24DA036410. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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