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. 2019 Oct 8;71(12):1879–1889. doi: 10.1111/jphp.13174

Phenolic compounds from nutmeg (Myristica fragrans Houtt.) inhibit the endocannabinoid-modulating enzyme fatty acid amide hydrolase

Abir T El-Alfy 1,✉, Ehab A Abourashed 1, Christina Patel 2, Nunmoula Mazhari 2, HeaRe An 2, Andrew Jeon 2
PMCID: PMC7938946  PMID: 31595522

Abstract

Objectives

The 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 (IC50). The most potent compound was investigated in the elevated plus maze (EPM) rodent anxiety model.

Key findings

Three compounds, licarin A (9), 5′-methoxylicarin A (8) and malabaricone C (6) were most active in inhibiting FAAH with IC50 of 7.02 μm ± 2.02, 4.57 μm ± 0.66 and 38.29 μm ± 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.

Keywords: fatty acid amide hydrolase, licarin A, malabaricone C, Myristica fragrans, nutmeg

Introduction

The endocannabinoid system is a complex neuromodulatory network involved in numerous physiological functions such as appetite, pain, reward, motor control, memory and cognition. The primary receptors involved are cannabinoid 1 receptor (CB1), predominantly located in the central nervous system, and cannabinoid 2 receptor (CB2) located in the periphery. Numerous endogenous ligands have been identified, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA); however, only AEA and 2-AG have been extensively studied. These endogenous cannabinoids target the CB1 and CB2 receptors, acting as retrograde messengers. They bind presynaptically to modulate various neurotransmitters such as serotonin (5-HT), noradrenaline, γ-aminobutyric acid (GABA) and glutamate. Following transporter-mediated neuronal uptake, the serine hydrolases, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) degrade AEA and 2-AG, respectively, lowering their concentrations.[1]

Due to psychotropic effects and potential misuse, which have been shown to result from direct activation, indirect modulation of the endocannabinoid system has become an appealing approach for discovery of drugs targeting pain and other CNS conditions. This approach has been shown to limit adverse effects, such as impaired mobility and disruption of thermoregulation, while keeping positive mood stabilizing and analgesic effects intact. However, indirect dual inhibition of FAAH and MAGL may also result in the same CB1 agonistic effects. Therefore, it appears that selective FAAH and MAGL inhibition may be a superior strategy to narrow the outcome of therapy and improve the safety profile of a potential agent.[2] In addition to synthetic compounds that have been developed as FAAH inhibitors, Alasmari et al.[3] have reported FAAH inhibition activity of mecamides, isolated from the Peruvian Maca plant and by flavonoids. Recent studies have also identified natural products that effectively inhibit MAGL enzyme, including acylglycerols isolated from Arasco and sunflower oils and phenylethanoid glycosides from Cistacnhe phelypaea plant.[4,5]

In addition to its common use as a kitchen spice, nutmeg (the kernel of Myristica fragrans Houtt., Myristicaceae) is used in traditional medicine to treat anxiety and depression, in addition to being utilized as an antibiotic, analgesic, aphrodisiac, antidiarrhoeal, antioxidant and lipid-lowering agent.[6,7] Likewise, when ingested in sufficient amounts, nutmeg elicits hallucination, euphoria and delusions.[8] Earlier accounts suggested that myristicin, the main constituent of nutmeg essential oil, was biotransformed to amphetamine-like metabolites that acted as CNS stimulants in humans.[9] However, this hypothesis was subsequently challenged by the failure to detect any such metabolites in animals and humans after ingestion of nutmeg.[10] A comprehensive review of the nutmeg chemistry and pharmacology has recently been published.[11]

Anecdotal reports of nutmeg being used as a cheap substitute for marijuana suggest that one or more of its secondary metabolites may potentially interact with the endocannabinoid system. Earlier investigation in our laboratory revealed that nutmeg may have utility in mood stabilization as its effects resemble delta-9-tetrahydrocannabinol (∆9-THC).[12] We reported varying behavioural activity in mice depending on solvents used to extract nutmeg, indicating various components of nutmeg might elicit different CNS activity. Follow-up studies examined the interaction of the in vivo active extracts identified by our group with the endocannabinoid system, showing no binding affinity to CB1 or CB2 receptors. However, significant inhibition of FAAH and MAGL enzymes was observed with these extracts.[13] In continuation to our previous findings, the most active solvent fraction was further fractionated to thirteen individual compounds (Figure 1), which were evaluated in vitro for FAAH and MAGL inhibition. The most active compounds were identified as licarin A (9), 5′-methoxylicarin A (8) and malabaricone C (6). Compound 8 was also evaluated in vivo in the elevated plus maze (EPM) mouse model of anxiety. Results collected from the current study demonstrate that the identified nutmeg compounds indirectly modulate the endocannabinoid system and can serve as lead entities for further development as potential therapeutic agents targeting the system. This study is the first to report and characterize the FAAH inhibition activity of the identified compounds and to offer a plausible explanation to the long-standing reputation of nutmeg as an inferior substitute for marijuana.

Figure 1.

Figure 1

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Chemical structures of secondary metabolites isolated from nutmeg active fraction including the most active compounds: licarin A (9), 5′-methoxylicarin A (8) and malabaricone C (6).

Materials and Methods

Plant material, test compounds and assay kits

Whole nutmeg kernels (M. fragrans Houtt., Myristicaceae) were acquired from Mond Trading Co. (Toronto, ON, Canada) and were reduced to fine powder using a coffee grinder. Total nutmeg extract was prepared by ultrasonicating 100 g of powdered nutmeg in methanol for 1 h followed by soaking overnight at room temperature. The filtered extract was stored in a freezer overnight then refiltered and concentrated under vacuum at 45 °C. An aliquot of the concentrated total extract (1 g) was subjected to successive solvent extract on a silica bed using n-hexane, dichloromethane, ethyl acetate and methanol (100 ml each). Each extract was dried under vacuum to yield four separate fractions. The total methanolic extract and its solvent fractions were fingerprinted by an analytical HPLC method developed in our laboratory.[14] The most active of the four fractions, ethyl acetate fraction, was further subjected to flash chromatography using a n-hexane-ethyl acetate gradient to generate 10 column fractions. The major constituents in each fraction were purified by semipreparative reversed-phase HPLC (Shimadzu Prominence System, Kyoto, Japan) to yield 13 compounds as described in an earlier report.[14] Evaluation of the isolated compounds for FAAH and MAGL inhibition was performed using enzyme assay inhibitor screening kits purchased from Cayman Chemical Company (Ann Arbor, USA). All assay kits were used to conduct in vitro enzyme inhibition evaluation of isolated compounds in 96-well plates. All solvents used for extraction and in vitro assays were of reagent grade and were acquired from Fisher Scientific (Fair Lawn, USA).

General assay procedures

Samples were mixed on a Thermo Scientific (Waltham, USA) Multi-Purpose Rotator (Model No. 2314, Dubuque, USA) and incubated, if needed, in a Thermo Scientific HeraTherm Incubator (Model No. 41214747, Germany). All plates were read on a BioTek Synergy 2 (Winooski, USA) plate reader, with data being analysed using BioTek Gen5 version 1.10.

A total of ten column subfractions collected from the ethyl acetate fraction were initially screened at 0.05 mg/ml for human recombinant FAAH enzyme inhibition and at 0.01 mg/ml for human recombinant MAGL enzyme inhibition.[13] Fractions that exhibited ≥50% enzyme inhibition were further separated into individual compounds. Total extraction, fractionation using solvents of increasing polarities and compound isolation by preparative chromatography were conducted following an in-house procedure.[14]

Isolated compounds were screened at 0.05 mg/ml for FAAH and 0.01 mg/ml for MAGL enzyme inhibition activity. Compounds that demonstrated ≥50% enzyme inhibition after 30 min of incubation were further evaluated for concentration-dependent inhibition. After initial FAAH activity was seen, 6 was assayed at concentrations ranging from 1.09 to 279.32 μm, 8 ranging from 1.10 to 140.45 μm and 9 ranging from 1.20 to 153.37 μm. None of the purified compounds showed significant MAGL inhibition; therefore, further dilutions were not performed. Each FAAH and MAGL inhibition assay was performed in three separate replicate experiments.

A time course analysis was performed for active subfractions and isolated compounds to assess duration of inhibition of FAAH and MAGL at timed readings of 30 min, 1, 2, 4, 6, 24 and 48 h.

FAAH enzyme inhibition assay

FAAH inhibition assay was performed using JZL 195 as a positive control in concentrations of 0.008–1 μm, using DMSO as a solvent. Manufacturer's protocol was followed to perform the assay. The assay buffer (125 mm Tri-HCl with 1 mm EDTA, pH 9.0) was used to dilute the FAAH human recombinant enzyme. Arachidonoyl amide, at a concentration of 400 mm, was then used as the FAAH substrate. Samples were mixed for 30 s and incubated at 37 °C for 30 min. The fluorescent byproduct 7-amino-4-methylcoumarin (AMC) released by the FAAH enzyme was detected and quantified at an excitation wavelength of 360 nm and emission wavelength of 460 nm.

MAGL enzyme inhibition assay

JZL 195 was also used as the positive control in the MAGL inhibition assay, however, at concentrations of 0.07–2.22 μm, diluted with DMSO. Manufacturer protocol was followed to perform the assay. MAGL human recombinant enzyme was diluted with assay buffer (10 mm Tris-HCl with 1 mm EDTA, pH 7.2) and the substrate used was a 4.25 mm concentration of an ethanolic solution of 4-nitrophenylacetate. The plate was mixed for 30 s and incubated at room temperature for 30 min. The plate was read at an absorption wavelength of 410 nm to detect the byproduct 4-nitrophenol.

Animals

Behavioural experiments were performed using 8-week-old male CD1 mice (Charles River Laboratories, Willmington, USA). The mice were housed in groups of five with a 12 h light/12 h dark cycle. Food and water were provided ad libitum. All mice were randomly selected for each treatment group (n = 7 animals/group). Procedures involving animals were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) and according to the National Institutes of Health Guide Care and Use of Laboratory Animals (Protocol number 10-006, Chicago State University, Chicago, USA).

Locomotor activity

Mice were injected intraperitoneally (i.p.) with either vehicle (ethanol : kolliphor EL : saline 1 : 1 : 18) or compound 8 (5 mg/kg or 120 mg/kg, dissolved in the same vehicle). Thirty minutes following injection, animals were individually placed in automated activity monitoring locomotor chambers (San Diego Instruments, San Diego, CA), where total locomotor activity was monitored and recorded for 30 min.

Elevated plus maze

Immediately following the locomotor activity chambers, animals were subjected to the EPM test. Each mouse was placed individually in the centre of the maze (Med Associates Inc., Fairfax, USA) and allowed 5 min for free exploration. The number of entries, explorations and the time spent in the open and closed arms were automatically recorded. Rodents have a natural aversion to open spaces and avoid entering the open arms while anxiolytic compounds cause an increase in the exploratory behaviour in the open arms. An open arm entry was recorded only when all the four paws of the mouse were into the open arm. The maze was thoroughly cleaned between each subject.

Data analysis

Per cent inhibition was calculated for each compound using the formula [(100% Activity − Sample Activity)/100% Activity] × 100. All data are presented as mean per cent inhibition ± standard error mean (SEM), unless otherwise noted. GraphPad Prizm (Version 5.03) (San Diego, USA) was used to analyse the non-linear curve fit concentration inhibition curves and to determine the IC50 values of the active MF compounds.

For the locomotor activity and EPM results, all values are represented as the mean ± SEM, with n = 7 mice/group. All data were analysed using one-way ANOVA followed by Dunnett's post hoc test using GraphPad Prism version 5.03 to determine significant difference from vehicle control at P < 0.05.

Results

Successive solvent extraction of total nutmeg extract followed by flash chromatography of the most active fraction (ethyl acetate) resulted in ten column subfractions. As depicted in Figure 2a, all ten subfractions showed greater than 50% FAAH enzyme inhibition after 30 min incubation, ranging from 60.72 ± 9.46 (MF55-10) to 87.63 ± 2.75 (MF55-7). Time course analysis showed enzyme recovery of all fractions, as a steady decline in activity is seen over a 48-h period (Figure 2b).

Figure 2.

Figure 2

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Inhibition of hrFAAH enzyme by column subfractions of ethyl acetate fraction of nutmeg; (a) Subfractions were prepared using flash chromatography of ethyl acetate fraction and screened for hrFAAH enzyme inhibition at 0.05 mg/ml; (b) Timed readings were recorded at 0.5, 1, 2, 4, 6, 24 and 48 h. Values are presented as the mean ± SEM of three replicates. Inhibition was considered significant at ≥50%.

On the other hand, MAGL inhibition assays revealed only four subfractions, MF 55-4 to MF-55-7, exhibiting significant (≥50%) enzyme inhibition, as seen in Figure 3a. MAGL inhibition increased over the first 6 h and it remained significant up to 24 h before showing enzyme recovery and eventually falling below 50% at 48 h (Figure 3b).

Figure 3.

Figure 3

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Inhibition of hrMAGL enzyme by column subfractions of ethyl acetate fraction of nutmeg; (a) Subfractions were prepared using flash chromatography of ethyl acetate fraction and screened for hrMAGL enzyme inhibition at 0.01 mg/ml; (b) Timed readings were recorded at 0.5, 1, 2, 4, 6, 24 and 48 h. Values are presented as the mean ± SEM of three replicates. Inhibition was considered significant at ≥50%.

As described in our earlier publication, a total of thirteen compounds were isolated from the tested subfractions. These compounds were identified as follows: elemicin (1), isoelemicin (2), 2-(3′-allyl-2′,6′-dimethoxy-phenyloxy)-1-methyl-5-methoxy-1,2-dihydrobenzofuran (3), myristicin (4), surinamensin (5), malabaricone C (6), 2-(3′-allyl-2′,6′-dimethoxy-phenyloxy)-1-acetoxy-(3,4-dimethoxyphenyl)-propane (7), 5′-methoxylicarin A (8), licarin A (9), malabaricone B (10), licarin C (11), 5′-methoxylicarin B (12) and licarin B (13).[14] Licarin A (9), 5′-methoxylicarin A (8) and malabaricone C (6) were the most active inhibitors of FAAH with per cent inhibition of 89.73 ± 2.43, 80.42 ± 2.90 and 64.85 ± 5.20, respectively, when tested at a concentration of 0.05 mg/ml (Figure 4).

Figure 4.

Figure 4

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Inhibition of hrFAAH enzyme by isolated nutmeg compounds. Thirteen compounds isolated from the ethyl acetate fraction were screened for hrFAAH enzyme inhibition at concentrations of 0.05 mg/ml. Values are represented as mean ± SEM of three replicates. Inhibition was considered significant at ≥50%.

Concentration-dependent assays were run with the positive control, JZL 195, yielding an IC50 value of 0.045 μm ± 0.002 as seen in Figure 5a. Likewise, an IC50 value of 7.02 μm ± 2.02 was determined for 9, 4.57 μm ± 0.66 for 8 and 38.29 μm ± 6.18 for 6 (Figure 5b–d).

Figure 5.

Figure 5

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hrFAAH IC50 curves for (a) JZL 195, (b) compound 6, (c) compound 8 and (d) compound 9. IC50 values were determined using Graph Pad Prism (version 5.03) from concentration-dependent assays performed at concentrations of (a) 0.008–1.00 μm, (b) 1.09–279.32 μm, (c) 1.10–140.45 μm and (d) 1.20–153.37 μm. Values are represented as the mean ± SEM of three replicates.

Time course analysis (Figure 6) showed enzyme recovery for all three compounds, similar to that seen in tested subfractions. Enzyme inhibition remained above 50% in all three compounds even after 24 h. Moreover, an increase in FAAH inhibition was seen over the first 6 h and returned to that of the initial readings after 24 h, continuing to trend down.

Figure 6.

Figure 6

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Time course of hrFAAH inhibition by active nutmeg compounds. Time course inhibition of hrFAAH enzyme by nutmeg compounds assayed at a concentration of 0.05 mg/ml. Timed readings recorded at 0.5, 1, 2, 4, 6, 24 and 48 h. Values are represented as the mean ± SEM of three replicates.

The positive control JZL 195 was shown to inhibit MAGL with an IC50 value of 0.71 μm ± 0.31. However, none of the isolated compounds demonstrated MAGL inhibitory activity ≥50% during initial screening. To evaluate the selectivity and specificity of compounds 6, 8 and 9 for FAAH inhibition, they were evaluated for MAGL inhibition at their respective FAAH IC50 concentrations. None of the compounds showed significant MAGL inhibition. Per cent MAGL inhibition recorded was 18.08 ± 10.67, 11.34 ± 3.55 and 16.08 ± 4.84 for compounds 6, 8 and 9, respectively.

Compound 8 was further evaluated for potential in vivo efficacy against anxiety. As shown in Figure 7, i.p. administration of compound 8 did not have significant effect on the locomotor activity of the animals. On the other hand, at 120 mg//kg dose, the compound exerted anxiolytic activity as evidenced by the significant increase in number of open arm entries (P < 0.5) (Figure 8b).

Figure 7.

Figure 7

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Effect of compound 8 on total locomotor activity in mice. Male CD1 mice (n = 7/group) were injected intraperitoneally (i.p.) with vehicle (ethanol : kolliphor : saline 1 : 1 : 18), 5 mg/kg compound 8 or 120 mg/kg compound 8. Thirty minutes following injection, animals were individually placed in activity monitoring chambers and total locomotor activity was automatically recorded for 30 min. Values are represented as the mean ± SEM of seven animals/group.

Figure 8.

Figure 8

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Effect of compound 8 on behaviour in the elevated plus maze (EPM) test. Male CD1 mice (n = 7/group) were injected intraperitoneally (i.p.) with vehicle (ethanol : kolliphor : saline 1 : 1 : 18) or compound 8 (5 or 120 mg/kg) and subjected to the locomotor activity monitoring for 30 min. Animals were then placed individually in the EPM and automatically recorded for 5 min. Activity in the closed arms (a) and open arms (b) are represented. Values are represented as the mean ± SEM of seven animals/group. *Statistical significance from vehicle group at P < 0.05 (one-way ANOVA followed by Dunnett's test).

Discussion

Nutmeg has been used both medicinally and recreationally for the treatment of anxiety and mood disorders. El-Alfy et al.[12] demonstrated that the administration of nutmeg extracts produces behavioural effects similar to ∆9-THC, a known exogenous cannabinoid and the major psychoactive constituent of marijuana. Later investigation revealed that these extracts did not have direct CB1 or CB2 receptor binding affinity.[13] To determine the effect of nutmeg on the endocannabinoid system, indirect modulation was thus assessed. Using an assay-guided fractionation approach coupled to in vitro assays of FAAH and MAGL inhibition, total nutmeg extract was fractionated using successive solvent extraction and each solvent extract was evaluated.[13] Flash chromatography was subsequently utilized to further fractionate the ethyl acetate active fraction. Significant FAAH inhibition was seen in all column subfractions of the ethyl acetate fraction. In a separate study, thirteen pure compounds were isolated from the ethyl acetate fraction using preparative HPLC.[14] After evaluating all isolated compounds, our current study identified three active compounds, 6, 8 and 9. Further investigation showed 8 to be the most potent FAAH inhibitor (IC50 4.57 μm ± 0.66), followed by 9 (IC50 7.02 μm ± 2.02) and 6 (IC50 38.29 μm ± 6.18). Although substantial inhibition was seen, the positive control JZL 195 was shown to be 100-fold more potent than any of the tested compounds. Conversely, only four subfractions of the ethyl acetate fraction possessed MAGL inhibitory activity slightly above 50%. Screening at higher concentrations was not feasible because of background interference with the assay. Time course studies conducted on the flash chromatography subfractions of ethyl acetate fraction showed FAAH enzyme inhibition activity subsiding gradually over the course of 48 h; at which no subfractions showed significant inhibition. In contrast to that seen with FAAH, time course inhibition of MAGL by the four, active column subfractions increased over the first 6 h, with MF 55-5 increasing over 24 h. MAGL inhibition remained significant for all active subfractions until 24 h and then tapered off and fell below 50% at 48 h. This suggests that FAAH and MAGL enzymes recover with time, thus preventing permanent inhibition and modulation of the endocannabinoid system. This is a favourable pharmacologic characteristic as perpetual inhibition of these endogenous enzymes could lead to deleterious dysregulation. Compound 8 was further evaluated for in vivo effect in the rodent model of anxiety, the EPM. The data revealed a significant anxiolytic effect at 120 mg/kg i.p. dose. Overall, these results provide convincing evidence that nutmeg compounds can indirectly modulate the endocannabinoid system, thus providing support for therapeutic potential in treating disorders such as mood and anxiety.

Several reports have revealed that nutmeg extracts exert cannabis-like effects while limiting the severity of extraneous effects. In vivo administration of n-hexane extracts displayed anxiolysis at doses of 10 mg/kg without impairing locomotion.[15] However, anxiogenic properties were seen with higher doses ranging from 30 to 100 mg/kg,[15,16] similar to the bimodal effect described with cannabis.[17] Another comparable cannabimimetic effect of interest is sedation and the amelioration of pain.[15] Murine studies of nutmeg extracts injected orally or intraperitoneally demonstrated antinociception at effective doses of 300 mg/kg[12] and 1 g/kg.[18] The effect of nutmeg on locomotor activity also resembled that of ∆9-THC.[12] Dose-dependent inhibition of locomotion was observed with the inhalation of nutmeg essential oils. However, it was ascertained from plasma concentrations that incoordination and reduced motor activity were attributed to specific nutmeg compounds: myristicin, safrole, 4-terpineol and elemicin.[19,20]

Although cannabimimetic effects elicited by nutmeg parallel those of ∆9-THC, the exact mechanism by which nutmeg interacts with the endocannabinoid system had not been fully described. Our earlier investigation into the effect of nutmeg on the endocannabinoid system revealed no interaction with CB1 or CB2 receptors. Instead, indirect modulation via FAAH and MAGL inhibition was observed.[13] Accordingly, the results of this study confirm the previous report that nutmeg indirectly modulates the endocannabinoid system via the inhibition of FAAH and MAGL enzymes. To date, no reports have been published about the endocannabinoid-related activity of compounds 6, 8 and 9. Identification of compounds inhibiting FAAH enzyme provides a specific answer to an old question about the CNS effect of nutmeg and an interesting insight into its mechanism of CNS activity. It also introduces new lead compounds for development as therapeutic agents. By analogy to recently published results by Jaiswal et al.,[21] we propose that compounds 6, 8 and 9 possess structural features necessary for effective binding to the active site of FAAH. In compounds 8 and 9, the propenyl benzofuran and substituted benzene have affinities towards the hydrophobic tunnel and the catalytic triad of FAAH, respectively. The same applies for compound 6 whereby the phenylalkyl and benzophenone moieties serve the same purpose as the benzofuran and substituted benzene moieties in compounds 8 and 9. Further modelling studies are needed to substantiate this speculation.

Apart from findings in this report, our most active compound, 8, has not been evaluated for other biological activity. Other in vitro studies suggested that 9 (isolated from Machilus thunbergii) may possess neuroprotective activity against glutamate-induced toxicity of rat cortical cells.[22] Additionally, synthetic 9 exhibited concentration-dependent antiparasitic activity.[23] On the other hand, many studies have demonstrated that 6 has potential pharmacological benefits in vascular disease,[24] promotion of healing, anti-inflammation and angiogenesis caused by stomach ulcers,[25–28] antioxidant activity[29] and cytotoxic activity against certain cancers.[30,31] It also has antibacterial and antifungal properties,[32–34] as well as antiparasitic leishmanicidal and nematocidal activity.[35,36]

Hydroalcoholic extracts of nutmeg were reported to inhibit other enzymes, such as acetylcholinesterase alkaline phosphatase and acid phosphatase in the nervous tissue of Lymnea acuminate.[37,38] Moreover, methanolic nutmeg extract has been implicated for gastrointestinal diseases as it effectively inhibits urease at an IC50 value of 78.19 ± 0.01 μg/ml and conferring protection against Helicobacter pylori infection.[39] Inhibition of elastase activity was also observed with methanolic nutmeg fractions screened at 1 mg/ml, possessing an IC50 value of 284.1 μg/ml.[40] These literature reports suggest that the active compounds identified may have non-specific action on alternative hydrolases within the body, possessing varying degrees of selectivity and potency on each enzyme. Future studies will focus on further investigating the enzyme inhibition selectivity of compounds 6, 8 and 9.

As mentioned, indirect modulation of the endocannabinoid system is a promising approach for drug development as it elicits cannabis-like effect while minimizing extraneous effects. Reversible FAAH inhibition has proven to be beneficial for analgesia[41,42] and anxiolysis[43,44] with minimal adverse outcomes. On the other hand, irreversible inhibition through FAAH double knockout produced hypomotility, catalepsy, hypothermia,[45] infertility,[46] weight gain and insulin resistance.[47] Therefore, enzyme recovery observed by all three active compounds in this study is advantageous as it may limit adverse effects, which could arise when administered in vivo. The lack of significant MAGL enzyme inhibition among 6, 8 and 9 is of additional benefit as dual inhibition of FAAH and MAGL has been associated with catalepsy and hypothermia. Conversely, selective MAGL inhibition reportedly resulted in hyperreflexia, hypomotility and physical dependence;[2] deleterious effects not observed in selective FAAH inhibition. Similarly, anandamide reuptake inhibitors caused memory impairment[48] and lacked analgesic properties as seen with FAAH inhibition. Hence, selective FAAH inhibition appears to offer the best approach for modulation, providing both analgesia and anxiolysis, while avoiding other negative cannabis-like effects such as hypomotility, thermodysregulation and dependence. To determine the potential value of the isolated nutmeg compounds, the most active FAAH inhibitor in this study, compound 8, was evaluated for anxiolytic activity in the EPM test. Compound 8 showed a dose-dependent reduction in anxiety as evidenced by a significant increase in the number of open arm entries. Such anxiolytic effect was not confounded by effects on locomotor function, whereby compound 8 did not alter total locomotor activity in the open field activity chamber test. Similar effects have been reported with other FAAH inhibitors.[43,44,49]

The afore-mentioned therapeutic properties resulting from FAAH inhibition suggest that nutmeg may have utility in treating anxiety disorders. The additional activation of the reward pathway through the endocannabinoid system and lack of dependence associated with FAAH inhibition also indicates these agents may be of prospective benefit in alleviating anxiety associated with substance abuse disorders. Therefore, 6, 8 and 9 have the potential to be used therapeutically. Of importance is the safety profile of these compounds. Behavioural studies have demonstrated no toxicity with nutmeg at doses up to 3 g/kg.[15] However, acute toxicity, denoted by hypoactivity, dizziness and unsteady gait, ensued with doses ≥4 g/kg.[18] These effects were attributed to myristicin;[8,50] a constituent other than the compounds of interest identified in this study. Thus, in vivo pharmacological evaluation of the active compounds in other animal models of anxiety and substance abuse withdrawal is impending. Based on FAAH inhibition potency, compound 8 seems to be the most viable candidate for these behavioural animal paradigms, followed by 9 then 6.

Conclusion

In conclusion, this study provides further evidence that nutmeg extracts target the endocannabinoid system indirectly by inhibiting both FAAH and MAGL enzymes. Such mechanism sheds light on the cannabis-like effects previously reported for nutmeg. When isolated, individual compounds primarily inhibit FAAH showing enzyme recovery within a 48-h time period. Their presence links the observed activity of nutmeg to pure chemical entities present in this spice. The isolated compounds are thus introduced as leads for structure-activity relationship and molecular modelling. In vivo studies of these compounds are also imperative to test the pharmacological effects with particular focus on anxiety, analgesia and substance use disorders. Additional studies are still needed to isolate and identify the compounds responsible for the MAGL inhibition activity previously reported for the ethyl acetate nutmeg extract.

Declarations

Conflict of interest

The Author(s) declare(s) no conflict of interest.

Funding

Research 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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