Alkylamides from Acmella oleracea: antinociceptive effect and molecular docking with cannabinoid and TRPV1 receptors

Abstract Alkylamides are secondary metabolites in Acmella oleracea and display wide applications in treating several diseases. Since alkylamides can inhibit pain, this work aims to evaluate the antinociceptive profile of A. Oleracea methanolic extracts used in vivo and in silico assays. The extracts inhibited the neurogenic and inflammatory phases of the formalin test, ratifying the antinociceptive effect of alkylamides. Furthermore, the results from molecular docking demonstrated the interaction of A. oleracea alkylamides with the CB1/CB2 and TRPV1 receptors. Additionally, the crude methanolic extract of flowers did not induce potential side effects related to the classical cannabinoid tetrad: hypolocomotion and catalepsy. In conclusion, this work confirms the potential of the alkylamides of A. Oleracea as antinociceptive agents and, for the first time, correlates its effects with the endocannabinoid and vanilloid systems through in silico assays. Graphical Abstract


Introduction
The interest in the alkylamide class of secondary metabolites has been increasing in biomedical research due to their several biological actions (Elufioye et al. 2020), such as immunomodulatory (Paulraj et al. 2015), larvicide (de Ara ujo et al. 2018;Ara ujo et al. 2020), antimicrobial (Arora et al. 2011;Arora and Vijay 2011;Thakur and Bhamare 2015), diuretic (Ratnasooriya et al. 2004;Yadav et al. 2011), analgesic (Silveira et al. 2018), and antioxidant (Tanwer et al. 2010;Lalthanpuii et al. 2017). The presence of alkylamides has already been described in species from the genera Achillea, Acmella, Echinacea, Heliopsis and Spilanthe, all belonging to the Asteraceae family (Elufioye et al. 2020). Literature highlights Acmella oleracea, a specie popularly known as jambu and widely consumed in northern Brazil, due to its chemical profile rich in alkylamides and its broad use in folk medicine (Simas et al. 2013). Spilanthol is the main alkylamide present in A. Oleracea leaves, being responsible for its pungent flavor appreciated in culinary. Furthermore, there are medicinal properties already described for this molecule including anesthetic, anti-inflammatory, and antinociceptive activities (Ramsewak et al. 1999;Favoreto and Gilbert 2010;Barbosa et al. 2016;De Freitas-Blanco et al. 2019).
Alkylamides are structurally related to endocannabinoids, such as anandamide. They are amides with a long polyunsaturated carbon chain as the acyl portion. Anandamide activates the endocannabinoid system by interacting with CB1 and CB2 receptors. The immunomodulatory property described for the species of Echinacea is related to the activation of CB2 receptors by alkylamides, modulating the expression of TNF-a in human monocytes and macrophages (Gertsch et al. 2004). Binding studies confirmed the interaction of alkylamides at the CB2 receptor (Raduner et al. 2006). CB1 and CB2 agonists induce antinociception in models of inflammatory hyperalgesia (Romero et al. 2013). Isobutylamides type alkylamides derived from Echinacea bind to CB1/CB2 receptors in the nanomolar and micromolar range. These molecules also increase intracellular Ca þ2 concentrations in cells expressing CB2 receptors, and this effect can be blocked by the CB2 antagonist SR144528 (Raduner et al. 2006).
Another pharmacological target explored for acute pain control is the vanilloid system. Several studies have shown that agonists of TRPV1 receptors like capsaicin are able to alleviate neuropathic pain by inducing nerve fibers desensitisation in a mechanism modulated by repeated activation of TRPV1 receptors (Derry et al. 2017). TRPV1 receptor antagonists like capsazepine also block nociception induced by intraplantar administration of formalin and capsaicin in mice (Sakurada et al. 2003). Several reports describe the ability of anandamide and other alkylamides to interact with TRPV1 (Ross 2003). Activation of TRPV1 receptors by alkylamides is often correlated with antinociceptive effects. For example, alkylamides obtained from A. oleracea attenuate pain sensation in the formalin test, and this effect is partially explained by TRPV1 receptor modulation (Dallazen et al. 2018). Moreover, in silico evaluation using molecular docking showed that spilanthol and several alkylamides isolated from Heliopsis longipes interact with the TRPV1 receptor (de la Rosa-Lugo et al. 2017).
Despite so many biological applications with promising results, the mechanisms by which A. oleracea alkylamides can produce their antinociceptive effects are yet unclear. This work proposes a possible correlation between the anti-inflammatory and antinociceptive activities of alkylamides and their chemical similarity with endogenous ligands of the cannabinoid system in such a way that cannabinomimetic effects would induce their antinociceptive activity. Therefore, we performed nociception and behavioral assays in vivo using the crude extract of aerial parts and flowers of A. oleracea, as well as in silico molecular docking to evaluate the interaction of these substances with CB1, CB2 receptors and, in a complementary way, TRPV1 receptors.

Results and discussion
The antinociceptive effects of crude methanol extract of flowers (CEF) and aerial parts (CEAP) were evaluated in the formalin test (Hunskaar and Hole 1987). The extracts were previously analysed by GC-MS and the presence of spilanthol, fatty acids such as palmitic acid, and steroids such as sitosterol and stigmasterol, were detected ( Figure  S1, Supplementary material). In the pharmacological evaluation, both extracts reduced the licking time in the first and second phases of the formalin test ( Figure S2), also known as neurogenic and inflammatory phases, respectively .
At the dose of 100 mg/Kg, CEF and CEAP inhibited the paw licking time at the neurogenic phase of the formalin test by 33.4% and 57.8% (68.3 ± 3.2 s for vehicle versus 45.5 ± 6.4 s for CEF and 28.8 ± 5.2 s for CEAP), respectively ( Figure S2A). Morphine at a dose of 10 mg/kg (i.p.) inhibited the neurogenic phase by 52.5% (32.4 ± 6.0 s). Morphine is a centrally acting analgesic that activates l-opioid receptors, which justifies the reduction in the nociceptive behavior found at this phase. In the second phase of the test, when inflammatory mediators such as cytokines and prostaglandins are produced, the paw licking time was inhibited by 59.4 and 53.5% (262,5 ± 47,1 s for vehicle versus 106.6 ± 20.1 s and 122.1 ± 15.0 s) for CEF and CEAP, respectively ( Figure  S2B). At a 10 mg/kg dose, indomethacin, a non-steroidal anti-inflammatory drug (NSAID), inhibited the inflammatory phase by 60.5% (103.8 ± 25.3 s). The decrease in the licking time in the first and second test phases demonstrates that the flower and aerial parts extracts of A. oleracea present an antinociceptive profile inhibiting the nociception produced by direct stimulation of nociceptive fibers in the first phase by formalin. In addition, this extract also presented an inhibitory effect in the nociception induced by the action of inflammatory mediators in the second phase of this test.
These results align with evidence described in previous works using alkylamides from A. oleracea. Ratnasooriya and Pieris (2005) demonstrated that the aqueous extract from jambu flowers decreased the paw licking time in both phases of the formalin test in mice. In another study, Dallazen et al. 2018 demonstrated the antiallodynic and antiedematogenic effect of the hexanic extract of jambu flowers administrated locally in mice paw in the carrageenan-induced acute inflammation model. Our study differs from the previous ones in some features, for example, the kind of extract of A. oleracea. In this work, we evaluated the methanolic extract of flowers and aerial parts in contrast to other studies that worked with hexanic, ethanolic or aqueous extracts. In addition, we adopted oral administration differing from intraplantar and intraperitoneal administration used in previous papers (Nomura et al. 2013;Dallazen et al. 2018). The maintenance of the antinociceptive effect of methanolic extract of A. Oleracea flowers when orally administered is an important finding since oral administration is the most used in treating pain states. Furthermore, this route of administration is cheap, accessible to self-administrate, and desirable for developing phytopharmaceutical products.
The antinociceptive and peripheral anti-inflammatory actions of several alkylamides, as anandamide, are described as a function of CB1, CB2 and TRPV1 receptors activation (Jara-Oseguera et al. 2008;Romero et al. 2013;Muller et al. 2020). Thus, it is plausible that the antinociceptive effect of alkylamides from A. oleracea can also be produced by modulation of these receptors, at least in part. To clarify this assumption, molecular docking studies were conducted on CB1, CB2 and TRPV1 receptors. The chosen alkylamides were anandamide (an endogenous agonist of CB1, CB2, and TRPV1 receptors), spilanthol (major alkylamide of CEAP, named ALK2), and five other alkylamides from A. oleracea described in our previous work (Simas et al. 2013) named as ALK1, ALK3, ALK4, ALK5, and ALK6. These structures are presented in Figure S3. Table  S1 shows the affinity energies computed by docking simulations for all the compounds with the receptors. The simulated affinity values for all alkylamides ranged from À6.8 to À8.5 kcal.mol À1 , similar to the calculated affinity for the CB1/CB2 agonist anandamide, À7.5 and À7.7 kcal.mol À1 , respectively. These data suggest that the alkylamides present in A. oleracea should be able to activate both CB1 and CB2 receptors. The simulated affinity values for TRPV1 receptor interactions ranged from À6.2 a À7.5 kcal.mol À1 including anandamide.
For the CB1 receptor, we chose a structure whose receptor is co-crystalised with the synthetic antagonist AM6538 (PDB ID: 5TZG) (Hua et al. 2016). X-ray data revealed reversible CB1-AM6538 complexation without forming a covalent bond. Although the receptor structure is in its inactive state, we were able to evaluate how representative agonists (such as anandamide) interact with CB1 receptors, pointing to relevant interactions for the agonism. We started by performing a redocking to validate our protocol. Superposition of crystallographic and redocked ligand (shown in Figure S4) resulted in a root mean square deviation (RMSD) of 0.263 Å, below the value accepted by literature (1.5 or 2.0 Å depending on the ligand size) (Hevener et al. 2009). Then, docking of anandamide resulted in interactions with Phe268 and Phe379 residues of CB1 ( Figure S5A), in line with experimental observations (Ahn et al. 2009;Hua et al. 2016). These interactions appeared for spilanthol ( Figure S5B), which adopted a Cshaped conformation. Likewise, Phe268 and Phe379 residues interacted with all the other docked alkylamides, except for ALK1, which displayed interaction only with Phe268. Figure S5 shows the molecular docking for the other alkylamides. It is important to highlight that these interactions established between Phe268 and Phe397 are all by dispersive van der Waals forces. Molecular docking suggests that these residues interact with the long-chain unsaturated acyl portion for anandamide, the other docked alkylamides, except for ALK3, which interactions occurred with the N-ethylbenzene portion.
For CB2 (PDB ID: 5ZTY) (Li et al. 2019), the superposition of the ligand AM10257 with the crystallographic structure resulted in an RMSD value of 0.503 Å. Li et al. (2019) demonstrated that interaction with the aromatic ring of Trp258 is directly involved with the antagonism of CB2, which is reproduced by our docking with the synthetic antagonists AM10257 ( Figure S6). Furthermore, Trp258 residue is not interacting with the agonists anandamide and MRI2594 ( Figure S7), in line with the literature (Li et al. 2019). For these molecules, docking showed van der Waals interactions with Phe106, Ile110, and Val113 amino acids and p-interaction with the aromatic ring of Phe117. Phe87, Phe91, Phe117, Phe183, and Phe281 residues also showed hydrophobic interactions for both agonists. Concerning the other alkylamides, we also observed hydrophobic interactions with Phe183 and Phe281 residues ( Figure S8). Additionally, most alkylamides interacted with Phe91, Phe106, Ile110, and Val113 residues. It is important to highlight that none of the alkylamides displayed interactions with Trp258 aminoacid residue.
For TRPV1 (PDB ID: 5IRX) (Gao et al. 2016), redocked resiniferatoxin (RTX) showed a good superposition with the experimental conformation of the ligand (RMSD of 1.645 Å). ( Figure S9). Moreover, we were able to reproduce interactions with aminoacid residues Thr550, Tyr511, Met547, and Arg557, mostly hydrophobic, identified by mutation studies as essential interactions for TRPV1 activation (Winter et al. 2013;Elokely et al. 2016). Docking with anandamide showed hydrogen bonds between the hydrogen atoms of the amide function and the terminal hydroxyl with Ser512 and Asn551. Hydrophobic interactions with Thr550, Tyr511, Met547, and Arg557 were also observed. For the other alkylamides, a similar interaction pattern was observed, except for the Arg557 interaction that was not observed for ALK1 and spilanthol ( Figure S10). Furthermore, for all structures presenting an aromatic ring (ALK3, ALK5, and ALK6), Tyr511 establishes a p-p T-shaped interaction.
Aiming to study whether A. Oleracea could induce other CB1 receptor-related effects in vivo, the open field and catalepsy tests in mice were conducted. These tests are part of the cannabinoid tetrad, composed of four in vivo biological effects induced by systemic administration of CB1 agonists: hypolocomotion, catalepsy, hypothermia, and analgesia (Martin et al. 1991;Compton et al. 1992). In the open field test, treatment with 100 mg/kg of CEF was unable to reduce the total distance traveled compared to the control group (control: 14,049 ± 1458 cm versus CEF: 11,036 ± 1363 cm) ( Figure S11A). Furthermore, CEF was not able to induce cataleptic behavior in mice (control: 0.81 ± 0.19 s versus CEF: 1.1 ± 0.19 s) as typically induced by CB1 agonists ( Figure S11B). These results demonstrated that the administration of CEF did not reproduce all the classical effects of CB1 receptor agonism in vivo. However, previous studies have already shown that CB1 agonists may induce biological effects at doses that do not alter rodents' mobility (Kruk-Slomka et al. 2015;Poleszak et al. 2020). Since only one dose was evaluated in these tests, these data do not exclude the potential involvement of CB1 receptors in the antinociceptive effect induced by CEF, especially the peripheral ones. Moreover, the observation that CEF has an antinociceptive effect at a dose devoid of motor effects can be considered an advantage of these alkylamides compared to other cannabimimetic potential analgesics.

Conclusion
This work shows for the first time that the alkylamides present in A. oleraceae have an acute antinociceptive effect after oral administration, confirming their adequate bioavailability. Moreover, these effects were observed at a dose devoided of potential cannabinomimetic motor side effects. Molecular docking studies showed interactions of these alkylamides with cannabinoids (CB1 and CB2) and TRPV1 receptors. We propose that A. oleracea alkylamides antinociceptive effects can be produced by a central and peripheric effect directly in nociceptors. Thus, the mechanism of action of alkylamides is complex and can trigger an anti-inflammatory and antinociceptive response in several physiological pathways. Therefore, further studies are needed.

Disclosure statement
No potential conflict of interest was reported by the authors.