Published online: 2019-06-24 Original Papers Thieme Reyes-Trejo Benito et al. Evidence for Involvement of … Planta Med Int Open 2019; 00: 00–00 Evidence for Involvement of TRPV1 Receptors and Potassium Channels in the Seizures Induced by α-Sanshool Authors Benito Reyes-Trejo1, Mario Noel Morales-Hernández1, Gloria Melisa González-Anduaga2, José Luis Balderas-López2, José Carlos Tavares-Carvalho3, Andrés Navarrete2 Affiliations ABStract 1 Agricultural High School Department, Natural Products α-Sanshool is an alkamide isolated from the stem bark of Zan- Laboratory, Chemistry Area, Autonomous University thoxylum liebmannianum, a Mexican medicinal plant known as Chapingo, Chapingo, Mexico Colopahtle. Our research group has reported that the intraperi- 2 Faculty of Chemistry, Department of Pharmacy, National toneal administration of α-sanshool induces tonic-clonic sei- Autonomous University of Mexico, University City, zures in mice. In the present study, we investigated the convul- Coyoacan 04510, Mexico City, Mexico sive effect of this alkamide and elucidated its mechanism of 3 Department of Biological Sciences and Health, action by comparing with well-known convulsive and anticon- Laboratory of Pharmaceutical Research, Pharmacy vulsive drugs in an in vivo approach. α-Sanshool showed a po- Course, Federal University of Amapa, Macapa, AP, Brazil tent (ED50 [CL 95 %] = 3.06 [2.92–3.22] mg/kg) and immediate (2 ± 2 s) seizure effect after the intraperitoneal administration Key words in mice. The convulsive effect of this alkamide was only ob- Zanthoxylum liebmannianum (Rutaceae), seizures, TRPV1, served for intraperitoneal administration; the oral route did not potassium channels, isobutylamides show any effect. α-Sanshool was less potent than strychnine (ED50 [CL 95 %] = 1.53 [1.44–1.62] mg/kg), but more effective received 27.09.2018 than bicuculline, 4-aminopyridine, affinin, and pentylenetet- revised 28.02.2019 razol, in that order. The seizures induced by α-sanshool were accepted 01.03.2019 reduced by capsazepine and diazoxide, suggesting the involve- ment of TRPV1 and potassium channels in the mechanism of Bibliography action of this compound. DOI https://doi.org/10.1055/a-0871-2496 Planta Med Int Open 2019; 6: 1–5 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2509-9264 Correspondence Dr. Andrés Navarrete Facultad de Química, Departamento de Farmacia Universidad Nacional Autónoma de México Ciudad Universitaria Coyoacán 04510 Ciudad de México México Tel.: + 52/55/56225291, Fax: + 52/55/56225 329 [email protected] Supporting Information for this article is available online at http://www.thieme-connect.de/products. Reyes-Trejo B et al. Evidence For Involvement Of … Planta Med Int Open 2019; 6: e23–e27 e23 Original Papers Thieme vator), ethosuximide (low-threshold T-type Ca2 + channels blocker), ABBREVIatIONS capsazepine (TRPV1 antagonist), and carbamazepine (voltage-gated sodium channels blocker). CNS central nervous system GC/MS gas chromatography coupled to mass spectrometry ICR Institute of Cancer research Results K2P 2-pore domain potassium channels ▶Fig. 2 shows the dose-response curves for seizures induced by KATP ATP-dependent potassium channels the intraperitoneal administration of increasing doses of the con- TRPV1 transient receptor potential vanilloid type 1 vulsive drugs affinin and α-sanshool. As seen in the plots, the sei- TRPA1 transient receptor potential ankyrin type 1 zure incidence increased with increasing dosage. α-Sanshool was less effective than strychnine (▶Table 1) but more potent than bi- cuculline, 4-aminopyridine, affinin, and pentylenetetrazol, in that Introduction Alkamides are common constituents in plants of the Echinaceaeae, O Zanthoxylum, Capsicum, Piperaceae, Spilanthes, Acmella, and Heliopsis genera [1]. Several plants of the genus Zanthoxylum (Rutaceae) are used N in the traditional medicine of native cultures from Africa, America, and N Asia [2]. These compounds have a diversity of biological activities, including insecticidal, antibacterial, antifungal, antiparasitic, analge- sic, and local anesthetic [3–5]. In the Brazilian Amazon region, Ac- ▶Fig. 1 Structure of α-sanshool. mella oleracea (L.) R.K. Jansen, which contains alkamides, has been reported as an aphrodisiac [6]. Also, some alkamides and the plants that provide them are used as flavoring in foods and beverages due to the unique tingling and numbing orosensations that they produce. For 7.0 example, the Brazilian alcoholic beverage Cachaça de Jambu contains an extract of A. oleracea, in which the alkamide spilanthol is a major metabolite [6]. In recent years, the academic and industrial interest for alkamides has remarkably increased, both for their medical and cosmetic applications and because of their biological mechanisms of 6.0 action [3]. Sanshools are the predominant alkamides in the Zanthoxy- lum genus [3]. The stimulation of transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (TRPV1) channels, the blockage of 2-pore domain potassium channels KCNK3 (TASK-1), KCNK9 (TASK-3) and KCNK18 (TRESK), as well as the antagonism of the cannabinoid recep- s 5.0 tors 1 (CB1) and 2 (CB2) and the agonist effect on CB2, have been Probit suggested as biomolecular targets for these compounds [3]. In a pre- vious study, we reported the isolation of α-sanshool (▶Fig. 1) from the stem bark of Zanthoxylum liebmannianum (Engelm.) P.Wilson (Rutaceae) and its anthelmintic effect in naturally infected sheep [4]. In that work, we also noticed that the intraperitoneal injection of this 4.0 compound induced tonic-clonic seizures in mice. Although low toxic- ity is generally associated with natural and synthetic alkamides, their convulsive effect in mice was recently reported [7]. This is of great significance due to the growing medical and cosmetic interest in plant extracts containing these compounds, even though little is known 3.0 about their toxic effects. Accordingly, we were interested in knowing the convulsive potency of α-sanshool, a natural alkamide. In this work, –0.5 0.00.5 1.01.5 2.02.5 we compared the seizures induced by α-sanshool with those caused Log D (mg/kg) by 4 known convulsive drugs: bicuculline (GABA antagonist), 4-ami- nopyridine (voltage-activated potassium channels KCND2/KCND3 ▶Fig. 2 Dose response curves for seizures induced by the intraperi- blocker), strychnine (glycine antagonist) and pentylenetetrazol toneal administration of α-sanshool (○), strychnine (▲), bicuculline (GABAergic blocker), and also with the effect of affinin (spilanthol), (■), 4-aminopyridine (●), affinin (□), and pentylenetetrazol (▼). The isolated from Heliopsis longipes (A. Gray) S.F. Blake (Asteraceae) roots. graph represents the decimal logarithm of the doses used for each drug vs. probit. Each symbol represents the probit, derivate from the We also investigated the inhibition of the α-sanshool-induced seizures cumulative frequency of animals that present seizures/total of ani- by diazepam and pentobarbital (positive allosteric modulators of mals (30) for each dose. GABAA receptors), diazoxide (ATP-sensitive potassium channels acti- e24 Reyes-Trejo B et al. Evidence for Involvement of … Planta Med Int Open 2019; 6: e23–e27 order. The latency to the first seizure induced by this alkamide was been suggested as a biomolecular target for alkamides [3]. There- extremely short, only 2 s (▶Table 1), with a mortality of 100 %. fore, the partial inhibition of the α-sanshool-induced convulsions Contrastingly, the oral administration of α-sanshool at the same by the TRPV1-antagonist capsazepine suggests that this alkamide doses than those used for the intraperitoneal injections did not in- induces the seizures, at least in part, by stimulating the TRPV1 duce seizures. The seizure incidence and the mortality induced by channels (▶Table 2). It is possible that convulsions induced by af- α-sanshool at 9 mg/kg were reduced by 84 % when administering finin, capsaicin, and the synthetic alkamide reported by de la Rosa capsazepine (50 mg/kg, s.c.). Diazoxide reduced them by 44 % (at et al. [7] also occur via the same mechanism. Additional experimen- 10 mg/kg, i.p.) and 50 % (at 100 mg/kg, i.p.). Diazepam and pento- tal data are required to assess this statement. barbital were effective (66 % reduction) only at very high doses. K2P channels have also been suggested as molecular targets for Carbamazepine and ethosuximide did not inhibit the α-sanshool- alkamides [3]. These channels are widely expressed in the central induced seizures (▶Table 2). and peripheral nervous system, and their blockage has been associ- ated with local anesthetic-induced seizures [11]. In a previous work, it was reported that the convulsions induced by the local anesthetic Discussion bupivacaine (50 mg/kg, i.p.), a blocker of K2P channels, were reduced α-Sanshool, a compound found in the stem bark of Z. liebmanni- by intraperitoneal administration of diazoxide (10 and 100 mg/kg), anum [4, 8], showed potent and immediate tonic-clonic seizures in which activates KATP channels [12]. Here, we found that diazoxide mice. This effect was partially inhibited by capsazepine, diazoxide reduced the α-sanshool induced seizures (▶Table 2) at the same and high doses of diazepam and pentobarbital (▶Table 2). The doses than those used to reduce the bupivacaine-induced seizures powerful (ED50 = 3 mg/kg) and immediate (2 s) seizure-inducing ef- [12]. Furthermore, seizures induced by 4-aminopyridine, a blocker fect showed by this compound differs from the pattern of the other of voltage-activated