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Antinociceptive effects of Salvia divinorum and bioactive salvinorins in experimental pain models in mice
Journal Pre-proof Antinociceptive effects of Salvia divinorum and bioactive salvinorins in experimental pain models in mice Lorenzo Leonel Tlacomulco-Flores, Myrna Déciga-Campos, María Eva GonzálezTrujano ME, Azucena Ibeth Carballo-Villalobos, Francisco Pellicer PII:
S0378-8741(19)30588-4
DOI:
https://doi.org/10.1016/j.jep.2019.112276
Reference:
JEP 112276
To appear in:
Journal of Ethnopharmacology
Received Date: 12 February 2019 Revised Date:
23 September 2019
Accepted Date: 3 October 2019
Please cite this article as: Tlacomulco-Flores, L.L., Déciga-Campos, M., González-Trujano ME, Marí.Eva., Carballo-Villalobos, A.I., Pellicer, F., Antinociceptive effects of Salvia divinorum and bioactive salvinorins in experimental pain models in mice, Journal of Ethnopharmacology (2019), doi: https:// doi.org/10.1016/j.jep.2019.112276. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Antinociceptive effects of Salvia divinorum and bioactive salvinorins in experimental pain models in mice
Pharmacological evaluation
Chromatographic analysis
Salvia divinorum leaves Salvinorin A
Crude extract, fraction and salvinorin’s mixture
1 Antinociceptive effects of Salvia divinorum and bioactive salvinorins in experimental pain models in mice
Lorenzo Leonel Tlacomulco-Floresa,b&, Myrna Déciga-Camposb&, María Eva GonzálezTrujano MEa*, Azucena Ibeth Carballo-Villalobosc, Francisco Pellicera.
a
Laboratorio
de
Neurofarmacología
de
Productos
Naturales.
Dirección
de
Investigaciones en Neurociencias. Instituto Nacional de Psiquiatría “Ramón de la Fuente”. Av. México-Xochimilco 101, Col. Sn Lorenzo Huipulco, 14370. Ciudad de México, México. b
Sección de Estudios de Posgrado e Investigación de la Escuela Superior de Medicina,
Instituto Politécnico Nacional. Plan de San Luis y Díaz Mirón s/n Col. Casco de Santo Tomás 11340, Ciudad de México, México. c
Departamento de Química Inorgánica y Nuclear. Facultad de Química, Universidad
Nacional Autónoma de México. Conjunto E. Circuito de la Investigación Científica. Ciudad Universitaria 04510, Ciudad de México, México.
This work was partially supported by the projects CONACYT-226454/256448. &
These authors contributed equally.
*Corresponding author: Instituto Nacional de Psiquiatría “Ramón de la Fuente” Av. México-Xochimilco No. 101, Col. San Lorenzo Huipulco Delegación Tlalpan 14370, Ciudad de México, México Tel.: (+52 55) 4160-5085 Fax: (+52 55) 5655-9980 E-mail address: [email protected]
2 Abstract Ethnopharmacological relevance. Salvia divinorum Epling & Játiva is a Mexican plant used not only in rituals but also in traditional medicine for pain relief. One of the most known bioactive compounds is salvinorin A, which acts centrally in kappa-type opioid receptors. Aim of the study. Despite its traditional use as a medicinal plant, there is not enough scientific investigation to reinforce its potential as analgesic. In this study, Salvia divinorum antinociceptive activity was evaluated in experimental models of nociceptive pain; the writhing test and formalin-induced licking behavior in mice. Material and Methods: Different Salvia divinorum extracts were prepared by maceration at room temperature in increased polarity (hexane, ethyl acetate and methanol). The ethyl acetate extract (EAEx) was chosen in order to be fractioned and to obtain a mixture of salvinorins. The antinociceptive effect of EAEx (3, 10, 30, and 100 mg/kg, i.p.) was compared with that of tramadol (a partial opioid agonist analgesic drug, 30 mg/kg, i.p.) and the mixture of salvinorins (30 mg/kg, i.p.). In addition, a participation of opioids (naloxone, NX 1 and/or 3 mg/kg, i.p.) and serotonin 5-HT1A receptors (WAY100635,
0.32
mg/kg,
i.p.)
was
investigated
as
possible
inhibitory
neurotransmission involved. Results. As a result, the EAEx produced significant and dose-dependent antinociceptive effect concerning salvinorins constituents. This effect was blocked in the presence of NX and WAY100635 in the abdominal test, but only by NX in the formalin-induced licking behavior. Whereas, the effect of salvinorins mixture involved opioids and serotonin 5HT1A receptors.
3 Conclusion. Data provide evidence of the potential of this species, where salvinorin A is in part responsible bioactive constituent involving participation of the opioids and/or 5HT1A serotonin receptors depending on the kind of pain model explored. Key words: Nociception, Opioids, Salvia divinorum Epling & Játiva, Salvinorins, serotonin. 1. Introduction Salvia divinorum belongs to the Salvia genus (Lamiaceae Family). This genus has a great diversity of species in the world (Martínez-Gordillo et al., 2013), but Mexico is one of the areas with greater distribution with more than 300 endemic species (CornejoTenorio and Ibarra-Manríquez, 2011; Martínez-Gordillo et al., 2013). Oneirogen is one of the effects recognized for this species (Valdés et al., 1983; Díaz, 2013; GonzálezTrujano et al., 2016), in part because of the presence of Salvinorin A (Ortega et al., 1982). This endemic plant from Mexico, essentially from the state of Oaxaca, has been used by Mazatecs in mystic rituals (Díaz, 2013). As traditional medicine, it is used to alleviate different ailments related to pain, such as stomachache, headache and rheumatism (Valdés et al., 1983). In fact, several Salvia species are recognized to produce pain relief, such as: S. officinalis, S. miltiorrhiza, S. mexicana, S. lerrifolia, S. africana-lutea, S. aethiopis, S. aegyptiaca (Imanshahidi and Hosseinzadeh, 2006). A great variety of compounds has been isolated from this genus, including phenolic acids, tannins, essential oils, flavonoids and terpenoids (mono, di, and triterpenes), many of them with biological activity (Wu et al., 2012). From the phytochemistry of S. divinorum at least 22 compounds of diterpene nature has been described, from these ten belong to the chemical group salvinorins (A to J) (Lee et al, 2005, Shirota et al., 2006, Kutrzeba et al., 2009), six to the divinatorins
4 (A to F) (Lee et al., 2005; Bigham et al., 2003; Shirota et al., 2006), four to the salvidivins (A to D) (Shirota et al., 2006), and two to the salvinicins (A and B) (Harding et al., 2005). From the salvinorins group, the salvinorin A is considered a principal bioactive molecule of this plant in part due to an agonist action on Kappa opioids and cannabinoids (CB1 and CB2) receptors (Roth et al., 2002; Capasso et al., 2006; 2008; Fichna et al., 2011). It has also been related to the release or modulation of monoaminergic neurotransmitters serotonin, dopamine and noradrenaline (Grilli et al., 2009; Pittaluga et al., 2013). Clinical experience with terpenes for relieving pain has demonstrated beneficial effects with generally non-serious adverse effects (Guimarães et al., 2013). Salvia divinorum is a great source of bioactive diterpenes like salvinorins; however, its pharmacological evaluation as a crude extract supporting its potential for pain relief as in folk medicine has not been explored. In this study, the antinociceptive effect of Salvia divinorum leaf extract, that was obtained under controlled hydroponic planting, was pharmacologically evaluated in pain models like the writhing test and formalin-induced licking behavior in mice to look for bioactive compounds and possible inhibitory neurotransmission involved.
2. Materials and methods 2.1. Animals Male and female Swiss albino mice (25-30 g) grouped in six animals were used for the study. Animals were housed at the facilities of the Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz and kept at constant room temperature (22±1°C) in a 12h/12h light/dark cycle. Experiments were carried out in accordance with the Ethical
5 Committee Guidelines laid down by the INP “Ramón de la Fuente” local authority (NC12.3280.0/August 22, 2017) and under the technical specifications of national (SAGARPA NOM-062-ZOO-1999) and international regulations regarding the care and use of animals for experimental procedures. All animals were fed with standard food and water ad libitum. 2.2. Drugs Tramadol was purchased by Grünenthal de México, S.A. de C.V. Naloxone and WAY100635 from Aldrich Química, S.A. de C.V. Acetic acid and formalin (J.T. Baker, USA). Salvinorin A (purity >99%) and salvinorin B (purity >93%) were purchased by Abcam Biochemicals (Cambridge, MA, USA) and Sigma (Sigma-Aldrich Co., St Louis, MO, USA), respectively, and used as standards. Control animals received the vehicle (0.5% Tween 80 in saline solution). Acetic acid and formalin were both used in 1% solutions as algesic substances to induce nociception. Drugs were freshly prepared on the day of the experiments; all treatments were administered via intraperitoneal (i.p.) in a volume of 0.1 ml/10 g body weight. Except at the beginning of the study, when the hexane, ethyl acetate and methanol crude extracts were evaluated to choose the most active using intragastric route of administration. 2.3. Plant material Leaves of Salvia divinorum Epling & Játiva-M (Lamiaceae) were obtained from Altiva Productos Hortícolas S.P.R de R.L. in Altiva Montecasino, Huitzilac, state of Morelos (latitude 19° 1´ 38.75´´ N/longitude 99° 16´ 16.08´´ W/elevation 2507 m above sea level) under the taxonomic identification of MSc Rogelio Silos Medina (Lot. INPJRF-1A/25-052012). All plants were grown in a hydroponic cultivation system under controlled
6 conditions of temperature, humidity and nutrients. The leaves were sorted out and then air-dried. 2.4. Extract preparation The extracts of different polarity were obtained from the dried and ground leaves (600 g) macerated at room temperature 22 ± 2°C for 48 h. The process began with a degreasing of the material with hexane (3.5 L, 3 times) followed by a second extraction with ethyl acetate (3.5 L, 3 times), and finally a third extraction using methanol (3.5 L, 3 times). After filtration, the solid residue was discarded, and liquid was concentrated under reduced pressure in a rotatory evaporator for the total removal of the solvent. The hexane solvent-free crude extract formed a dark green semisolid (4.96 g, 0.83%); the ethyl acetate extract (EAEx) was also a dark green semisolid (16.79 g, 2.8%), and the methanolic extract a dark brown syrupy (59.88 g, 9.98%). EAEx was selected to explore antinociceptive activity since it contains compounds like salvinorin A. Thus, it was fractionated by chromatographic open column to isolate a salvinorin mixture in approximately 3 mg/g of crude ethyl acetate extract, in which salvinorin A was identified by ultra-performance liquid chromatography (UPLC) (Fig. 1). Thus, it would be expected that 50 mg of salvinorin mixture were included in the plant material explored. 2.5 UPLC analysis The EAEx (10 mg/ml), salvinorin mixture or individual salvinorin A and B standards were diluted in methanol, filtered through a 0.2 mm filter (GHP, acrodisc 13, Waters), and then injected directly (10 µl injection volume). The mobile phase consisted of acidified water at 0.1% phosphoric acid (A) and methanol (B) (HPLC grade). The initial gradient elution was 50% A: 50% B to reach 100% B (15 min).
System regeneration was
achieved at 50% A: 50% B. The flow rate was 1 ml/min with an elution curve of 4. An
7 Acquity UPLC Waters with a PDA eλ detector controlled by EmPower 3TM software was equipped with a Symmetry C18 100Å (150 mm x 4.6 mm, 5µm) column (Waters, made in Ireland) and the thermostat was set at 43°C. The salvinorin peak profile was identified at UVλ max 210 nm. 2.6. Pharmacological evaluation 2.6.1 Writhing test Number of writhes was recorded in 5 min-intervals during a 30 min-period after injection of 1% acetic acid at volume of 10 ml/kg, i.p. Abdominal writhing was considered as nociceptive behavior defined as an exaggerated extension of the abdomen combined with the outstretching of the hind limbs according to Collier et al., 1968. To choose the most active extract, independent groups of mice received one of the three extracts (hexane, EAEx or methanol) using esophageal administration, then latency to the first writhe and AUC (0-30 min) were evaluated in this test. From the results of the previous evaluation, the EAEx was selected as active to continue experiments not only in the writhing test but also in the formalin-induced licking behavior in mice. In these experiments an intraperitoneal injection was used to optimize the extract, fractions and pure compounds to reach the antinociceptive response. Then, animals received the EAEx at 3, 10, 30, and 100 mg/kg, i.p., a salvinorin mixture (30 mg/kg, i.p.), or the reference drug (tramadol, 30 mg/kg, i.p.) 30 minutes before the algesic administration. Control animals received vehicle by the same route and volume of administration. 2.6.2. Formalin-induced licking behavior Mice were injected in the subplantar area of the right hind paw with 20 µl diluted of 1%
8 formalin by using a 30-gauge needle. The number of licking was obtained as nociceptive behavior in the first min and each 5 min during a period of 30 min after formalin administration. According to the Hunskaar et al. (1985), two stages were obtained from this temporal course curves taken as neurogenic (0-10 min) and inflammatory phases (10-30 min). Thirty min before the intraplantar injection of formalin, animals were administered varying doses of EAEx (30 and 100 mg/kg, i.p.), or with a salvinorin mixture (30 mg/kg, i.p.) or the reference drug at the same dosage. Control animals received vehicle. 2.6.3 Inhibitory neurotransmission In order to investigate participation of opioids and 5-HT1A serotonin receptors in the antinociceptive activity of S. divinorum, a significant antinociceptive response of the EAEx was explored in the presence of the antagonists: naloxone (NX, an opioid antagonist, 1 and/or 3 mg/kg, i.p.), and WAY100635 (WAY, a selective 5-HT1A antagonist, 0.32 mg/kg, i.p.). 2.7 Statistical analysis Temporal course curves were plotted to describe the response of several doses of S. divirorum EAEx in comparison to the vehicle group. Statistical differences were analyzed using a two-way repeated-measures analysis of variance (ANOVA) followed by Sidak´s multiple comparisons test for factors like time and treatment. From temporal course curves, the area under the curve (AUC) was obtained using the trapezoidal rule and data are presented in bars as the mean ± SEM of 6 repetitions, then statistical differences were analysed using a one-way ANOVA followed by Dunnett´s t test. ANOVA on ranks followed by Dunn´s test or Student´s t test was also applied according
9 to data. A value of p<0.05 was considered significant. Statistical analysis was done using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Antinociceptive response in the writhing test Figure 2 shows the temporal course curves of the abdominal stretches induced by 1% acetic acid, i.p., in mice receiving vehicle or the individual extracts in increasing polarity (hexane, EAEx and methanol at 100, 300 and 400 mg/kg) by oesophagi route of administration (p.o.) in comparison to the vehicle group (Fig. 2A). In this preliminary screening, the EAEx showed better significative antinociceptive response in mice (Fig. 2B). The same graphic shows the antinociceptive effect of several doses of S. divinorum EAEx (3 to 100 mg/kg, i.p.) and the salvinorin mixture (30 mg/kg, i.p.). The lowest doses of the extract (3 and 10 mg/kg, ip) and the mixture of salvinorins (30 mg/kg, i.p) produced a decrease in stretches with respect to the vehicle (Fig. 2C). These effects were not greater than those obtained with tramadol (TR30, 30 mg/kg, i.p.), used as reference drug. In contrast, the effect of the two highest doses (30 and 100 mg/kg) of the extract resembled that obtained with tramadol that decreased nociceptive response up to 50% in mice (Fig. 2C). Nociception expressed as AUC described significant effects of the S. divinorum EAEx from a dosage of 10 mg/kg, i.p. and in a dose-dependent manner, it produced similar effect than tramadol at the same dosage (30 mg/kg, i.p.) (Fig. 2D). The dosage of 30 mg/kg of the extract was the effective dose 50%. The maximum response was obtained with 100 mg/kg producing 92% of antinociceptive response. The salvinorin
10 mixture (MIX30) produced 30% of antinociceptive response at a dosage of 30 mg/kg in the acetic acid test (Fig. 2D). 3.2. Antinociceptive response in the formalin-induced licking behaviour Figure 3 shows the temporal course curves of the antinociceptive behaviour induced by 1% formalin by intraplantar administration in mice receiving vehicle. This graphic also showed the total abolition of the antinociceptive effect in the presence of tramadol (TR30, 30 mg/kg, i.p.) in mice in neurogenic (0-10 min) and inflammatory phases (10-30 min) (Fig. 3A). All doses of S. divinorum EAEx (10 to 100 mg/kg, i.p.) and the salvinorin mixture (MIX30, 30 mg/kg, i.p.) produced inhibition in the behavioural nociceptive response, major emphasis was noticed in the inflammatory phase (Fig. 3A). Data from AUC describe that nociceptive response in the neurogenic phase was significantly diminished by the extract from a dosage of 10 mg/kg (Fig. 3A). An increase in doses (30 and 100 mg/kg, i.p.) improved the antinociceptive effect (Fig. 3A). Salvinorin mixture did not reach significance at a dose of 30 mg/kg in this phase (Fig. 3B). Additionally, behavioural nociceptive response in the inflammatory phase was significantly decreased in a dose-dependent manner from a dosage of 10 mg/kg, i.p. of S. divinorum EAEx (Fig. 3C). Significant effect was obtained with the salvinorin mixture in the inflammatory phase that resembled those observed with the extract at the minimal dose of 10 mg/kg and tramadol at the same dosage of salvinorins (Fig. 3C). 3.3. Inhibitory neurotransmission In the writhing test, it was observed that administration of NX (1 mg/kg, i.p.) did not show antinociceptive effect per se respect to the response observed in the vehicle group. In
11 contrast, NX avoided the significant antinociceptive effect of the S. divinorum EAEx (30 mg/kg, i.p.) and the salvinorin mixture (30 mg/kg, i.p.) (Fig. 4A). In a similar manner, the presence of WAY100635 (WAY 0.32 mg/kg, i.p.) did not modify the nociceptive response after individual administration, whereas antinociceptive response obtained with the EAEx or salvinorins mixture at 30 mg/kg, i.p. was totally inhibited when combined with this 5-HT1A antagonist (Fig. 4A). Regarding to the formalin-induced licking behavior, both antagonists per se did not modify significantly the nociceptive response in the neurogenic (Fig. 4B) and inflammatory phases (Fig. 4C). Nevertheless, in the case of the antinociceptive response of the EAEx, it was partially blockage when combined with NX at 1 mg/kg. A complete inhibition of the antinociceptive response in both phases of the formalin test was obtained in the presence of NX at 3 mg/kg (Figs. 4B and 4C). Whereas no modification in the antinociceptive effect of the EAEx was obtained in the presence of WAY100635 (0.32 mg/kg, i.p.) in these two phases of the formalin-induced licking behavior in mice (Figs. 4B and 4C). Regarding the antinociceptive effect of salvinorins mixture, it was observed that in an equivalent manner, the antagonists NX (3 mg/kg, i.p.) and WAY100635 (0.32 mg/kg, i.p.) prevented its antinociceptive effect in both the writhing test (Fig. 4A) and formalininduced licking behavior in mice (Fig. 4B and 4C).
4. Discussion Results of this study show that an ethyl acetate extract of S. divinorum produces antinociceptive effect, mainly because of the presence of salvinorin constituents, by involving inhibitory neurotransmission mediated by opioids and 5-HT1A serotonin
12 receptors. The experimental models used to induce behavioral responses in this study are considered the first choice to detect medicinal plants to relieve pain. These tests together have complemented each other. In this sense, they reflect several peripheral and central mechanisms by which the nociception is generated. Visceral nociception involves somatic and central neurotransmission from organs such as the heart, veins, respiratory tract, colon, bladder, and uterus (Cervero, 1996; Cervero and Laird, 1999; Gebhart, 1994). Peptides as the substance P and CCK are responsible for transmission from visceral afferent fibers (Perry and Lawson, 1998), which excitability is extended to the spinal cord (Laird et al., 1995; Roza et al., 1998). Thus, the fact that S. divinorum reduces the number of writhes correlated with a decrease in the transmission of nociceptive activity at peripheral and central level. The effect of S. divinorum was as that obtained with tramadol, a weak opioid with high clinical efficacy and central action used as a reference drug (Guillen et al., 2000). The antinociceptive effect of this S. divinorum extract was corroborated using the formalin-induced licking behavior in mice, which allowed the evaluation of the neurogenic and inflammatory stages in the nociceptive process (Hunskaar et al., 1985). The dose-dependent antinociceptive effect of S. divinorum in the inflammatory phase of this test agrees with previous data using the carrageenan test (Simón-Arceo et al., 2017). It has been established that the most known mechanism by which non-steroidal anti-inflammatory drugs (NSAIDs) induce antinociception is mostly by inhibiting cyclooxygenases (COX) and the concentration of prostaglandins, but recent investigations have reported anti-inflammatory actions of NSAIDs though COXindependent like inducing the downregulation of L-selectin, inhibition of nuclear factor
13 kappa B and proinflammatory cytokines and nitric oxide synthase activity inhibition (Miranda et al., 2019). It is known that nociceptive response induced by an injection of diluted acetic acid in the writhing test or in the formalin-induced licking behaviour involves not only the presence of chemical mediators such as prostaglandins but also bradykinin, acetylcholine and substance P as activators of Aδ and C fibers in sensory nerves to rise pain during the inflammation process (Cervero and Laird, 2004; Estrup et al., 2009). Present results contribute to the pharmacological characterization of the antinociceptive spectrum of activity of this species; as well as the salvia genus since other species have been found to produce antinociceptive effect, such as S. officinalis, S. miltiorrhiza, S. mexicana, S. lerrifolia, S. africana-lutea, S. aethiopis, and S.aegyptiaca (Imanshahidi and Hosseinzadeh, 2006), which mechanism of action might involve inhibition of several targets evoking pain and inflammation depending on the type of pain. Reports about the antinociceptive effects of S. divinorum extracts are deficient in literature (Simón-Arceo et al., 2017). Whereas, the nature of the bioactive compounds participating in the antinociceptive effect of this species are partially known. For example: several salvinorins can be isolated from a medium polar extract of S. divinorum leaves, to identify mainly the presence of salvinorin A (Lee et al., 2005). The antinociceptive effect of this bioactive compound in an individual form has already been reported in the writhing test (McCurdy et al., 2006) and the formalin-induced licking behavior (Aviello et al., 2011), being significant at doses from 0.5 to 2 mg/kg, i.p., in mice. Thus, our results agree that this molecule is in great manner responsible for the activity of this medicinal plant supporting its use for alleviating pain in folk medicine
14 (Valdés et al., 1983). Phytochemical studies of plants from salvia genus have reported the presence of phenolic acids, tannins, essential oils, flavonoids, and other terpenoids (mono, di, and triterpenes) (Wu et al., 2012). However, for the specific case of S. divinorum, investigation of this chemical variety is lacking. Its phytochemical corresponds to the presence of neoclerodane diterpenes like salvinorin A (Lee et al, 2005; Shirota et al., 2006; Kutrzeba et al., 2009; Casselman et al., 2014), as well as triterpenes like oleanolic acid and stigmasterol (Casselman et al., 2014), or the monoterpene-lactone (−)-loliolide (Valdés, 1986; Casselman et al., 2014). Antinociceptive and anti-inflammatory effects has already been reported for oleanolic acid (Singh et al., 1992; Martínez et al., 2012) and stigmasterol (Githinji et al., 2012) in writhing and/or inflammatory evaluation in rodents. In fact, in this study a preliminary evaluation of the S. divinorum leaf extracts by enteral administration demonstrated significant antinociceptive activity in non-polar (hexane) and polar (methanol) extracts in an equivalent manner than EAEx. Likewise, it has been reported that salvinorin A may be inactivated by the gastrointestinal system (Siebert, 1994). This information together suggest that other bioactive metabolites participate in the medicinal properties of this plant and it will be interesting to explore these considerations in future studies. Regarding to the inhibitory neurotransmission explored in the presence of the medium polarity extract and the abundant salvinorins mixture, it was confirmed the participation of opioids and 5-HT1A receptors. In the case of opioid receptors, it was observed a prevention in the antinociceptive effect of the extract and in the salvinorins mixture in the writhing test and in both phases of the formalin-induced licking behavior in mice. Preliminary data on this receptor’s involvement was reported using nor-
15 binaltorphimine (1 mg/kg, i.p.), a kappa opioid antagonist, which significantly prevented the antihyperalgesic responses of the S. divinorum EAEx on the thermal and mechanical stimuli in an inflammatory and neuropathic pain model in rats (Simón-Arceo et al., 2017). In our study, naloxone (1 mg/kg, i.p., a non-selective opioid antagonist) avoided the antinociceptive responses of EAEx and the salvinorins mixture (including abundant presence of salvinorin A) on the chemical algesic effect induced in the writhing and formalin test in mice. The antinociceptive effect reported for salvinorin A in different pain models has implicated not only participation of the opioid receptors like kappa-type, but also cannabinoids mediated by CB1 receptors; for example: in experimental colitis in mice (Fichna et al., 2012), as well as in writhing test (Aviello et al., 2017) o in inflammatory responses induced by formalin or carrageenan (McCurdy et al., 2011; Aviello et al., 2017) in mice. Our data reinforced the involvement of opioid receptors in the antinociceptive effects of S. divinorum medium polarity extract containing salvinorins-like constituents. The serotonin 5-HT1A receptors are one of the specific mediators in the control of pain. They participate as part of the descending inhibitory serotonergic spinal-raphe projections from the nucleus raphe magnus forming an endogenous descending painmodulating network (Millan, 2002). Its activation may differentially influence pain responses in a dependent manner on the type of algesic stimulus (Sałat et al., 2017). In agreement to this report, in our study the blockage of these receptors inhibited antinociceptive effects in the writhing test, but not in the formalin-induced licking behavior in mice in the presence of the S. divinorum ethyl acetate extract. Nevertheless, it was observed an involvement of this receptors in the effect of the salvinorins mixture in both tests. These data suggest a preferential action in the opioid mechanisms.
16 As previous mentioned, loliolide is other interesting bioactive terpene of S. divinorum leaves (Valdés, 1986), which has been reported to inhibit activity on the serotonin transporter (Neergaard et al., 2010). The use of selective serotonin reuptake inhibitors (SSRIs), to increase serotonin (5-HT) within the brain like some antidepressants, is nowadays a common choice of successful treatment for high prevalence of chronic pain in the population that has limited efficacious therapy (Martin et al., 2017). The triterpene oleanolic acid is also a metabolite found in S. divinorum, it interrelates with the 5-HT1A receptors (Fajemiroye et al., 2015) and the opioid system (Maia et al., 2006), both associated with the descending and pain-inhibiting pathway (Millan, 2002; Hernandez-Leon et al., 2017). All this information together gives evidence of the potential effect of S. divinorum involving likely different bioactive constituents and inhibitory mechanisms of action for pain.
5. Conclusion S. divinorum medium polarity extract produced a dose-dependent antinociceptive effect at central and peripheral levels in pain models. The effect of the extract was observed due to the presence of salvinorins, mainly salvinorin A. Antinociceptive effect of S. divinorum involved both the serotonin 5-HT1A and opioids receptors as inhibitory neurotransmission depending on the type of nociception. Our data contribute to the ethnopharmacological knowledge of this species and the Salvia genus.
Conflict of interest statement
The authors declare no conflict of interest.
17
Author contributions Dr. González-Trujano ME ([email protected]) and Dr. Pellicer F ([email protected]) contributed in the experimental design, pharmacological experiments, data analysis, manuscript preparation and financial support for this study. Dr. Déciga-Campos M ([email protected]) and MSc Tlacomulco-Flores LL ([email protected]) contributed in carrying out the pharmacological experiments according to the experimental design, data analysis and preparation of the manuscript. Dr. Azucena Ibeth Carballo-Villalobos ([email protected]) participated in the experimental design, data analysis and manuscript preparation.
Acknowledgements We are thankful to Dr Alberto Hernandez Leon, MSc Mariana Yetlanezy HernándezArámburo and the student Guillermo Vázquez Díaz for their technical assistance. We thank Prof. Marissa González for proof-reading English version of the manuscript. This work was partially supported by projects of CONACYT [226454 and 256448] and INP [NC093223.0, NC123280.0, and NC17073.0]. Leonel Flores-Tlacomulco thanks the fellowship by CONACYT No. 745888.
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Legends of the figures Figure 1. Representative chromatogram showing the profile of the salvinorins mixture in which the most abundant was the presence of salvinorin A as compared to the standard at retention time of 4.36 min. Wavelength 210 nm. Figure 2. Nociception in the time-course curves was expressed as writhes (A and C) and as the AUC obtained from those temporal course curves (B and D). Organic extracts hexane (Hex), ethyl acetate (EAEx) or methanol (MeOH) were preliminary tested at 100, 300 and 400 mg/kg using via oral. Then, the nociceptive agent (1% acetic acid) was intraperitoneally (i.p.) injected in mice. EAEx was also tested in different doses in comparison to the reference drug tramadol (TR30: 30 mg/kg, i.p.) and a mixture of salvinorins at the same dosage (MIX30, i.p.). Data are expressed as the mean ± S.E.M. of at least six animals. Statistical differences were analyzed using a two-way repeatedmeasures ANOVA followed by Tukey´s multiple comparisons in the temporal course
25 curves. To compare AUC treatments vs vehicle (VEH) a one-way ANOVA followed by Dunnett´s test was used. *P<0.05, **P<0.01, ***P<0.001. Figure 3. Time-course curves of nociceptive behavior (A) and nociception expressed as AUC of the temporal course curves in the neurogenic (B) and inflammatory (C) phases after the nociceptive agent injection (1% formalin) in mice. Data are expressed as the mean ± S.E.M. of at least six animals. To compare AUC treatments vs vehicle (VEH) a one-way ANOVA followed by Dunnett´s test was used. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 4. Effect of naloxone (NX, 1 and 3 mg/kg, i.p.) and WAY100635 (WAY0.32, 0.32 mg/kg, i.p.) in the antinociceptive effect of the S. divinorum EAEx (30 mg/kg, i.p.) and salvinorins mixture (MIX30, 30 mg/kg, i.p.) in the writhing test (A), as well as in the neurogenic (B) and inflammatory (C) phases of the formalin test in mice. Data are expressed as the AUC of nociception (the writhes or shakings, respectively). Bars represent the mean ± S.E.M. of at least six animals. Significance was determined by ANOVA followed by Tukey’s test. *P<0.05, **P<0.01, ***P<0.001 in comparison to the vehicle (VEH) group. #P<0.05 Student´s t test.
ABBREVIATIONS: Ethyl Acetate Extract (EAEx); Salvinorin Mixture (MIX); Naloxone (NX); Vehicle (VEH); Ultra Performance Liquid Chromatography (UPLC); Area Under the Curve (AUC); Analysis of Variance (ANOVA).
A
Writhing test 1000 750
#
500
** 250
VE H W NX A 1 Y0 EA .32 Ex M 30 IX 30 N X1 + EA N EA Ex X1 Ex 30 3 + W 0 + MI X A Y0 WA 30 .3 2 Y0. 3 + M 2 IX 30
0
Dose (mg/kg, i.p.)
Figure 4, Tlacomulco-Flores et al., 2019
Figure 1 Tlacomulco-Flores et al., 2019
Fig
ure 2. Flores-Tlacomulco et al., 2019
Figure 3, Tlacomulco-Flores et al., 2019 M
30
10
3
30
IX 30
10 0
TR
VE H
Nociception (AUC0-10 min)
S0378-8741(19)30588-4
DOI:
https://doi.org/10.1016/j.jep.2019.112276
Reference:
JEP 112276
To appear in:
Journal of Ethnopharmacology
Received Date: 12 February 2019 Revised Date:
23 September 2019
Accepted Date: 3 October 2019
Please cite this article as: Tlacomulco-Flores, L.L., Déciga-Campos, M., González-Trujano ME, Marí.Eva., Carballo-Villalobos, A.I., Pellicer, F., Antinociceptive effects of Salvia divinorum and bioactive salvinorins in experimental pain models in mice, Journal of Ethnopharmacology (2019), doi: https:// doi.org/10.1016/j.jep.2019.112276. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Antinociceptive effects of Salvia divinorum and bioactive salvinorins in experimental pain models in mice
Pharmacological evaluation
Chromatographic analysis
Salvia divinorum leaves Salvinorin A
Crude extract, fraction and salvinorin’s mixture
1 Antinociceptive effects of Salvia divinorum and bioactive salvinorins in experimental pain models in mice
Lorenzo Leonel Tlacomulco-Floresa,b&, Myrna Déciga-Camposb&, María Eva GonzálezTrujano MEa*, Azucena Ibeth Carballo-Villalobosc, Francisco Pellicera.
a
Laboratorio
de
Neurofarmacología
de
Productos
Naturales.
Dirección
de
Investigaciones en Neurociencias. Instituto Nacional de Psiquiatría “Ramón de la Fuente”. Av. México-Xochimilco 101, Col. Sn Lorenzo Huipulco, 14370. Ciudad de México, México. b
Sección de Estudios de Posgrado e Investigación de la Escuela Superior de Medicina,
Instituto Politécnico Nacional. Plan de San Luis y Díaz Mirón s/n Col. Casco de Santo Tomás 11340, Ciudad de México, México. c
Departamento de Química Inorgánica y Nuclear. Facultad de Química, Universidad
Nacional Autónoma de México. Conjunto E. Circuito de la Investigación Científica. Ciudad Universitaria 04510, Ciudad de México, México.
This work was partially supported by the projects CONACYT-226454/256448. &
These authors contributed equally.
*Corresponding author: Instituto Nacional de Psiquiatría “Ramón de la Fuente” Av. México-Xochimilco No. 101, Col. San Lorenzo Huipulco Delegación Tlalpan 14370, Ciudad de México, México Tel.: (+52 55) 4160-5085 Fax: (+52 55) 5655-9980 E-mail address: [email protected]
2 Abstract Ethnopharmacological relevance. Salvia divinorum Epling & Játiva is a Mexican plant used not only in rituals but also in traditional medicine for pain relief. One of the most known bioactive compounds is salvinorin A, which acts centrally in kappa-type opioid receptors. Aim of the study. Despite its traditional use as a medicinal plant, there is not enough scientific investigation to reinforce its potential as analgesic. In this study, Salvia divinorum antinociceptive activity was evaluated in experimental models of nociceptive pain; the writhing test and formalin-induced licking behavior in mice. Material and Methods: Different Salvia divinorum extracts were prepared by maceration at room temperature in increased polarity (hexane, ethyl acetate and methanol). The ethyl acetate extract (EAEx) was chosen in order to be fractioned and to obtain a mixture of salvinorins. The antinociceptive effect of EAEx (3, 10, 30, and 100 mg/kg, i.p.) was compared with that of tramadol (a partial opioid agonist analgesic drug, 30 mg/kg, i.p.) and the mixture of salvinorins (30 mg/kg, i.p.). In addition, a participation of opioids (naloxone, NX 1 and/or 3 mg/kg, i.p.) and serotonin 5-HT1A receptors (WAY100635,
0.32
mg/kg,
i.p.)
was
investigated
as
possible
inhibitory
neurotransmission involved. Results. As a result, the EAEx produced significant and dose-dependent antinociceptive effect concerning salvinorins constituents. This effect was blocked in the presence of NX and WAY100635 in the abdominal test, but only by NX in the formalin-induced licking behavior. Whereas, the effect of salvinorins mixture involved opioids and serotonin 5HT1A receptors.
3 Conclusion. Data provide evidence of the potential of this species, where salvinorin A is in part responsible bioactive constituent involving participation of the opioids and/or 5HT1A serotonin receptors depending on the kind of pain model explored. Key words: Nociception, Opioids, Salvia divinorum Epling & Játiva, Salvinorins, serotonin. 1. Introduction Salvia divinorum belongs to the Salvia genus (Lamiaceae Family). This genus has a great diversity of species in the world (Martínez-Gordillo et al., 2013), but Mexico is one of the areas with greater distribution with more than 300 endemic species (CornejoTenorio and Ibarra-Manríquez, 2011; Martínez-Gordillo et al., 2013). Oneirogen is one of the effects recognized for this species (Valdés et al., 1983; Díaz, 2013; GonzálezTrujano et al., 2016), in part because of the presence of Salvinorin A (Ortega et al., 1982). This endemic plant from Mexico, essentially from the state of Oaxaca, has been used by Mazatecs in mystic rituals (Díaz, 2013). As traditional medicine, it is used to alleviate different ailments related to pain, such as stomachache, headache and rheumatism (Valdés et al., 1983). In fact, several Salvia species are recognized to produce pain relief, such as: S. officinalis, S. miltiorrhiza, S. mexicana, S. lerrifolia, S. africana-lutea, S. aethiopis, S. aegyptiaca (Imanshahidi and Hosseinzadeh, 2006). A great variety of compounds has been isolated from this genus, including phenolic acids, tannins, essential oils, flavonoids and terpenoids (mono, di, and triterpenes), many of them with biological activity (Wu et al., 2012). From the phytochemistry of S. divinorum at least 22 compounds of diterpene nature has been described, from these ten belong to the chemical group salvinorins (A to J) (Lee et al, 2005, Shirota et al., 2006, Kutrzeba et al., 2009), six to the divinatorins
4 (A to F) (Lee et al., 2005; Bigham et al., 2003; Shirota et al., 2006), four to the salvidivins (A to D) (Shirota et al., 2006), and two to the salvinicins (A and B) (Harding et al., 2005). From the salvinorins group, the salvinorin A is considered a principal bioactive molecule of this plant in part due to an agonist action on Kappa opioids and cannabinoids (CB1 and CB2) receptors (Roth et al., 2002; Capasso et al., 2006; 2008; Fichna et al., 2011). It has also been related to the release or modulation of monoaminergic neurotransmitters serotonin, dopamine and noradrenaline (Grilli et al., 2009; Pittaluga et al., 2013). Clinical experience with terpenes for relieving pain has demonstrated beneficial effects with generally non-serious adverse effects (Guimarães et al., 2013). Salvia divinorum is a great source of bioactive diterpenes like salvinorins; however, its pharmacological evaluation as a crude extract supporting its potential for pain relief as in folk medicine has not been explored. In this study, the antinociceptive effect of Salvia divinorum leaf extract, that was obtained under controlled hydroponic planting, was pharmacologically evaluated in pain models like the writhing test and formalin-induced licking behavior in mice to look for bioactive compounds and possible inhibitory neurotransmission involved.
2. Materials and methods 2.1. Animals Male and female Swiss albino mice (25-30 g) grouped in six animals were used for the study. Animals were housed at the facilities of the Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz and kept at constant room temperature (22±1°C) in a 12h/12h light/dark cycle. Experiments were carried out in accordance with the Ethical
5 Committee Guidelines laid down by the INP “Ramón de la Fuente” local authority (NC12.3280.0/August 22, 2017) and under the technical specifications of national (SAGARPA NOM-062-ZOO-1999) and international regulations regarding the care and use of animals for experimental procedures. All animals were fed with standard food and water ad libitum. 2.2. Drugs Tramadol was purchased by Grünenthal de México, S.A. de C.V. Naloxone and WAY100635 from Aldrich Química, S.A. de C.V. Acetic acid and formalin (J.T. Baker, USA). Salvinorin A (purity >99%) and salvinorin B (purity >93%) were purchased by Abcam Biochemicals (Cambridge, MA, USA) and Sigma (Sigma-Aldrich Co., St Louis, MO, USA), respectively, and used as standards. Control animals received the vehicle (0.5% Tween 80 in saline solution). Acetic acid and formalin were both used in 1% solutions as algesic substances to induce nociception. Drugs were freshly prepared on the day of the experiments; all treatments were administered via intraperitoneal (i.p.) in a volume of 0.1 ml/10 g body weight. Except at the beginning of the study, when the hexane, ethyl acetate and methanol crude extracts were evaluated to choose the most active using intragastric route of administration. 2.3. Plant material Leaves of Salvia divinorum Epling & Játiva-M (Lamiaceae) were obtained from Altiva Productos Hortícolas S.P.R de R.L. in Altiva Montecasino, Huitzilac, state of Morelos (latitude 19° 1´ 38.75´´ N/longitude 99° 16´ 16.08´´ W/elevation 2507 m above sea level) under the taxonomic identification of MSc Rogelio Silos Medina (Lot. INPJRF-1A/25-052012). All plants were grown in a hydroponic cultivation system under controlled
6 conditions of temperature, humidity and nutrients. The leaves were sorted out and then air-dried. 2.4. Extract preparation The extracts of different polarity were obtained from the dried and ground leaves (600 g) macerated at room temperature 22 ± 2°C for 48 h. The process began with a degreasing of the material with hexane (3.5 L, 3 times) followed by a second extraction with ethyl acetate (3.5 L, 3 times), and finally a third extraction using methanol (3.5 L, 3 times). After filtration, the solid residue was discarded, and liquid was concentrated under reduced pressure in a rotatory evaporator for the total removal of the solvent. The hexane solvent-free crude extract formed a dark green semisolid (4.96 g, 0.83%); the ethyl acetate extract (EAEx) was also a dark green semisolid (16.79 g, 2.8%), and the methanolic extract a dark brown syrupy (59.88 g, 9.98%). EAEx was selected to explore antinociceptive activity since it contains compounds like salvinorin A. Thus, it was fractionated by chromatographic open column to isolate a salvinorin mixture in approximately 3 mg/g of crude ethyl acetate extract, in which salvinorin A was identified by ultra-performance liquid chromatography (UPLC) (Fig. 1). Thus, it would be expected that 50 mg of salvinorin mixture were included in the plant material explored. 2.5 UPLC analysis The EAEx (10 mg/ml), salvinorin mixture or individual salvinorin A and B standards were diluted in methanol, filtered through a 0.2 mm filter (GHP, acrodisc 13, Waters), and then injected directly (10 µl injection volume). The mobile phase consisted of acidified water at 0.1% phosphoric acid (A) and methanol (B) (HPLC grade). The initial gradient elution was 50% A: 50% B to reach 100% B (15 min).
System regeneration was
achieved at 50% A: 50% B. The flow rate was 1 ml/min with an elution curve of 4. An
7 Acquity UPLC Waters with a PDA eλ detector controlled by EmPower 3TM software was equipped with a Symmetry C18 100Å (150 mm x 4.6 mm, 5µm) column (Waters, made in Ireland) and the thermostat was set at 43°C. The salvinorin peak profile was identified at UVλ max 210 nm. 2.6. Pharmacological evaluation 2.6.1 Writhing test Number of writhes was recorded in 5 min-intervals during a 30 min-period after injection of 1% acetic acid at volume of 10 ml/kg, i.p. Abdominal writhing was considered as nociceptive behavior defined as an exaggerated extension of the abdomen combined with the outstretching of the hind limbs according to Collier et al., 1968. To choose the most active extract, independent groups of mice received one of the three extracts (hexane, EAEx or methanol) using esophageal administration, then latency to the first writhe and AUC (0-30 min) were evaluated in this test. From the results of the previous evaluation, the EAEx was selected as active to continue experiments not only in the writhing test but also in the formalin-induced licking behavior in mice. In these experiments an intraperitoneal injection was used to optimize the extract, fractions and pure compounds to reach the antinociceptive response. Then, animals received the EAEx at 3, 10, 30, and 100 mg/kg, i.p., a salvinorin mixture (30 mg/kg, i.p.), or the reference drug (tramadol, 30 mg/kg, i.p.) 30 minutes before the algesic administration. Control animals received vehicle by the same route and volume of administration. 2.6.2. Formalin-induced licking behavior Mice were injected in the subplantar area of the right hind paw with 20 µl diluted of 1%
8 formalin by using a 30-gauge needle. The number of licking was obtained as nociceptive behavior in the first min and each 5 min during a period of 30 min after formalin administration. According to the Hunskaar et al. (1985), two stages were obtained from this temporal course curves taken as neurogenic (0-10 min) and inflammatory phases (10-30 min). Thirty min before the intraplantar injection of formalin, animals were administered varying doses of EAEx (30 and 100 mg/kg, i.p.), or with a salvinorin mixture (30 mg/kg, i.p.) or the reference drug at the same dosage. Control animals received vehicle. 2.6.3 Inhibitory neurotransmission In order to investigate participation of opioids and 5-HT1A serotonin receptors in the antinociceptive activity of S. divinorum, a significant antinociceptive response of the EAEx was explored in the presence of the antagonists: naloxone (NX, an opioid antagonist, 1 and/or 3 mg/kg, i.p.), and WAY100635 (WAY, a selective 5-HT1A antagonist, 0.32 mg/kg, i.p.). 2.7 Statistical analysis Temporal course curves were plotted to describe the response of several doses of S. divirorum EAEx in comparison to the vehicle group. Statistical differences were analyzed using a two-way repeated-measures analysis of variance (ANOVA) followed by Sidak´s multiple comparisons test for factors like time and treatment. From temporal course curves, the area under the curve (AUC) was obtained using the trapezoidal rule and data are presented in bars as the mean ± SEM of 6 repetitions, then statistical differences were analysed using a one-way ANOVA followed by Dunnett´s t test. ANOVA on ranks followed by Dunn´s test or Student´s t test was also applied according
9 to data. A value of p<0.05 was considered significant. Statistical analysis was done using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Antinociceptive response in the writhing test Figure 2 shows the temporal course curves of the abdominal stretches induced by 1% acetic acid, i.p., in mice receiving vehicle or the individual extracts in increasing polarity (hexane, EAEx and methanol at 100, 300 and 400 mg/kg) by oesophagi route of administration (p.o.) in comparison to the vehicle group (Fig. 2A). In this preliminary screening, the EAEx showed better significative antinociceptive response in mice (Fig. 2B). The same graphic shows the antinociceptive effect of several doses of S. divinorum EAEx (3 to 100 mg/kg, i.p.) and the salvinorin mixture (30 mg/kg, i.p.). The lowest doses of the extract (3 and 10 mg/kg, ip) and the mixture of salvinorins (30 mg/kg, i.p) produced a decrease in stretches with respect to the vehicle (Fig. 2C). These effects were not greater than those obtained with tramadol (TR30, 30 mg/kg, i.p.), used as reference drug. In contrast, the effect of the two highest doses (30 and 100 mg/kg) of the extract resembled that obtained with tramadol that decreased nociceptive response up to 50% in mice (Fig. 2C). Nociception expressed as AUC described significant effects of the S. divinorum EAEx from a dosage of 10 mg/kg, i.p. and in a dose-dependent manner, it produced similar effect than tramadol at the same dosage (30 mg/kg, i.p.) (Fig. 2D). The dosage of 30 mg/kg of the extract was the effective dose 50%. The maximum response was obtained with 100 mg/kg producing 92% of antinociceptive response. The salvinorin
10 mixture (MIX30) produced 30% of antinociceptive response at a dosage of 30 mg/kg in the acetic acid test (Fig. 2D). 3.2. Antinociceptive response in the formalin-induced licking behaviour Figure 3 shows the temporal course curves of the antinociceptive behaviour induced by 1% formalin by intraplantar administration in mice receiving vehicle. This graphic also showed the total abolition of the antinociceptive effect in the presence of tramadol (TR30, 30 mg/kg, i.p.) in mice in neurogenic (0-10 min) and inflammatory phases (10-30 min) (Fig. 3A). All doses of S. divinorum EAEx (10 to 100 mg/kg, i.p.) and the salvinorin mixture (MIX30, 30 mg/kg, i.p.) produced inhibition in the behavioural nociceptive response, major emphasis was noticed in the inflammatory phase (Fig. 3A). Data from AUC describe that nociceptive response in the neurogenic phase was significantly diminished by the extract from a dosage of 10 mg/kg (Fig. 3A). An increase in doses (30 and 100 mg/kg, i.p.) improved the antinociceptive effect (Fig. 3A). Salvinorin mixture did not reach significance at a dose of 30 mg/kg in this phase (Fig. 3B). Additionally, behavioural nociceptive response in the inflammatory phase was significantly decreased in a dose-dependent manner from a dosage of 10 mg/kg, i.p. of S. divinorum EAEx (Fig. 3C). Significant effect was obtained with the salvinorin mixture in the inflammatory phase that resembled those observed with the extract at the minimal dose of 10 mg/kg and tramadol at the same dosage of salvinorins (Fig. 3C). 3.3. Inhibitory neurotransmission In the writhing test, it was observed that administration of NX (1 mg/kg, i.p.) did not show antinociceptive effect per se respect to the response observed in the vehicle group. In
11 contrast, NX avoided the significant antinociceptive effect of the S. divinorum EAEx (30 mg/kg, i.p.) and the salvinorin mixture (30 mg/kg, i.p.) (Fig. 4A). In a similar manner, the presence of WAY100635 (WAY 0.32 mg/kg, i.p.) did not modify the nociceptive response after individual administration, whereas antinociceptive response obtained with the EAEx or salvinorins mixture at 30 mg/kg, i.p. was totally inhibited when combined with this 5-HT1A antagonist (Fig. 4A). Regarding to the formalin-induced licking behavior, both antagonists per se did not modify significantly the nociceptive response in the neurogenic (Fig. 4B) and inflammatory phases (Fig. 4C). Nevertheless, in the case of the antinociceptive response of the EAEx, it was partially blockage when combined with NX at 1 mg/kg. A complete inhibition of the antinociceptive response in both phases of the formalin test was obtained in the presence of NX at 3 mg/kg (Figs. 4B and 4C). Whereas no modification in the antinociceptive effect of the EAEx was obtained in the presence of WAY100635 (0.32 mg/kg, i.p.) in these two phases of the formalin-induced licking behavior in mice (Figs. 4B and 4C). Regarding the antinociceptive effect of salvinorins mixture, it was observed that in an equivalent manner, the antagonists NX (3 mg/kg, i.p.) and WAY100635 (0.32 mg/kg, i.p.) prevented its antinociceptive effect in both the writhing test (Fig. 4A) and formalininduced licking behavior in mice (Fig. 4B and 4C).
4. Discussion Results of this study show that an ethyl acetate extract of S. divinorum produces antinociceptive effect, mainly because of the presence of salvinorin constituents, by involving inhibitory neurotransmission mediated by opioids and 5-HT1A serotonin
12 receptors. The experimental models used to induce behavioral responses in this study are considered the first choice to detect medicinal plants to relieve pain. These tests together have complemented each other. In this sense, they reflect several peripheral and central mechanisms by which the nociception is generated. Visceral nociception involves somatic and central neurotransmission from organs such as the heart, veins, respiratory tract, colon, bladder, and uterus (Cervero, 1996; Cervero and Laird, 1999; Gebhart, 1994). Peptides as the substance P and CCK are responsible for transmission from visceral afferent fibers (Perry and Lawson, 1998), which excitability is extended to the spinal cord (Laird et al., 1995; Roza et al., 1998). Thus, the fact that S. divinorum reduces the number of writhes correlated with a decrease in the transmission of nociceptive activity at peripheral and central level. The effect of S. divinorum was as that obtained with tramadol, a weak opioid with high clinical efficacy and central action used as a reference drug (Guillen et al., 2000). The antinociceptive effect of this S. divinorum extract was corroborated using the formalin-induced licking behavior in mice, which allowed the evaluation of the neurogenic and inflammatory stages in the nociceptive process (Hunskaar et al., 1985). The dose-dependent antinociceptive effect of S. divinorum in the inflammatory phase of this test agrees with previous data using the carrageenan test (Simón-Arceo et al., 2017). It has been established that the most known mechanism by which non-steroidal anti-inflammatory drugs (NSAIDs) induce antinociception is mostly by inhibiting cyclooxygenases (COX) and the concentration of prostaglandins, but recent investigations have reported anti-inflammatory actions of NSAIDs though COXindependent like inducing the downregulation of L-selectin, inhibition of nuclear factor
13 kappa B and proinflammatory cytokines and nitric oxide synthase activity inhibition (Miranda et al., 2019). It is known that nociceptive response induced by an injection of diluted acetic acid in the writhing test or in the formalin-induced licking behaviour involves not only the presence of chemical mediators such as prostaglandins but also bradykinin, acetylcholine and substance P as activators of Aδ and C fibers in sensory nerves to rise pain during the inflammation process (Cervero and Laird, 2004; Estrup et al., 2009). Present results contribute to the pharmacological characterization of the antinociceptive spectrum of activity of this species; as well as the salvia genus since other species have been found to produce antinociceptive effect, such as S. officinalis, S. miltiorrhiza, S. mexicana, S. lerrifolia, S. africana-lutea, S. aethiopis, and S.aegyptiaca (Imanshahidi and Hosseinzadeh, 2006), which mechanism of action might involve inhibition of several targets evoking pain and inflammation depending on the type of pain. Reports about the antinociceptive effects of S. divinorum extracts are deficient in literature (Simón-Arceo et al., 2017). Whereas, the nature of the bioactive compounds participating in the antinociceptive effect of this species are partially known. For example: several salvinorins can be isolated from a medium polar extract of S. divinorum leaves, to identify mainly the presence of salvinorin A (Lee et al., 2005). The antinociceptive effect of this bioactive compound in an individual form has already been reported in the writhing test (McCurdy et al., 2006) and the formalin-induced licking behavior (Aviello et al., 2011), being significant at doses from 0.5 to 2 mg/kg, i.p., in mice. Thus, our results agree that this molecule is in great manner responsible for the activity of this medicinal plant supporting its use for alleviating pain in folk medicine
14 (Valdés et al., 1983). Phytochemical studies of plants from salvia genus have reported the presence of phenolic acids, tannins, essential oils, flavonoids, and other terpenoids (mono, di, and triterpenes) (Wu et al., 2012). However, for the specific case of S. divinorum, investigation of this chemical variety is lacking. Its phytochemical corresponds to the presence of neoclerodane diterpenes like salvinorin A (Lee et al, 2005; Shirota et al., 2006; Kutrzeba et al., 2009; Casselman et al., 2014), as well as triterpenes like oleanolic acid and stigmasterol (Casselman et al., 2014), or the monoterpene-lactone (−)-loliolide (Valdés, 1986; Casselman et al., 2014). Antinociceptive and anti-inflammatory effects has already been reported for oleanolic acid (Singh et al., 1992; Martínez et al., 2012) and stigmasterol (Githinji et al., 2012) in writhing and/or inflammatory evaluation in rodents. In fact, in this study a preliminary evaluation of the S. divinorum leaf extracts by enteral administration demonstrated significant antinociceptive activity in non-polar (hexane) and polar (methanol) extracts in an equivalent manner than EAEx. Likewise, it has been reported that salvinorin A may be inactivated by the gastrointestinal system (Siebert, 1994). This information together suggest that other bioactive metabolites participate in the medicinal properties of this plant and it will be interesting to explore these considerations in future studies. Regarding to the inhibitory neurotransmission explored in the presence of the medium polarity extract and the abundant salvinorins mixture, it was confirmed the participation of opioids and 5-HT1A receptors. In the case of opioid receptors, it was observed a prevention in the antinociceptive effect of the extract and in the salvinorins mixture in the writhing test and in both phases of the formalin-induced licking behavior in mice. Preliminary data on this receptor’s involvement was reported using nor-
15 binaltorphimine (1 mg/kg, i.p.), a kappa opioid antagonist, which significantly prevented the antihyperalgesic responses of the S. divinorum EAEx on the thermal and mechanical stimuli in an inflammatory and neuropathic pain model in rats (Simón-Arceo et al., 2017). In our study, naloxone (1 mg/kg, i.p., a non-selective opioid antagonist) avoided the antinociceptive responses of EAEx and the salvinorins mixture (including abundant presence of salvinorin A) on the chemical algesic effect induced in the writhing and formalin test in mice. The antinociceptive effect reported for salvinorin A in different pain models has implicated not only participation of the opioid receptors like kappa-type, but also cannabinoids mediated by CB1 receptors; for example: in experimental colitis in mice (Fichna et al., 2012), as well as in writhing test (Aviello et al., 2017) o in inflammatory responses induced by formalin or carrageenan (McCurdy et al., 2011; Aviello et al., 2017) in mice. Our data reinforced the involvement of opioid receptors in the antinociceptive effects of S. divinorum medium polarity extract containing salvinorins-like constituents. The serotonin 5-HT1A receptors are one of the specific mediators in the control of pain. They participate as part of the descending inhibitory serotonergic spinal-raphe projections from the nucleus raphe magnus forming an endogenous descending painmodulating network (Millan, 2002). Its activation may differentially influence pain responses in a dependent manner on the type of algesic stimulus (Sałat et al., 2017). In agreement to this report, in our study the blockage of these receptors inhibited antinociceptive effects in the writhing test, but not in the formalin-induced licking behavior in mice in the presence of the S. divinorum ethyl acetate extract. Nevertheless, it was observed an involvement of this receptors in the effect of the salvinorins mixture in both tests. These data suggest a preferential action in the opioid mechanisms.
16 As previous mentioned, loliolide is other interesting bioactive terpene of S. divinorum leaves (Valdés, 1986), which has been reported to inhibit activity on the serotonin transporter (Neergaard et al., 2010). The use of selective serotonin reuptake inhibitors (SSRIs), to increase serotonin (5-HT) within the brain like some antidepressants, is nowadays a common choice of successful treatment for high prevalence of chronic pain in the population that has limited efficacious therapy (Martin et al., 2017). The triterpene oleanolic acid is also a metabolite found in S. divinorum, it interrelates with the 5-HT1A receptors (Fajemiroye et al., 2015) and the opioid system (Maia et al., 2006), both associated with the descending and pain-inhibiting pathway (Millan, 2002; Hernandez-Leon et al., 2017). All this information together gives evidence of the potential effect of S. divinorum involving likely different bioactive constituents and inhibitory mechanisms of action for pain.
5. Conclusion S. divinorum medium polarity extract produced a dose-dependent antinociceptive effect at central and peripheral levels in pain models. The effect of the extract was observed due to the presence of salvinorins, mainly salvinorin A. Antinociceptive effect of S. divinorum involved both the serotonin 5-HT1A and opioids receptors as inhibitory neurotransmission depending on the type of nociception. Our data contribute to the ethnopharmacological knowledge of this species and the Salvia genus.
Conflict of interest statement
The authors declare no conflict of interest.
17
Author contributions Dr. González-Trujano ME ([email protected]) and Dr. Pellicer F ([email protected]) contributed in the experimental design, pharmacological experiments, data analysis, manuscript preparation and financial support for this study. Dr. Déciga-Campos M ([email protected]) and MSc Tlacomulco-Flores LL ([email protected]) contributed in carrying out the pharmacological experiments according to the experimental design, data analysis and preparation of the manuscript. Dr. Azucena Ibeth Carballo-Villalobos ([email protected]) participated in the experimental design, data analysis and manuscript preparation.
Acknowledgements We are thankful to Dr Alberto Hernandez Leon, MSc Mariana Yetlanezy HernándezArámburo and the student Guillermo Vázquez Díaz for their technical assistance. We thank Prof. Marissa González for proof-reading English version of the manuscript. This work was partially supported by projects of CONACYT [226454 and 256448] and INP [NC093223.0, NC123280.0, and NC17073.0]. Leonel Flores-Tlacomulco thanks the fellowship by CONACYT No. 745888.
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Legends of the figures Figure 1. Representative chromatogram showing the profile of the salvinorins mixture in which the most abundant was the presence of salvinorin A as compared to the standard at retention time of 4.36 min. Wavelength 210 nm. Figure 2. Nociception in the time-course curves was expressed as writhes (A and C) and as the AUC obtained from those temporal course curves (B and D). Organic extracts hexane (Hex), ethyl acetate (EAEx) or methanol (MeOH) were preliminary tested at 100, 300 and 400 mg/kg using via oral. Then, the nociceptive agent (1% acetic acid) was intraperitoneally (i.p.) injected in mice. EAEx was also tested in different doses in comparison to the reference drug tramadol (TR30: 30 mg/kg, i.p.) and a mixture of salvinorins at the same dosage (MIX30, i.p.). Data are expressed as the mean ± S.E.M. of at least six animals. Statistical differences were analyzed using a two-way repeatedmeasures ANOVA followed by Tukey´s multiple comparisons in the temporal course
25 curves. To compare AUC treatments vs vehicle (VEH) a one-way ANOVA followed by Dunnett´s test was used. *P<0.05, **P<0.01, ***P<0.001. Figure 3. Time-course curves of nociceptive behavior (A) and nociception expressed as AUC of the temporal course curves in the neurogenic (B) and inflammatory (C) phases after the nociceptive agent injection (1% formalin) in mice. Data are expressed as the mean ± S.E.M. of at least six animals. To compare AUC treatments vs vehicle (VEH) a one-way ANOVA followed by Dunnett´s test was used. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 4. Effect of naloxone (NX, 1 and 3 mg/kg, i.p.) and WAY100635 (WAY0.32, 0.32 mg/kg, i.p.) in the antinociceptive effect of the S. divinorum EAEx (30 mg/kg, i.p.) and salvinorins mixture (MIX30, 30 mg/kg, i.p.) in the writhing test (A), as well as in the neurogenic (B) and inflammatory (C) phases of the formalin test in mice. Data are expressed as the AUC of nociception (the writhes or shakings, respectively). Bars represent the mean ± S.E.M. of at least six animals. Significance was determined by ANOVA followed by Tukey’s test. *P<0.05, **P<0.01, ***P<0.001 in comparison to the vehicle (VEH) group. #P<0.05 Student´s t test.
ABBREVIATIONS: Ethyl Acetate Extract (EAEx); Salvinorin Mixture (MIX); Naloxone (NX); Vehicle (VEH); Ultra Performance Liquid Chromatography (UPLC); Area Under the Curve (AUC); Analysis of Variance (ANOVA).
A
Writhing test 1000 750
#
500
** 250
VE H W NX A 1 Y0 EA .32 Ex M 30 IX 30 N X1 + EA N EA Ex X1 Ex 30 3 + W 0 + MI X A Y0 WA 30 .3 2 Y0. 3 + M 2 IX 30
0
Dose (mg/kg, i.p.)
Figure 4, Tlacomulco-Flores et al., 2019
Figure 1 Tlacomulco-Flores et al., 2019
Fig
ure 2. Flores-Tlacomulco et al., 2019
Figure 3, Tlacomulco-Flores et al., 2019 M
30
10
3
30
IX 30
10 0
TR
VE H
Nociception (AUC0-10 min)