Role of β-Caryophyllene in the Antinociceptive and Anti-Inflammatory Effects of Tagetes lucida Cav. Essential Oil

Tagetes lucida Cav. (Asteraceae) is an ancient medicinal plant commonly used to alleviate pain. Nevertheless, scientific studies validating this property are lacking in the literature. Animal models of pain were used to evaluate the antinociceptive and anti-inflammatory activities of T. lucida essential oil (TLEO) and a bioactive metabolite. The chemical constitution and possible toxicity of the extract and the mechanism of action of β-caryophyllene were also explored. Temporal course curves and dose–response graphics were generated using TLEO (0.1–10 mg/kg or 3.16–31.62 mg/kg) and β-caryophyllene (3.16–10 mg/kg). Metamizole (80 mg/kg) and indomethacin (20 mg/kg) were used as reference drugs in the formalin assay and writhing test in rats and mice, respectively. The β-caryophyllene mechanism of action was explored in the presence of naloxone (1 mg/kg), flumazenil (10 mg/kg), WAY100635 (0.16 mg/kg), or nitro-l-arginine methyl ester (L-NAME) (20 mg/kg) in the formalin test in rats. GC/MS analysis demonstrated the presence of geranyl acetate (49.89%), geraniol (7.92%), and β-caryophyllene (6.27%). Significant and dose-dependent antinociceptive response was produced by TLEO and β-caryophyllene without the presence of gastric damage. In conclusion, β-caryophyllene was confirmed as a bioactive compound in the T. lucida analgesic properties by involving the participation of receptors like opioids, benzodiazepines, and Serotonin 1A receptor (5-HT1A), as well as nitric oxide.


Introduction
Essential oils are natural products of aromatic plants traditionally used all over the world for thousands of years. Their effectiveness is recommended by healers and shamans throughout history in all cultures [1], for example: for treatment of infection, as an anti-inflammatory, and for relaxing or stimulating therapy [2]. Essential oils are very complex and contain several compounds, many of them in traces. The combination of all ingredients can produce the final activity of an essential oil. Nevertheless, one extracted component may be an important element in the efficacy of the entire extract
The analyses and identification following NMR spectroscopy of the Tagetes lucida essential oil revealed the presence of 22 compounds, representing 100% of the total content of volatiles, with 69.18% monoterpenes, 29.10% sesquiterpenes, and 1.71% non-identified compounds. The qualitative composition of volatile compounds in aerial parts was determined by GC/MS analysis (Table 1 and Figure 1; see also Supplementary Materials), listed in order of their elution together with their retention times. The major volatile compounds were geranyl acetate (1,49.89%), geraniol (2,7.92%), β-caryophyllene (3, 6.27%), (−)-β-cubebene (4, 7.72%), and cis-β-ocimene (5, 5.60%). It is worth mentioning that compound 2 could be obtained via an elimination reaction of the acetoxy group from geranyl acetate using the temperature conditions in the chromatograph. In part, this could explain its lower percentage determined by GC versus the quantity determined by NMR.   (4), and E-β-ocimene (5) as main constituents of the T. lucida Cav. essential oil.

Antinociceptive Activity in the Writhing Test
It was considered to integrate the effects of both treatments to show that the essential oil and the bioactive metabolite produced a similar profile in the temporal course ( Figure 2A) and in the dose-response graph ( Figure 2B). A maximal number of writhes was observed in mice receiving the vehicle; this point was reached after the first 10 min of the nociceptive agent injection (Figure 2A). The T. lucida essential oil (0.1, 1.0, and 10 mg/kg, i.p.) produced significant antinociceptive activity by reducing writhing behavior in a dose-dependent manner, reducing nociception (75.39%) at dosage of 10 mg/kg, i.p., in a similar fashion to β-caryophyllene at doses of 3.16 and 10 mg/kg (Figure 2A,B) (time: F 6,222 = 29.68, p < 0.0001; treatment: F 6,37 = 49.99, p < 0.0001; interaction: F 36,222 = 8.68, p < 0.0001). Data expressed as the AUC over a period of 30 min allowed observing the significant reduction in the nociception of the essential oil and one of its metabolites, resembling that obtained with the analgesic metamizole (80 mg/kg, i.p.) ( Figure 2B) (F 7,42 = 12.52, p < 0.0001).

Antinociceptive Activity in the Formalin Test
In this test, the temporal course curves of the shaking behavior in rats receiving the essential oil showed a significant reduction at doses of 10 and 31.62 mg/kg in neurogenic and inflammatory phases of the formalin ( Figure 3A-C) (time: F 6,174 = 34.74, p < 0.0001; treatment: F 4,29 = 11.77, p < 0.0001; interaction: F 24,174 = 5.23, p < 0.0001). No significant diminution was observed in the dosage of 3.16 mg/kg of this treatment ( Figure 3A-C), whereas a significant and dose-dependent response in the neurogenic phase between doses of 10 and 31.62 mg/kg, i.p. allowed determining a half-maximal effective dose (ED 50 ) = 14.54 mg/kg, i.p. (F 4,30 = 6.12, p = 0.001) ( Figure 3C). On the other hand, a significant but non-dose-dependent effect was obtained in the inflammatory stage (F 4,30 = 6.02, p = 0.001) ( Figure 3C).
The bioactive metabolite β-caryophyllene also produced a significant reduction in the nociceptive behavior all along the temporal course ( Figure 4A-C). These results allowed observing that β-caryophyllene (10 mg/kg) produced almost a 95% analgesic-like effect in the neurogenic phase ( Figure 4B). Regarding the inflammatory phase ( Figure 4C), β-caryophyllene at 5.62 and 10 mg/kg produced antinociception of 34% and 73%, respectively.  .62, and 10 mg/kg) in comparison to the anti-inflammatory drug indomethacin (Ind: 20 mg/kg) and vehicle (Veh) in the formalin test in rats. Two-way ANOVA followed by Tukey's post-hoc test. One-way ANOVA followed by Dunnett's test. # p < 0.05, *** p < 0.001.

Median Lethal Dose (LD 50 )
The LD 50 was calculated as 316 mg/kg, i.p. in mice. This dosage produced mortality in 50% of mice treated with the T. lucida essential oil. Animals receiving 100 or 1000 mg/kg, i.p. showed a reduced ambulatory activity that was recovered after 10 and 25 min of the treatment administration, respectively. Twenty-four hours after treatment, mice administrated with 1000 mg/kg died (100% mortality), whereas those receiving 100 mg/kg showed 0% mortality. Mice surviving gained weight and did not show pathological changes in the macroscopic examination after euthanasia subsequent to the observation for 14 days.

Discussion
The Asteraceae family is endemic to the American continent. Mexico is an important center of origin and diversity for tribes, subtribes, genera, and species of this botanical family, contributing around 30% of the diversity of Asteraceae. The Tagetes genus belongs to the Tageteae tribe and, until now, 56 species distributed in America were reported; about 24 to 28 of them are in Mexico, but the knowledge in such species can be qualified as scanty and scattered [16].
T. lucida belongs to the Asteraceae family. It is an ancient medicinal species that, according to the literature and recent verbal reports of merchants and healers of Mexican regions, is used to treat several gastrointestinal affections involving colic and ulcer, as well as to alleviate musculoskeletal pain [6]. Because of the ethnobotanical reports for T. lucida, in this study, we explored the analgesic-like response using the formalin test in rats, a nociceptive pain model that allowed exploring central (neurogenic) and peripheral (inflammatory) nociception, as well as the writhing test in mice, which mimics abdominal pain.
Aromatic properties of Tagetes depend on the presence of essential oils. In the case of T. lucida Cav., the phytochemical studies of its essential oil were already reported after gas chromatography/mass spectrometry analysis. It is important to mention that the presence of methyl chavicol (estragole) was observed as a major constituent among other volatile compounds in T. lucida species, such as those cultivated in Guatemala (33.9%) [17], Costa Rica (95-97% aerial parts) [18], Italia (93.8% leaf and 78.2% flowers) [19], Cuba (96.8%) [10], Colombia (92.1%) [20], and Egypt (90%) [11]. However, this compound was not found in the T. lucida oil extracted from a species from Mexican regions, as corroborated in this study. The low or very low presence of methyl chavicol (<11.7%) agrees with other groups exploring this species [21].
Estragole is a natural oil constituent in several species. Studies using oral, intraperitoneal, or subcutaneous administration of this constituent reported carcinogenic effects in mice, with controversial results reported for its mutagenicity [22]. In fact, metabolites of estragole are considered stronger hepatocarcinogens due to the formation of hepatic DNA adducts in vivo and in vitro [23]. An absence of this toxic constituent in Mexican species is important to mention because of the frequent medicinal use of the plant for several ailments [6].
In our study, T. lucida essential oil produced a significant and dose-dependent pharmacological antinociceptive response in the nociceptive experimental pain model. The most abundant constituents found in this species were geranyl acetate, followed by the presence of geraniol and β-caryophyllene; minor abundance was found for (−)-β-cubebene, (E)-β-ocimene, γ-elemene, linalool, and β-pinene, as well as traces of others.
Geranyl acetate (100 and 200 mg/kg, i.p.) and geraniol (12.5-50 mg/kg, i.p. and 50-200 mg/kg, p.o.) were previously reported to possess significant antinociceptive activity in the writhing and glutamate tests in mice exploring antioxidant activity [29], as well as the inhibition of Na+ voltage-sensitive sodium channels [30], suggesting involvement in glutamatergic neurotransmission. No neurological alteration was produced by geranyl acetate, since motor performance was not modified in the rotarod test after at dosage of 200 mg/kg, i.p. [29]. In addition, geraniol produced antiallodynic and anti-hyperalgesic effects in neuropathic pain in rats, reducing inducible nitric oxide synthase and N-methyl-d-aspartate receptor 1 expression, as well as protein levels of TNF-α in the injured region [31].
Another interesting and important metabolite in the T. lucida essential oil is β-caryophyllene, which is considered a potent selective agonist of the cannabinoid receptor subtype 2 (CB2). Its antinociceptive activity in a neuropathic pain model in mice reported not only the involvement of CB2 receptors in the central nervous system (CNS) but also inhibition of p38 MAPK/NF-κB activation, as well as reducing cytokine release [32]. No psychoactive responses are associated with CNS effects of β-caryophyllene; in contrast, it is considered a neuroprotective compound [33]. In addition, it was reported as a significant inhibitor of the inflammatory phase of formalin using oral administration [34]. In our study, it was observed that opioid and benzodiazepine receptors are also involved in the antinociceptive mechanism of action of this terpene, mainly at a central level. On the other hand, in addition to these receptors, the 5-HT 1A serotonin receptor and nitric oxide showed partial participation in both phases of the formalin test in rats. Traditional healers recommend T. lucida aerial parts, not only in hot baths but also orally consumed as an infusion or hydroalcoholic extract. In a preliminary study, we explored a polar extract and the identified flavonoids and coumarins as antinociceptive compounds of this species [35]. In addition, our present data complement the potential of this medicinal species by exploring the essential oil and a bioactive metabolite, as well as likely constituents in the hot preparations of this plants. Altogether, these constituents and others might be acting in a synergistic manner to support the potential analgesic-like effect of T. lucida preparations in traditional medicine for alleviating pain.
The essential oil was tested for a possible toxicological effect obtaining an LD 50 in mice of 316 mg/kg, i.p. The results showed that T. lucida produced lower toxicity in comparison to other Tagetes species already reported, with a toxic dose of 99.6 mg/kg for T. erecta and 112 mg/kg for T. patula, per os (p.o.) [36].
Gastric damage is a well-known collateral adverse effect in NSAIDs because of the pharmacological mechanism of action. Indistinct inhibition of cyclooxygenase 1 (COX-1) and 2 (COX-2) by this anti-inflammatory drug is the cause of ulcers in the muscular layer of the stomach [37]. Our data agree with a preventive gastric damage instead of ulceration preliminary reported for the T. patula essential oil [38]. These results reinforce the medicinal use of Tagetes species and bioactive constituents of their essential oils for gastrointestinal diseases.

Plant Material
Aerial parts of Tagetes lucida Cav. (Asteraceae) were collected in San Cristóbal, Municipality of Ecatepec Mexico State, Mexico. People use this plant during the time of flowering, and it is commonly collected from the field or bought in the markets. Since flowering occurs from July to December, the vegetal material for this study was collected in September 2016. Taxonomist Ana Rosa López identified the species with voucher specimen No. 82038 deposited at the Herbarium of the Biology Department of the Universidad Autónoma Metropolitana-Iztapalapa.

Essential Oil Preparation
The essential oil from aerial parts of Tagetes lucida was prepared by steam distillation for three hours using air-dried and ground aerial parts (749 g of plant material and 9.0 L of distilled water) using a modified Clevenger-type apparatus. From this procedure, 2 mL of the pure essential oil was obtained (yield 0.26%), which was dried over anhydrous sodium sulfate and stored in an amber bottle at −4 • C until further analysis.

Equipment and Chromatographic Conditions
NMR spectra were acquired on a Varian Unity INOVA at 400 MHz ( 1 H) and 100 MHz ( 13 C). The major volatile compound was identified based on NMR spectroscopy ( 1 H, 13 C, COSY, HSQC, HMBC, Varian, Santa Clara, CA, USA).
Analyses by GC/MS were carried out in an Agilent 7890 A series gas chromatograph (Agilent Technology, Palo Alto, CA, USA) coupled with a JEOL JMS-GC Mate II mass spectrometer (JEOL USA, Peabody, MA, USA). Compounds were separated on an Agilent Technology DB-5MS (30 m × 0.25 mm, film thickness 0.25 µm) capillary column, using the following GC oven temperature program: 1 min at 40 • C, followed by 8 • C/min up to 300 • C, held for 6 min. Helium was used as the carrier gas at a flow rate of 1 mL/min. The electronic ionization energy was 70 eV, and the mass range scanned was 1-600 uma. Scan rate was 1 spec/s. Transfer line and ionization chamber temperatures were 300 • C. Data acquisition and processing were performed with TSS pro 3.0 software system (Shrader Analytical and Consulting Laboratories, Detroit, MI, USA); mass spectra were compared with the National Institute of Standards and Technology-2 (NIST-2) data base. The components were identified based on the comparison of the relative retention indices calculated using a series of n-alkanes (C-8-C-20) and the mass spectra from the spectrometer database (NIST library), followed by comparison with published data.

Animals
Female Swiss Webster mice (25-30 g) and female Wistar rats (180-200 g) used in the study were provided by Instituto Nacional de Psiquiatría "Ramón de la Fuente Muñiz". Animals were kept at a controlled temperature of (22 ± 1 • C) with a light/dark cycle of 12 h, and they were fed ad libitum with standard water and food. The study was carried out according to the Official Mexican Norm for the care and handling animal (NOM-062-ZOO-1999), and the international rules of care and use for laboratory animals. Experimental activity was done in the institute following the specifications issued by the Committee of Ethics and Research of the Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz with the approval of the project numbers NC-123280.0 and NC-17073.0 (CONBIOETICA-09-CEI-010-20170316).

Experimental Design
Antinociceptive Activity Groups of at least six mice or rats received the vehicle or different doses of the T. lucida essential oil or β-caryophyllene as the possible bioactive metabolite. After 30 min, a nociceptive agent was injected to induce writhing or shaking behavior, as described below.
Writhing test. Abdominal nociception was induced by an i.p. injection of 1% acetic acid; this test is characterized by abdominal contractions known as writhes, described as an exaggerated extension of the abdomen combined with the outstretching of hind limbs [39]. The essential oil (0.1, 1.0, and 10 mg/kg, i.p.), β-caryophyllene (3.16 and 10 mg/kg, i.p.), and metamizole (reference drug, 80 mg/kg, i.p.) were evaluated in this test. Immediately after acetic acid administration, the number of writhes was recorded for 1 min every 5 min during 30 min. Data expressed as temporal course curves were utilized to observe changes in the maximal number of writhes induced in mice. A dose-response graph was plotted using the area under the curve (AUC) to determine the significant antinociceptive dose of the essential oil or β-caryophyllene (see timeline in Figure 7A). Formalin test. After a habituation of 20 min, rats were administered with the essential oil (3.16, 10, and 31.62 mg/kg, i.p.), β-caryophyllene (3.16, 5.62, and 10 mg/kg, i.p.), or the reference drug indomethacin (20 mg/kg, i.p.). Thirty minutes later, chemical nociception was induced as follows: immediately after 50 µL injection in the sub plantar area of the right hind paw with 1% formalin by using a 30-gauge needle, each rat was placed into a glass cylinder provided with mirrors to enable a total panorama of the nociceptive behavior [40]. The number of shakings observed in the injected paw was taken as the nociceptive response. Two periods of high shaking activity were considered. The first one was present immediately after injection and lasted 5 min; this is known as the early phase (neurogenic phase). A second period was observed 5-30 min after formalin injection, denominated the late phase (inflammatory phase). Control animals received the vehicle with the same route and time of administration (see timeline in Figure 7B). The temporal course curves and dose-response graphs were plotted. The area under the curve (AUC: shaking per min) was calculated to determine the significant antinociceptive dose of the essential oil and β-caryophyllene.

Gastric Damage as an Adverse Effect
Rats receiving vehicle or treatment with the reference drug indomethacin (20 mg/kg, i.p.) or the Tagetes lucida essential oil (10 and 31.62 mg/kg, i.p.) and β-caryophyllene (3.16 and 10 mg/kg, i.p.) alone were explored for possible gastric damage.
For gastric lesion observation, all rats were euthanized inside of a CO 2 chamber, and then the stomachs were dissected. Then, the stomachs were filled with 10 mL of 10% formaldehyde to fix the tissue for 10 min. Finally, the stomachs were opened by the greater curvature and rinsed with distilled water to eliminate their content. A scanning of the tissue was obtained for qualitative analysis of the possible gastric ulcer lesions [41] (see timeline in Figure 7C).

Acute Toxicity
The method estimated the dose of the essential oil that would kill 50% of the animal population via an intraperitoneal route of administration (i.p.). Mice were treated with a maximal dosage of 2000 mg/kg, i.p. according to the indication of the Organisation for Economic Co-operation and Development (OECD) (2001). If 100% of the treated mice died, doses of 1000 mg/kg and 100 mg/kg were then tested. Mice surviving were kept under observation for the following 14 days, and their weights were registered; at the end of the study, a macroscopic evaluation was done. The median lethal dose was calculated according to the report of Lorke [42] (see timeline in Figure 7D).

Statistical Data Analysis
To describe nociceptive behavior (dose-response) in the writhing and formalin tests, the temporal course curves of nociception were calculated as the area under the curve (AUC) using the trapezoidal rule. Data are expressed as the mean ± standard error of the mean (SEM) of at least six repetitions. Temporal course curves were analyzed by two-way repeated-measures analysis of variance (ANOVA) followed by Tukey's post hoc test. Dose-response data were examined by one-way ANOVA followed by Dunnett's test or Tukey´s test using GraphPad Prism software (GraphPad Software INC, La Jolla, CA, USA), version 8.0. A p-value <0.05 was considered statistically significant.

Conclusions
In conclusion, the NMR and GC/MS methods allowed the analysis of T. lucida essential oil to demonstrate the presence of geranyl acetate as the major compound, followed by geraniol and β-caryophyllene, where the latter was confirmed as a participant bioactive metabolite in the antinociceptive activity of this Tagetes species. Pharmacological in vivo data gave evidence not only of the antinociceptive effects of this plant but also its safety as analgesic, since it did not produce gastric damage, unlike clinic drugs, suggesting its potential for pain therapy.