Antinociceptive local activity of 4-allyl-1-hydroxy- 2-methoxybenzene (eugenol) by the formalin test: an anti-inflammatory effect

Eugenol has been employed for decades as a condiment, an antimycotic, an antibacterial, an antiviral, and an antioxidant, and it is one of the natural analgesics most frequently utilized for pain and inflammation. Our objective was to determine the analgesic/anti-inflammatory effect of eugenol compared with diclofenac, naproxen, and tramadol using the formalin test. The formalin method was used in 6to 10-week-old Wistar rats (weighing 250 g each) divided into six groups: saline (0.9%); formalin (5%); diclofenac (250 μg/kg); naproxen (400 μg/kg); tramadol (500 μg/kg), and eugenol (1,400 μg/kg), in the intraplantar part of the hind-end trunk of the rats, with n = 5 per group. Eugenol diminished 44.4% of nociceptive behavior in phase 1 and 48% in phase 2 (p ≤0.05 vs formalin). Eugenol was shown to be 1.14 times more effective than diclofenac, but 1.62 and 1.75 times less effective than naproxen and tramadol, respectively, in phase 1 and 1.45 times less effective than diclofenac and naproxen and 1.66 less effective than tramadol in phase 2 (p ≤0.05). These data suggest that eugenol possesses moderate activity in the acute pain phase and greater activity in inflammatory-type pain, and both effects are comparable to those produced by diclofenac and are less than the effects produced by naproxen and tramadol in the formalin test.


INTRODUCTION
For centuries, popular culture has utilized plant extracts to alleviate a wide range of pathologies, principally pain. Thus, according to a World Health Organization (WHO) report, between 70 and 80% of the world population depends on some herbal sources as their main therapeutic option (OMS, 2012).
Eugenol (4-allyl-2-methoxyphenol; C10H12O2) is a natural product with broad therapeutic properties that is extracted from Eugenia cariophyllata and other plants. Eugenol belongs to the phenlypropanoid family and has a molecular weight (MW) of 164.2 g/mol with a pKa = 10.19 at 25°C (Charan-Raja et al., 2015). This plant has been used as an antioxidant (it inhibits the generation of active oxygen species in endothelial cells), antimycotic, antiviral, antiparasitic, antibiotic, and in deinsectation (Kong et al., 2014c). Studies in humans have shown that eugenol possesses antiplatelet activity due to the inhibition of cyclooxygenase (COX)-dependent thromboxane A2 (TXA2) (Raghavendra and Naidu, 2009). It has been considered to be an anti-inflammatory agent due to its participation in the inhibition of cyclooxygenase type 2 (COX2) expression and, consequently, of prostaglandins (PG), which are derived from COX2 (Murakami et al., 2012). PG and leukotrienes (LT) are mediators of the inflammatory response; thus, they increase blood flow and vascular permeability and, at physiological concentrations, sensitize nerve endings and attenuate pain and inflammation (Faezeh et al., 2015).
iD It has been noted that eugenol at low concentrations exerts a reducing effect on synaptic transmission of the neuromuscular zone, thus being capable of producing anesthesia (Charan-Raja et al., 2015). However, Dal Bó and colleagues (2013) demonstrated that the analgesic mechanism of action can involve the opioid system, glutaminergic receptors (Kainate and AMPA), and the inhibition of tumor necrosis factor alpha (TNF-α); therefore, it can be an important component in the treatment of acute pain.
In models of pain and inflammation, eugenol has demonstrated its effectiveness in the diminution of behavior induced by acetic acid and the hot-fire platelet test in mice to an extent comparable with the effects of celecoxib and indomethacin (Apparecido et al., 2009).
Moreover, eugenol is effective in some treatments for dental alveolar osteitis, presentations of pastes that contain a vegetal fiber impregnated with iodoform, butoform, eugenol, olive oil, and a topical anesthetic (Morales, 2011). Among some of the applications in the pharmaceutical industry, we can highlight topical creams and emulsions for the oral administration of medicaments used as topical and oral anesthesia (Hu, Arocha, Pineda, 2014). One of the specialties that has the most experience in the use of eugenol is odontology, given that it has played a very important role in dental preparations applied on deep cavities (Pavithra, 2014). An example of this is the use of the zinc oxide/ eugenol (ZOE) combination, which is frequently employed as obturation material in the treatment of pulpotomy and pulpectomy in temporary dentition (Fucks and Peretz, 2016;González-Lara et al., 2014). The aim of this work was to determine the antinociceptive activity of eugenol compared with diclofenac, naproxen, and tramadol by the formalin method in rats.

Animals
We utilized male Wistar rats 6 to 8 weeks of age, with an approximate weight of 250 g each, which were acclimatized at a regulated temperature of 25°C with a 12:12 h light:dark cycle and free access to food and water. Each experimental group consisted of n = 5. Once the tests were carried out, the animals were sacrificed according to the ethical guidelines for the investigation of pain in experimental animals of the International Association for the Study of Pain (IASP, 2014). The experimental models were maintained under standard bioterium conditions.

Measurement of nociceptive response
Prior to the experiment, the animals were acclimatized for a 60-min period for 3 days and for 1 h prior to the experiment. Nociception was measured under the administration of formalin at 5% (50 µL) and the quantification of the number of paw flinches/shakes during 1 h for 5-min periods in two phases: the first comprised 0 to 15 min, and the second, 15 to 60 min. The antinociceptive effect was evaluated with the administration of the treatments 20 min before the intraplantar administration of formalin at 5%.
For evaluation of the analgesic effect of the drugs, we calculated the percentage of antinociception by means of the following formula:

Experimental design
The animals were injected with a total solution of 50 µL per experiment with the following concentrations: formalin 5%, saline solution 0.9%, eugenol 1,400 µg/kg, diclofenac 250 µg/kg, naproxen 400 µg/kg, and tramadol 500 µg/kg, in independent groups and a volume of 25 µL at the local level in the intraplantar part of the hind-end trunk of the rats (Table I; proportion v/v).

Statistical analysis
We calculated the average and standard error (SE) of each group with an n = 5 for the time courses; after this, we applied the Shapiro Wilks normality test, the one-way analysis of variance (ANOVA), followed by a post hoc Tukey test (p ≤0.05). All of the data were modeled using the Origin ver. 8.0 statistical software package.
from 15 min to 60 min, there was a decrease of up to 34 paw flinches/shakes with respect to the group with formalin 5% (p ≤0.05); see Figure 1(a). Administration of diclofenac (250 µg/kg), as seen in Figure 1(b), demonstrated a diminution from 28 to 12 paw flinches/ shakes at 15 min and from 12 to 5 paw flinches/shakes from min 15 to min 60 (p ≤0.05) vs. formalin 5%. The group with naproxen (400 µg/kg) presented 6-11 paw flinches/shakes from min 5 and remained at these values until min 60 of evaluation (p ≤0.05 vs. formalin 5%); see Figure 1(c). On administering tramadol (500 µg/kg), from min 5, we observed only 5-8 paw flinches/shakes, and this The treatments are administered twenty minutes before the administration of formalin 5 %.

Non-steroidal anti-inflammatory drugs (NSAIDS)
are considered first-choice analgesics for slight-tomoderate pain; however, for moderate-to-severe pain, the joint use of NSAIDS and low-dose opioids is considered, according to WHO criteria in 2012. Additionally, mention has been made of the use of tramadol for slight-to-severe pain; however, in view of the natural-type therapeutic options, eugenol had been widely utilized within the empirical context by means of some scientific criteria, as in the case of odontology (Apparecido et al., 2009;Escobar-Garcia et al., 2016). In this regard, Dal Bó and coworkers (2013) evaluated the effects of eugenol at 3-300 mg/kg, per os (p.o.) for 60 min, or intraperitoneally (i.p.) for 30 min, finding that it inhibited 82 ± 10% and 90 ± 6% of nociceptive behavior, respectively, in the acetic acid acute pain model in mice. Our data reflect the decrease in the number of paw flinches/shakes as 48% with eugenol vs. the 5% formalin group (p ≤0.05), but from min 15 of the formalin test, as seen in figure 1(a), the behavior was more marked in inflammatory pain due to its effect in phase 2, as seen in figure 2, with a tendency to diminish the acute pain phase (44.4% without statistical significance, phase 1 vs. formalin 5%); see figure 3.
These data are also in agreement with those described by Apparecido and colleagues in 2009, in which the authors demonstrated that eugenol at an oral dose of 400 µg/kg presented antinociceptive effects in a carrageenan model. Eugenol diminished edema 2-4 h after the administration of carrageenan in 41.1% of subjects, very similar to the effect produced by indomethacin and celecoxib. Garcia de Alba Garcia and coworkers (2012) noted that eugenol likewise reduced synaptic transmission at the neuromuscular junction, where nerve fibers play an important role in the generation of the inflammatory response, because sensory nerves in dental pulp contain vasoactive peptides such as substance P (SP), the peptide related to the calcitonin gene, and others. For their part, Faezeh and colleagues (2015) reported that oil of clove is a potent inhibitor of thromboxane production and platelet aggregation in human blood in vitro: PG as well as leukotrienes (LT) are important mediators in the inflammatory response.
The fact that eugenol effectiveness during phase 1 of the formalin test was 44.4% and diclofenac was 38.8% (see figure 2) and that in phase 2, diclofenac effectiveness was 70% (see figure 3) and eugenol was 48% might be explained based on activity in the nervous system and on the vascular components of the inflammatory response. These results indicate a potential for eugenol FIGURE 2 -Graphic of the phase 1 of the formalin test, the data representing Ẋ ± SEM of an n = 5. *P ≤0.05 vs. the 5% formalin group. as an analgesic as well as an anti-inflammatory, to an extent comparable with diclofenac and naproxen. In the literature, there are very few data in this regard. In this context, Yano and colleagues (2006) demonstrated that, in mice, methyleugenol (10 µg/kg) isolated from Asiasari radix significantly diminished the duration of paw licking and biting time in phase 2 of the formalin test without affecting phase 1. This behavior is similar to that found in our experiments for eugenol and comparable to that produced by diclofenac, which itself has demonstrated an effect in the formalin test (Picazo, Castañeda-Hernández, Ortiz, 2006) and the thermal model of inflammation (Hasani et al., 2011).
Within this context, eugenol presents a 1.62-times lesser effect in phase 1 (44.4 vs. 72.2%; see figure 2) and a 1.45-times lesser effect in phase 2 than naproxen (48 vs. 70%; see figure 3). In this regard, Mendoza et al. (2013), in a rat model of osteoarthritis, found that naproxen diminished inflammatory pain by 41.6%, which is even less than our finding in the formalin test during phases 1 and 2 (72.2 and 70%, respectively; p ≤0.05 vs. formalin 5%; see figures 2 and 3). Liang and colleagues (2013) proposed that the anti-inflammatory activity of eugenol is similar to that of naproxen and exhibits less ulcerogenic activity. In addition, they synthesized a prodrug denoted as the naproxen eugenol ester, with good anti-inflammatory results and fewer adverse reactions. Zhao and colleagues (2005) proposed a formulation of ibuprofen-eugenol ester as an oral suspension with promising effects in the field of analgesia and inflammation, which is in agreement with our findings of a more marked activity of eugenol in phase 2.
Tramadol conferred a 1.75 and 1.66 times greater phase 1 and 2 effect, respectively, than eugenol. It diminished the behavior by 66% and nearly 77.7% in phase 1, and 80% of phase 2, in the formalin test for eugenol (p ≤0.05 vs. formalin 5%) in our study; see figures 2 and 3. It appears, according to other studies, that eugenol presents an antinociceptive effect that can be due to central and peripheral control mechanisms (Kurian et al., 2006), which can be mediated by adrenergic α2, opioid (but not serotoninergic) mechanisms (Park et al., 2011), glutamatergic receptors (kainate and AMPA), inhibition of TNF-α (Dal Bó et al., 2013), calcium channel and vanilloid receptor modulation (Kurian et al., 2006), and in neuropathic pain, the antinociceptive effect is provided by N-methyl-D-aspartate receptors (NMDAr) (Aoshima, Hamamoto, 1999). However, eugenol does not have a similar effect to that of tramadol at the dose employed.
Despite the scientific evidence and experience in the use of eugenol, no total acceptance for its use has been achieved. Notwithstanding this, in the dental area, it has been utilized (Fucks, Peretz, 2016;Escobar-Garcia et al., 2016). An example of this is pulpar therapy in temporary dentition (González-Lara et al., 2014). Other studies argue about the toxic effects in certain cellular types, such as that reported by Escobar-Garcia and colleagues (2016), in which the authors assert that different concentrations of eugenol produce high toxicity in dental pulp fibroblasts in temporary dentition.
On the other hand, eugenol has been shown to have anti-cancerogenic properties. It is widely used in the treatment of dental caries and periodontal diseases, and it has been shown to diminish allergic asthma and produce anticonvulsant and anti-stress activities (Kurian et al., 2006).
With the experimental evidence, it is clear that eugenol (4-allyl-2-methoxyphenol) has considerable potential from the viewpoint of inflammatory pain in models such as that of formalin. The data substantiate the empirical knowledge of its use, complement the experience of its employment in the clinic, and can support its use in natural therapy as an option with a scientific rationale.

CONCLUSION
The data suggest that eugenol possesses moderate antinociceptive activity in acute pain and more activity in inflammatory-type pain (phase 1 and 2 of the formalin test) at the doses employed. Both effects are comparable to those produced by diclofenac but inferior to the effects produced by naproxen and tramadol in the formalin model.