Karanfil yağının göz ameliyatı yapacak düzeylerde bölgesel uyuşturma etkisini gösteren bilimsel çalımadır. Karanfil yapının topikal anestezik etkisi ile korneaya müdahale yapılabilmiştir.
Effects of topical and systemic administration of Eugenia caryophyllata buds essential oil on corneal anesthesia and analgesia
Clinical studies suggest that essential oil of Eugenia caryophyllata (Clove) buds (EOEC) is efficacious in the treatment of dental pain. In the present study, we investigated the analgesic and local anesthetic effects of EOEC and its possible mechanisms of action in acute corneal pain in rats. EOEC was extracted by hydro-distillation in a Clevenger type apparatus from clove buds. The acute corneal pain was induced by applying a drop (40 µl) of 5 M NaCl solution on the corneal surface, and the numbers of eye wipes were counted during the first 30 s. The mechanical sensation of the cornea was evaluated by calibrated Von Frey filaments. Systemic administration of EOEC (100 and 200 mg/kg, SC) and morphine (2.5 and 5 mg/kg, IP) produced a significant antinociceptive effect in acute corneal pain. Pretreatment with naloxone or atropine prevented the EOEC-induced analgesia. However, L-arginine and methylene blue did not change the suppressive effect of EOEC on corneal pain response. Topical application of EOEC, eugenol and lidocaine significantly decreased corneal sensitivity. Combination treatments of eugenol (25 µg) with lidocaine (0.5%) and EOEC (50 µg) with lidocaine (0.5%) also significantly suppressed corneal sensitivity. Systemic administration of EOEC produced analgesia in the acute corneal pain through mechanisms that involved both opioidergic and cholinergic systems. In addition, topical instillation of EOEC, eugenol, and lidocaine produced local anesthesia in the rat cornea. Sub-anesthetic doses of EOEC or eugenol produced a significant local anesthetic effect when concurrently used with the sub-anesthetic dose of lidocaine.
Keywords: Eugenia caryophyllata, Analgesic, Corneal pain, Lidocaine, Local anesthesia, Eugenol
Eugenia caryophyllata, well known as Clove, is a tree from the Myrtaceae family with a height ranging from 10 to 20 meters that is widely cultivated in Madagascar, Tanzania, Sri Lanka, Brazil and Indonesia (1). Clove oil and its essential oil are traditionally used in aromatherapy and for the relief of headaches, joint pains, toothaches and also as an oral antiseptic (2,3). In addition, eugenol as a major component of clove essential oil has different kinds of biological activities, including antifungal (4), antiallergic (5), and antioxidant (6) properties. Essential oil of E. caryophyllata and its main constituent eugenol have been recognized as a safe, effective and inexpensive anesthetic for fishes and amphibians (7). Also, the analgesic effect of eugenol in different models of pain has been well documented (8,9,10,11,12).
Pain arises from the cornea would be very powerful and incapacitating. Corneal nociceptor density has been estimated to be 20-40 times greater than dental pulp and 300 to 600 times higher than skin (13). These polymodal nociceptors mostly respond to a range of noxious stimuli such as cold, heat, high threshold touch, chemicals, and protons. Moreover, there is a wide range of conditions including dry eye, post-herpetic neuralgia, trigeminal neuralgia, contaminated environ-ments, contact lens wear and new surgical techniques for the correction of refractive defects that cause ocular discomfort and pain (14).
Due to lack of understanding and recognition of acute and chronic trigeminal pain mechanisms, there are some difficulties in the management of these kinds of pains (15).
Because of less adverse and more beneficial effects, herbal therapies have a great advantage over common painkillers like opioids and nonsteroidal anti-inflammatory drugs when side effects are taken into account (16).
The essential oil of E. caryophyllata buds (EOEC) is used in dental care as an analgesic, local anesthetic and oral antiseptic. Therefore, the present study was aimed to investigate the systemic antinociceptive and local anesthetic effects of EOEC in rat cornea. Also, to clarify the possible analgesic mechanisms of EOEC, we used morphine, naloxone (nonselective opioid receptor antagonist), atropine (nonselective muscarinic receptor antagonist), L-arginine (nitric oxide pathway precursor), and methylene blue (a non-specific inhibitor of NO/guanylyl cyclase) in the acute model of corneal chemical pain. Moreover, corneal touch thresholds and also duration of corneal local anesthesia were determined after topical instillation of EOEC and eugenol (as an active ingredient of EOEC), alone and in co-application with lidocaine.
MATERIAL AND METHODS
All study experiments and animal care procedures were approved by the Veterinary Ethics Committee of the Faculty of Veterinary Medicine, University of Tabriz (9 June 2014, Ref. No: D/2014.16) and were performed in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals (17).
Adult male Wistar rats weighing 230-260 g were randomly housed in polyethylene cages with ad libitum access to food and water in a room with controlled temperature (22 ± 1°C) and under a 12-h light-dark cycle (lights on from 07:00 a.m.). Six rats were used in each group. All experiments were performed between 11:00 a.m. and 15:00 p.m. Each rat received one systemic and one topical administration of solution and seven days was allowed between each trial.
Morphine sulfate was purchased from Tolid Darou Co (Tehran, Iran). Atropine sulfate and naloxone hydrochloride, Tween 80, lidocaine and eugenol were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). L-arginine and methylene blue were purchased from Merck Chemicals (Darmstadt, Germany). For the systemic and topical studies all drugs and chemicals were dissolved in physiological saline. An emulsion of essential oil and eugenol were prepared using Tween 80 and saline (0.5%, v/v) as solvent. Polysorbate 80 (Tween 80) is a nonionic surfactant and emulsifier that enables EOEC and eugenol to be easily emulsified in the saline. All solutions were adjusted to pH 7.4 before use.
Plant material and essential oil extraction
The buds of E. caryophyllata were purchased from a Phyto-medical company (Parsi Teb Co, Tabriz, Iran) and were subsequently authenticated by Dr A. Ebrahimi, a botanist at the Herbarium of Faculty of Pharmacy (Tbz-Fph), Medical University of Tabriz, Tabriz, Iran. The dried buds of the plant were ground into a fine powder. The essential oil was extracted from powdered buds by hydro-distillation in a Clevenger type apparatus (Soham Scientific Co, Soham, UK) for 3 h and produced 8% (v/w) yield. Yielded essential oil was dried over anhydrous sodium sulfate until the last traces of water were removed, and then stored in dark glass bottles at 4°C. Gas chromatography mass spectrometry (GC-MS) analysis of EOEC has already been performed in our laboratory and reported (18).
Acute corneal pain induction (Eye wiping test)
Each rat was placed on a 50 × 50 × 1 cm wooden table and after a 15 min habituation period, one drop (40 µl) of 5 M NaCl solution was topically applied onto the surface of the cornea using a sampler (Transferpette® S 10-100 µl Brand Co, Germany). After topical application of 5 M NaCl solution, rats always wiped their eyes with their forepaw and sometimes rapidly scratched their eyes with the hind paw. The numbers of the eye wipes performed with ipsilateral forepaw were counted over a period of 30 s. Each burst of hind paw scratches was counted as one wipe as well (19,20).
The first eye wiping test (pre drug wiping test) of each rat was measured 10 min before all chemicals administration. The second eye wiping test was performed 30 or 40 min after drug administration, depending on the type of treatment. The maximal possible effect (% MPE) of drugs and essential oil was calculated for eye-wipes according to the following formula:
% MPE = 100 × [(post drug wipes count – pre drug wipes count)/(0 – pre drug wipe count)]
Administration of chemicals for evaluation of acute corneal pain
The rats were injected with vehicle (Tween 80, 0.5% in saline, 1 mL/kg, subcutaneously (SC)), EOEC (50, 100 and 200 mg/kg, SC), and morphine sulfate (1.25, 2.5 and 5 mg/kg, intraperitoneally (IP) 30 min before induction of the pain. Naloxone (1 mg/kg, IP), atropine (1 mg/kg, IP), L-arginine (100 and 300 mg/kg, IP) and methylene blue (5 and 10 mg/kg, IP) were administrated 40 min before the last eye wiping test. Each animal was injected only once in the eye wiping test.
Von Frey test for assessing corneal touch threshold
The Von Frey test was employed to examine mechanical sensitivity thresholds of the cornea (21). Animals were gently held and restrained by hand during the testing period. Using a set of calibrated von Frey filaments (VFF) (Stoelting Co., Wood Dale, Illinois, USA), animal withdrawal responses to the corneal touch include blinking and head withdrawal (abrupt head movement) were counted as a positive response. The corneal touch threshold (CTT) was defined as the lowest force of the filaments that produced at least three withdrawal responses in five tests in control animals (21,22,23).
Local anesthetic assay using Von Frey filaments
Dose-dependent study of CTT
To assess behavioral corneal responses in the dose-dependent study, Von Frey filaments of ascending stiffness (0.008, 0.02, 0.04, 0.16, 0.4, 0.6, 1, and 2 g) were used to evaluate mechanical sensitivity of cornea (in the central cornea).
Vehicle (Tween 80, 0.5% dissolved in saline), EOEC (25, 50, 200 µg/eye), eugenol (25, 50 and 100 µg/eye), lidocaine (0.5% and 2%, 40 µl/eye) and combination of EOEC (25 µg) or eugenol (25 µg) with lidocaine (0.5%, 40 µl/eye) were topically applied to the cornea in two consecutive 20 µl volumes (total volume of 40 µl) and animals gently restrained by hand to prevent wiping of solution for 1 min in each application. Five to eight min after solution instillation to the cornea, Von Frey tests were started for evaluation of CTT (24).
Time-dependent study of local anesthesia duration
In the time-dependent study, we used only greatest filament (2 g) for assessment of the duration of corneal anesthesia induced by EOEC (200 µg), Eugenol (100 µl), lidocaine 2% and combination of EOEC (25 µg) or eugenol (25 µg) with lidocaine (0.5%). Touch test procedures were performed 5 min before and 5, 10, 15, 20, 25, 30, and 40 min after topical instillation of all solutions.
Statistical differences were determined by one-way analysis of variance (ANOVA) followed by Tukey HSD post hoc test used for dose dependent studies and also two-way ANOVA with repeated measures and Bonferroni post hoc which used to assess the effects of time and drug in time course study using IBM® SPSS® software version 19 (IBM company, USA). In figures, all values are expressed as Mean ± SEM. A value of P<0.05 was considered statistically significant.
Effect of EOEC and morphine administration on acute corneal pain
In the present study, subcutaneous administration of EOEC at doses of 100 and 200 mg/kg but not at a dose of 50 mg/kg showed an inhibitory effect on eye wiping response (33.84% and 39.91% respectively, P<0.05, Fig. 1A) compared to vehicle treated group. In addition, morphine (1.25, 2.5 and 5 mg/kg, IP) produced an analgesic effect on eye wiping response (30.82%, 41.24% and 74.1% respectively, P<0.05 and P<0.01, Fig. 1B) compared to vehicle treated group.
Effect of naloxone, atropine, and L-arginine on EOEC-induced analgesia in the acute corneal pain
Administration of naloxone (1 mg/kg, IP) alone had no effect on eye wiping response, but pretreatment of animals with naloxone inhibited the antinociceptive effect of EOEC (200 mg/kg) and morphine (5 mg/kg) in the eye wiping response (-1.89% and 14.62% respectively, P<0.01, Fig. 2) in comparison with the EOEC 200 mg/kg and morphine 5 mg/kg groups respectively. On the other hand, administration of atropine (1 mg/kg, IP) alone had no effect on eye wiping response. However, pretreatment of animals with atropine inhibited the antinociceptive effect of EOEC (200 mg/kg) in the eye wiping response (2.22%, P<0.01, Fig. 3) in comparison with the EOEC 200 mg/kg.
Administration of L-arginine (100 and 300 mg/kg, IP) alone had no significant effects on eye wiping response. Pretreatment of animals with L-arginine failed to prevent the antinociceptive effect of EOEC at 200 mg/kg (38.65%, P<0.05, Fig. 4A). In addition, methylene blue (5 and 10 mg/kg, IP) alone had no significant effects on pain response and also pretreatment of animals with methylene blue (5 mg/kg, IP) did not produce any significant effect on EOEC (200 mg/kg) induced antinociception (34.22%, P<0.05, Fig. 4B).
Local anesthetic activity of topical EOEC, eugenol, and lidocaine
The CTT was determined to be 0.4 g (calibrated force) in the vehicle treated cornea (Fig. 5A). In the dose-dependent study, EOEC at doses of 50 µg/eye increased (P<0.05) corneal touch threshold to 0.6 g (calibrated force). EOEC at a dose 0f 200 µg/eye significantly produced a local anesthetic effect (P<0.001) for all tested forces in comparison with the vehicle group (Fig. 5A).
Eugenol at doses of 50 and 100 µg/eye significantly (P<0.05) increased corneal touch threshold to 1 and 2 g calibrated force respectively (Fig. 5B). Topical application of lidocaine at the dose of 2% (40 µl/eye) but not 0.5% produced a potent local anesthetic effect (P<0.001) for all tested forces in comparison with the vehicle group (Fig. 5C). Moreover, co-administration of low (sub-anesthetic) dose of EOEC or eugenol with the low (sub-anesthetic) dose of lidocaine 0.5% produced significant (P<0.001) local anesthetic effect for all tested forces in comparison with the vehicle group (Fig. 5D).
In time course study, the duration of effect was assessed using greatest filament (2 g) by comparing differences over time relative to the baseline for each solution. At baseline, there were no differences in corneal response values between any of the treatment groups. Topical administration of EOEC at 200 µg and eugenol at 100 µg significantly (P<0.001 and P<0.05, respectively) produced the local anesthetic effect against 2 g force produced by VFF until 15 min and 10 min after instillation, respectively, however, lidocaine 2% produced local anesthesia against the same force persisting until 25 min after instillation (P<0.001 and P<0.01) (Figs. (Figs.6A6A and and6B6B).
Co-administration of low (sub-anesthetic) dose of EOEC (25 µg) with the low (sub-anesthetic) dose of lidocaine (0.5%) produced significant (P<0.001 and P<0.01) local anesthesia until 30 min after instillation (Fig. 6C). Co-administration of low (sub-anesthetic) dose of eugenol (25 µg) with the low (sub-anesthetic) dose of lidocaine (0.5%) produced significant (P<0.0001 and P<0.001) local anesthesia until 15 min after instillation (Fig. 6C).
Topical administration of one drop 5 M NaCl solution to the corneal surface produced acute chemical pain responses in this study. It has been shown that the application of NaCl, capsaicin and nicotine on the corneal surface produce a vigorous response in the nociceptive neurons in the trigeminal subnucleus caudalis in rat (25). Thin myelinated A-delta and unmyelinated C type fibers responds to chemical, mechanical and thermal noxious stimuli on the corneal surface (14). It has been reported that hyper-osmotic solutions like NaCl activating transient receptor potential vanilloid subtype 1 (TRPV1) and transient receptor potential melastatin subtype 8 (TRPM8) receptors in corneal nociceptors could induce chemical nociception (26,27). Using hypertonic saline to explore acute corneal pain was adapted by Frazifard et al. (2005) as a new model to assess acute pain in cornea and thereafter this test has been frequently used with success in several studies (19,20,28).
In the present study, IP injection of morphine suppressed eye wiping response in the acute corneal pain and also pretreatment with naloxone prevented this suppressive effect. These results showed that the analgesic effect of morphine is mediated by naloxone-sensitive mechanism in this model of pain.
Our results indicated the antinociceptive effect of EOEC (100 and 200 mg/kg) on hypertonic saline induced corneal pain in rats. In addition, these data provided some evidence for determination of possible mechanisms of antinociceptive action of EOEC in this model of nociception. The antinociceptive effect induced by the EOEC (200 mg/kg, SC) was significantly inhibited by both naloxone and atropine. Moreover, the administration of methylene blue and L-arginine did not alter EOEC-induced analgesia. These findings suggest that the analgesic effect of EOEC in this study may be mediated through opioidergic and cholinergic systems, but not via L-arginine/NO/cyclic GMP pathway.
It was reported that oral administration of eugenol produced naloxone-sensitive anti-nociception in the acetic acid-induced writhing test (8).
Analgesia produced by EOEC in the acute corneal pain appears to depend on its cholinergic activity, since EOEC effect was completely antagonized by the muscarinic cholinergic receptor antagonist atropine. In vitro studies of clove oil and eugenol have shown potent anti-acetylcholinesterase property (29,30). Since cholinergic system activation by muscarinic agonist or cholinesterase inhibitors have shown to produce analgesic effects, anti-acetylcholinesterase activity of EOEC and eugenol might also be in agreement with its mechanisms of their antinociception (31,32).
On the other hand, our previous experiments showed that SC administration of EOEC significantly reduced pain and inflammation in formalin-induced orofacial pain and xylene-induced ear edema, respectively (18).
The results of the present study showed that topical application of EOEC and eugenol like lidocaine (as a positive control) reduced corneal mechanical sensitivity in rats. The patterns of corneal anesthesia produced by application of high doses of EOEC and eugenol were similar to the anesthetic pattern of lidocaine. Co-administration of low (sub-anesthetic) dose of EOEC or eugenol with the low (sub-anesthetic) dose of lidocaine produced a significant local anesthetic effect. It seems that a concur effect between EOEC or eugenol with lidocaine is responsible for their local anesthetic effects when administered concomitantly. Co-administration of sub-anesthetic dose of lidocaine with EOEC produced a long lasting anesthesia in comparison with co-administration of lidocaine with a sub-analgesic dose of eugenol. This effect may be due to the coexisting of other components with eugenol in the E. caryophyllata essential oil.
We observed a high concentration of eugenol (54.86%) and β-caryophyllene (20.19%) in the EOEC composition (18). Eugenol is a phenylpropene derivative, well known as a local anesthetic (33), analgesic (8,11), and anti-inflammatory agent (34). The analgesic and local, anesthetic effects of eugenol may be modulated by its inhibitory effect on voltage-gated Na+ channels (35) and on high voltage-activated Ca2+ channels (36), also by weak activation of TRPV1 receptors (37). As eugenol activates transient receptor potential ankyrin subtype 1 (TRPA1) receptors expressed in trigeminal ganglion neurons in rodents and humans, it is possible that activation of TRPA1 by eugenol contributes to its antinociceptive activity (38). The inhibition of voltage-gated Na+ channels and activation of TRPV1 produced by eugenol are very similar to the effects of local anesthetics such as lidocaine (39).
β-caryophyllene, a sesquiterpene with CB2 cannabinoid receptor agonist activity, also exist in the E. caryophyllata essential oil (18,40). Cannabinoid receptor CB2 and its selective agonists are accepted as a new pharmacological target for the treatment of painful disorders (41). Moreover, topical administration of β-caryophyllene produced a strong local anesthetic effect in the rabbit conjunctival reflex test (42).
The presence of another sesquiterpene, α-humulene, in EOEC was confirmed and reported by us (18). Fernandes et al. reported that oral treatment with α-humulene and (-)-trans-caryophyllene produced noticeable inhibitory effects in different inflammatory experimental models in mice and rats (43).
In conclusion, the present results suggest that the EOEC produced antinociception in the acute chemical corneal pain through mechanisms that involved both opioidergic and/or the cholinergic systems, but not via L-arginine/NO/cyclic GMP pathway, supporting the traditional usage of the plant to treat various painful processes in the trigeminal nerve territory. Topical application of EOEC, eugenol and lidocaine reduced corneal sensitivity, but maximal anesthesia and duration differed between chemicals. Sub-anesthetic doses of EOEC or eugenol produced a good local anesthetic effect when used concurrently with the sub-anesthetic dose of lidocaine.
We would like to thank Dr. Amir Erfanparast at the faculty of veterinary medicine of Urmia University for English editing of this paper. This study was financially supported by the Vice Chancellor for Research and Technology of University of Tabriz, I.R. Iran via grant NO. 7353.
1. Arung ET, Matsubara E, Kusuma IW, Sukaton E, Shimizu K, Kondo R. Inhibitory components from the buds of clove (Syzygium aromaticum) on melanin formation in B16 melanoma cells. Fitoterapia. 2011;82:198–202. [PubMed]
2. Soto CG, Burhanuddin CG. Clove oil as a fish anaesthetic for measuring length and weight of rabbit fish (Siganus lineatus) Aquaculture. 1995;136:149–152.
3. Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi AB, Mahmoud R, Mahdouani K, et al. The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother Res. 2007;21:501–506. [PubMed]
4. Pinto E, Vale-Silva L, Cavaleiro C, Salgueiro L. Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species. J Med Microbiol. 2009;58:1454–1462. [PubMed]
5. Corrêa MFP, Melo GO, Costa SS. Substâncias de origem vegetal potencialmente úteis na terapia da Asma. Rev Bras Farmacogn. 2008;18:785–797.
6. Gülçin I, Elmastas M, Aboul-Enein HY. Antioxidant activity of clove oil – A powerful antioxidant source. Arabian J Chem. 2012;5:489–499.
7. Kildeaa MA, Allanb GL, Kearney RE. Accumulation and clearance of the anaesthetics clove oil and AQUI-S from the edible tissue of silver perch (Bidyanus bidyanus) Aquaculture. 2004;232:265–277.
8. Park SH, Sim YB, Lee JK, Kim SM, Kang YJ, Jung JS, et al. The analgesic effects and mechanisms of orally administered eugenol. Arch Pharm Res. 2011;34:501–507. [PubMed]
9. Lionnet L, Beaudry F, Vachon P. Intrathecal eugenol administration alleviates neuropathic pain in male Sprague-Dawley rats. Phytother Res. 2010;24:1645–1653. [PubMed]
10. Daniel AN, Sartoretto SM, Schmidt G, Caparroz-Assef SM, Bersani-Amado CA, Cuman RKN. Anti- inflammatory and antinociceptive activities A of eugenol essential oil in experimental animal models. Rev Bras Farmacogn. 2009;19:212–217.
11. Kurian R, Arulmozhi DK, Veeranjaneyulu A, Bodhankar SL. Effect of eugenol on animal models of nociception. Indian J of Pharmacol. 2006;38:341–345.
12. Okhubo T, Shibata M. The selective capsaicin antagonist capsazepine abolishes the antinociceptive action of eugenol and guaiacol. J Dent Res. 1997;76:848–851. [PubMed]
13. Rosenthal P, Baran I, Jacobs DS. Corneal pain without stain: is it real? Ocul Surf. 2009;7:28–40. [PubMed]
14. Belmonte C, Acosta MC, Gallar J. Neural basis of sensation in intact and injured corneas. Exp Eye Res. 2004;78:513–525. [PubMed]
15. Miranda HF, Sierralta F, Prieto JC. Synergism between NSAIDs in the orofacial formalin test in mice. Pharmacol Biochem Behav. 2009;92:314–318. [PubMed]
16. Zdunić G, Godevac D, Milenković M, Vucićević D, Savikin K, Menković N, et al. Evaluation of Hypericum perforatum oil extracts for an antiinflammatory and gastroprotective activity in rats. Phytother Res. 2009;23:1559–1564. [PubMed]
17. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 1983;16:109–110. [PubMed]
18. Khalilzadeh E, Hazrati R, Vafaei Sayah Gh. Evaluation of chemical composition, anti-inflammatory and anti-nociceptive effects of Eugenia caryophyllata buds essential oil. J Herbal Drugs. 2014;5:68–76.
19. Farazifard R, Safapour F, Sheibani V, Javan M. Eyewiping test: a sensitive animal model for acute trigeminal pain studies. Brain Res Brain Res Protoc. 2005;16:44–49. [PubMed]
20. Khalilzadeh E, Hazrati R, Vafaie Sayah G, Hasannejad H. Opioidergic and cholinergic but not nitric oxide
pathways are involved in antinociceptive activity of Vitex agnus-castus essential oil in the acute trigeminal model of pain in rat. Asian J Pharm Clin Res. 2015;8:283–286.
21. Ren KE. An improved method for assessing mechanical allodynia in therat. Physiol Behav. 1999;67:711–716. [PubMed]
22. Kim J, Kim NS, Lee KC, Lee HB, Kim MS, Kim HS. Effect of topical anesthesia on evaluation of corneal sensitivity and intraocular pressure in rats and dogs. Vet Ophthalmol. 2013;16:43–46. [PubMed]
23. Zagon IS, Sassani GW, Immonen JA, McLaughlin PJ. Ocular surface abnormalities related to type 2 diabetes are reversed by the opioid antagonist naltrexone. Clin Experiment Ophthalmol. 2014;42:159–168. [PubMed]
24. Bates B, Mitchell K, Keller JM, Chan C, Swaim WD, Yaskovich R, et al. Prolonged analgesic response of cornea to topical resiniferatoxin, a potent TRPV1 agonist. Pain. 2010;149:522–528. [PMC free article] [PubMed]
25. Carstens E, Kuenzier N, Handwerker HO. Activation of neurons in rat trigeminal subnucleus caudalis by different irritant chemicals applied to the oral and ocular mucosa. J Neurophysiol. 1998;80:465–492. [PubMed]
26. Hirata H, Meng ID. Cold-sensitive corneal afferents respond to a variety of ocular stimuli central to tear production: implications for dry eye disease. Invest Ophthalmol Vis Sci. 2010;5:3969–3976. [PMC free article] [PubMed]
27. Pan Z, Wang Z, Yang H, Zhang F, Reinach PS. TRPV 1 activation is required for hypertonicity stimulated inflammatory cytokine release in human corneal epithelial cells. Invest Ophthalmol Vis Sci. 2011;52:485–493. [PMC free article] [PubMed]
28. Tamaddonfard E, Khalilzadeh E, Hamzeh-Gooshchi N, Seiednejhad-Yamchi S. Central effect of histamine in a rat model of acute trigeminal pain. Pharmacol Rep. 2008;60:219–224. [PubMed]
29. Dalai MK, Bhadra S, Chaudhary SK, Bandyopadhyay A, Mukherjee PK. Anti-cholinesterase activity of the standardized extract of Syzygium aromaticum L. Pharmacogn Mag. 2014;10:S276–S282. [PMC free article] [PubMed]
30. Dohi S, Terasaki M, Makino M. Acetylcholinesterase inhibitory activity and chemical composition of commercial essential oils. J Agric Food Chem. 2009;57:4313–4318. [PubMed]
31. Cozanitis DA, Friedmann T, Furst S. Study of the analgesic effects of galanthamine, a cholinesterase inhibitor. Arch Int Pharmacodyn Ther. 1983;266:229–238. [PubMed]
32. Naguib M, Yaksh TL. Characterization of muscarinic receptor subtypes that mediate antinociception in the rat spinal cord. Anesth Analg. 1997;85:847–853. [PubMed]
33. Park CK, Kim K, Jung SJ, Kim MJ, Ahn DK, Hong SD, et al. Molecular mechanism for local anesthetic action of eugenol in the rat trigeminal system. Pain. 2009;144:84–94. [PubMed]
34. Hashimoto S, Uchiyama K, Maeda M, Ishitsuka K, Furumoto K, Nakamura Y. In vivo and in vitro effects of zinc oxide-eugenol (ZOE) on biosynthesis of cyclo-oxygenaseproducts in rat dental pulp. J Dent Res. 1988;67:1092–1096. [PubMed]
35. Park CK, Li HY, Yeon KY, Jung SJ, Choi SY, Lee S, et al. Eugenol inhibits sodium currents in dental afferent neurons. J Dent Res. 2006;85:900–904. [PubMed]
36. Lee MH, Yeon K-Y, Park C-K, Li HY, Fang Z, Kim MS, et al. Eugenol inhibits calcium currents in dental afferent neurons. J Dent Res. 2005;84:848–851. [PubMed]
37. Yang BH, Piao ZG, Kim YB, Lee CH, Lee JK, Park K, et al. Activation of vanilloid receptor 1 (VR1) by eugenol. J Dent Res. 2003;82:781–785. [PubMed]
38. Chung G, Im ST, Kim YH, Jung SJ, Rhyu MR, Oh SB. Activation of transient receptor potential ankyrin 1 by eugenol. Neuroscience. 2014;261:153–160. [PubMed]
39. Leffler A, Fischer MJ, Rehner D, Kienel S, Kistner K, Sauer SK, et al. The vanilloid receptor TRPV 1 is activated and sensitized by local anesthetics in rodent sensory neurons. J Clin Invest. 2008;118:763–776. [PMC free article] [PubMed]
40. Gertsch J, Leonti M, Raduner S, Racz I, Chen JZ, Xie XQ, et al. Beta-caryophyllene is a dietary cannabinoid. Proc Natl Acad Sci USA. 2008;105:9099–9104. [PMC free article] [PubMed]
41. Anand P, Whiteside G, Fowler CJ, Hohmann AG. Targeting CB2 receptors and the endocannabinoid system for the treatment of pain. Brain Res Rev. 2009;60:255–266. [PMC free article] [PubMed]
42. Ghelardini C, Galeotti N, Mannelli LDC, Mazzanti G, Bartolini A. Local anaesthetic activity of β-caryophyllene. Il Farmaco. 2001;56:387–389. [PubMed]
43. Fernandes ES, Passos GF, Medeiros R, Cunha FMD, Ferreira J, Campos MM, et al. Anti-inflammatory effects of compounds alpha-humulene and (-)-trans-caryophyllene isolated from the essential oil of Cordia verbenacea. Eur J Pharmacol. 2007;569:228–236. [PubMed]