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Docking, characterization and investigation of [beta]-cyclodextrin complexed with citronellal, a monoterpene present in the essential oil of Cymbopogon species, as an anti-hyperalgesic agent in chronic muscle pain model.


Background: Citronellal (CT) is a monoterpene with antinociceptive acute effect. [beta]-Cyclodextrin ([beta]CD) has enhanced the analgesic effect of various substances.

Hypothesis/Purpose: To evaluate the effect of CT both complexed in [beta]- cyclodextrin (CT-[beta]CD) and non-complexed, in a chronic muscle pain model (CMP) in mice.

Study Design: The complex containing CT in [beta]CD was obtained and characterized in the laboratory. The antihyperalgesic effect of CT and CT-[beta]CD was evaluated in a pre-clinical in vivo study in a murine CMP.

Methods: The complex was characterized through differential scanning calorimetry, derivative thermogravimetry, moisture determination, infrared spectroscopy and scanning electron microscopy. Male Swiss mice were pre-treated with CT (50 mg/kg, po), CT-[beta]CD (50 mg/kg, po), vehicle (isotonic saline, po) or standard drug (tramadoM mg/kg, ip). 60 min after the treatment and then each 1 h, the mechanic hyperalgesia was evaluated to obtain the time effect. In addition, the muscle strength using grip strength meter and hyperalgesia were also performed daily, for 7 days. We assessed by immunofluorescence for Fos protein on brains and spinal cords of mice. The involvement of the CT with the glutamatergic system was studied with molecular docking.

Results: All characterization methods showed the CT-[beta]CD complexation. CT- induced anti-hyperalgesic effect lasted until 6 h (p < 0.001) while CT-[beta]CD lasted until 8 h (p < 0.001 vs vehicle and p < 0.001 vs CT from the 6th h). CT-[beta]CD reduced mechanical hyperalgesia on all days of treatment (p < 0.05), without changing muscle strength. Periaque-ductal gray (p < 0.01) and rostroventromedular area (p < 0.05) showed significant increase in the Fos protein expression while in the spinal cord, there was a reduction (p <0.001). CT showed favorable energy binding (-5.6 and -6.1) to GluR2-S1S2J protein based in the docking score function.

Conclusion: We can suggest that (ICO improved the anti-hyperalgesic effect of CT, and that effect seems to involve the descending pain-inhibitory mechanisms, with a possible interaction of the glutamate receptors, which are considered as promising molecules for the management of chronic pain such as CMP.





Chronic pain



1. Introduction

Chronic pain is a pathologic painful condition lasting longer than three months, classified as a worldwide health problem that results in enormous social and economic damage (Azevedo et al., 2012). In Canada, the prevalence of people with chronic pain is about 18.9% in adult people (Schopflocher et al., 2011). In the United States, in 2008, about 100 millions of adult people had chronic pain, with annual costs ranging between US$ 560 and US$ 635 billions of dollars in 2010, and additional costs of health, caused by chronic pain, of US$ 261 to US$ 300 billions of dollars (Gaskin and Richard, 2012). Among the causes of chronic pain stands out fibromyalgia (FM), a type of chronic muscle pain (CMP) that is a syndrome of chronic pain present in about 5% of the world population. This syndrome is manifested through widespread musculoskeletal pain, besides sensitivity alterations. Skin hyperalgesia and momentary pain attacks are also reported by the patients (Gauffin et al., 2013).

Despite problems faced because of FM, only 30-35% of the drug therapy offers some benefit, besides the high costs and side effects, such as nausea, weight gain and tachycardia (Mist et al., 2013), bringing forth the need to search for new drugs. Recently, FM and neuropathic pain have been described as a "dysfunctional pain" with many gaps in the pharmacological treatment (Nagakura, 2015), which has created a class of "neglected pains" for specific analgesic treatment. Thus, the pharmacological treatment of chronic pain, such as FM and neuropathic pain, remains a challenge for modern medicine and brings patients, who suffer a lifetime with symptoms, a sensation of being in a bottomless pit.

For thousands of years, scientists and pharmaceutical industry have used natural products (NP) as a search source for new drugs or their precursors, aimed at treating diseases or symptomatology that have not yet had an effective treatment (Harvey et al., 2015). Between 2005 and 2010, the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) approved 19 medicaments derived from natural products,, such as Trabectedin (Yondelis[TM]) and Cannabidol (Sativex[R]), for the cancer and painful treatment, respectively (Mishra and Tiwari, 2011).

Monoterpenes are among the major constituents (about 90%) of the essential oil of several plants, and they possess pharmacological activities already described in the literature (Brito et al., 2012; Menezes et al., 2010). The antinociceptive activity of several monoterpenes such as borneol (Almeida et al., 2013), linalool (Quintans-Junior et al., 2013) and citronellol (Brito et al., 2012) have been described. Furthermore, monoterpenes, as menthol and thymol, are present in various formulations with analgesic effect and available in the market, as Salonpas (Kamatou et al., 2013; McAllister and Burnett, 2015).

In this context, citronellal (CT) is a monoterpene present in the essential oil of several aromatic plants, such as Corymbia citriodora and plants of the gender Cymbopogon, as C. nardus and C. winterianus, isolated with steam distillation, solvent extraction or recently with the ultrasound technique (Kortvelyessy, 1985; Quintans-Junior et al., 2008). Anticonvulsant (M.S. Melo et al., 2011), anti-inflammatory (de Santana et al., 2013) and antinociceptive effects (Melo et al., 2010; L. Quintans-Junior et al., 2011) have been already described for the CT, with evidence of actions on the glutamatergic system (Quintans-Junior et al., 2010), extremely important in the maintenance of chronic pain, and in the opioid system (Melo et al., 2010; L. Quintans-Junior et al., 2011). However, there are no reports on the possible central nervous system (CNS) areas involved in its pharmacological effects (Lenardao et al., 2007) or its properties in dysfunctional pain conditions, such as FM or CMP.

Cydodextrins (CDs) are cyclic products of the starch enzymatic hydrolysis by some microorganisms (Loftsson and Duchene, 2007) that have been used to improve water solubility and also to reduce the number of doses and toxic effects (Oliveira et al., 2009; Schaffazick et al., 2003). According to the International Union of Pure and Applied Chemistry (IUPAC), numerous drugs of clinical importance have [beta]-cydodextrins ([beta]CD) present in their composition, such as the cephalosporin, piroxicam and tiaprofenic acid (Szejtli, 2005). Recently, our research group has focused on the complexation of NP, mainly non-polar compounds such as terpenoids, with [beta]CD (Nascimento et al., 2015; Quintans-Junior et al., 2013; Santos et al., 2015; Siqueira-Lima et al., 2014), and we have reported that this complexation was able to improve the effect of these substances mainly for FM or CMP approaches (Nascimento et al., 2014; Quintans et al., 2013). Moreover, predinical and clinical evidence have demonstrated that the complexation in CDs is capable to improve pharmacological effect of analgesic and anti-inflammatory drugs (Brito et al., 2015; Oliveira et al., 2015).

Following this rational approach, since there is the need to search for new drugs for the treatment of chronic pain and that [beta]CD has been used to enhance the effect of drugs available on the market, we aimed to investigate the possible action of CT complexed in [beta]CD (CT-[beta]CD) on the experimental model of noninflammatory chronic muscle pain (a CMP model), considered to be an animal model for FM (Nascimento et al., 2015), and the possible involvement of CT in CNS areas which may explain its analgesic properties.

2. Materials and methods

2.1. Chemicals

Alexa Fluor 555 (Life Technologies, Carlsbad, California, USA); Bovine Serum Albumin (BSA) (Santa Cruz, USA); ([+ or -])-Citronellal (Sigma, Brazil); c-Fos polyclonal antibody (Santa Cruz Biotechnology, Dallas, Texas, USA); Gelatin (Synth, Brazil); Ketamine (Cristalia, Brazil); Phosphate Buffered Saline (Neon, Brazil); Sodium Chloride (Dinamica, Brazil); Sucrose (Synth, Brazil); Tramadol (Merck, Brazil); Xylazine (Cristalia, Brazil); [beta]-Cydodextrin (Sigma, Brazil).

2.2. Preparation of the inclusion complex

The complex was prepared according to the procedure described by Pinto et al.,(2005), which consisted of the following steps: 1) Physical Mixture (PM): CT 97% (154.25 g/mol) and [beta]-CD 98% (1135 g/mol) were mixed (1:1 mol ratio) mechanically at ambient conditions and 2) Co-evaporation (CE): CT and [beta]CD were mixed (1:1 mol ratio) in 60 ml of water under constant stirring (36 h) in 400 rpm (Quimis Q 261A21, Brazil) until equilibrium. Hence, the samples were dried in a glass desiccator, and after that were removed and stored in amber vials.

The complex obtained that presented the best effectiveness in characterization tests was used in the pharmacological tests.

2.3. Physical and chemical characterization of the complex

2.3.1. Thermal analysis

The thermal analysis was studied using differential scanning calorimetry (DSC) and thermogravimetry/derivative thermogravimetry (TG/DTG). The DSC curves were obtained on DSC-50 cell (Shimadzu) using aluminum sample holder with 2 mg of sample in an atmosphere of nitrogen (50ml.[min.sup.-1]) and heating rate of 10 [degrees]C [min.sup.-1] in the range temperature from 25 to 500 [degrees]C. The DSC cell was checked with indium (m.p. 156.6 [degrees]C, [DELTA]Hfus = 28.54 [Jg.sup.-1]) and zinc (m.p. 419.6 [degrees]C). The TG/DTG curves were obtained in a thermobalance model TGA 50 (Shimadzu) using a temperature range of 25-900 [degrees]C in a platinum sample holder with ~3 mg of sample, under dynamic nitrogen atmosphere (50 [ml.min.sup.-1]) and heating rate of 10 [degrees]C.[min.sup.-1]. The thermogravimetric system was calibrated using a Ca[C.sub.2][O.sub.4]x[H.sub.2]O standard substance in conformity to the American Society for Testing and Materials pattern (ASTM, 1993).

2.3.2. Moisture determination

The moisture contents of the [beta] CD, CT, PM and CE were determined by the Karl Fisher method using a KF 1000 Analyzer (Brazil) and iodine solution, in the presence of pyridine (and sulfur dioxide) as titrating solution. The analyses were carried out in triplicate.

2.3.3. Fourier transform infrared spectroscopy (FTIR)

The infrared absorption data were obtained in the range of 4000-400 [cm.sup.-1] in KBr pellets using an FTIR Bomen spectrophotometer, model MB-120, at room temperature.

2.3.4. Scanning electron microscopy (SEM)

The dried products were mounted and visualized with a JEOL Model JSM-7410-F scanning electron microscope, at an accelerated voltage of 1 kV.

2.4. Pharmacological evaluation

2.4.1. Animals

Forty-two adult (3-month-old) male albino Swiss mice (28-32 g) were randomly housed (n = 7/per group) in appropriate cages at 21 [+ or -] 2 [degrees]C with a 12-h light: dark cycle (light from 06:00 to 18:00), with free access to food (Purina[R], Brazil) and tap water. All experiments were carried out between 09:00 am and 02:00 pm in a quiet room. Experimental protocols were approved by the Animal Care and Use Committee at the Federal University of Sergipe (CEPA/UFS # 45/13). The ethical principles established by the Brazilian Society for Laboratory Animal Science (SBCAL) and by the National Institutes of Health (NIH) were respected. All tests were carried out by the same blinded visual observer and all efforts were made to minimize the number of animals used as well as to minimize any discomfort. Vehicle and experimental drug were performed orally by gavage.

2.4.2. Acid saline-induced chronic muscular pain

Initially, the mice were evaluated regarding the mechanical threshold of paw withdrawal (digital von Frey) for the measurement of its baseline (no pain stimulus). Immediately after that, the animals were anesthetized with a mixture of Ketamine (100 mg/kg) and xylazine (10 mg/kg) and subjected to an injection of 20 [micro]l of sterile acid saline (pH 4.0), into the right gastrocnemius muscle, on the day 0 (1st injection) and again on the day 5 (2nd injection). This procedure produces bilateral mechanical hyperalgesia which radiates from the muscle to the paw for a period up to 4 weeks (Sluka et al., 2001).

2.4.3. Evaluation of the time effect of CT or CT-[beta]CD

Twenty-four hours after the 2nd injection of sterile acidic saline (pH 4.0), 21 animals were treated with CT (50 mg/kg, po), CT-[beta]CD (50 mg/kg, po) or vehicle (isotonic saline, po) and 60 min after, submitted to evaluation in digital von Frey, 1 h each, until there was not significant effect of animals treated with the substances studied when compared to the vehicle. Measurements were performed 5 times to obtain an arithmetic mean of the values. The CT dose was chosen following a previous study of our group (Melo et al., 2010; 2011; Quintans-Junior et al., 2010; De Santana et al., 2013).

2.4.4. Evaluation of the mechanical hyperalgesia

After confirming mechanical hyperalgesia, 21 mice (n = 7/group) were daily treated with CT-[beta]CD (50 mg/kg, po), vehicle (distilled water, po), or Tramadol (TRAM 4 mg/kg, ip), as standard drug, which is a [mu]-opioid receptor agonist with monoamine-reuptake inhibitor properties (MacLean and Schwartz, 2015; Nascimento et al., 2014; Russell et al., 2000). Sixty min after treatment, they were evaluated for mechanical hyperalgesia by digital analgesimeter (digital von Frey) and muscle strength (grip strength meter) for 7 consecutive days. Measurements were performed 5 times, by digital von Frey, or 3 times, by grip strength meter, to obtain an arithmetic mean of the values.

2.4.5. Immunofluorescence for fos

To evaluate the action in the CNS, animals with chronic muscle pain and treated with CT-[beta]CD (50 mg/kg, po) or vehicle (isotonic saline, po) were perfused. Their brains and spinal cords were collected and cryoprotected for immunofluorescence processing to Fos protein (Brito et al., 2013). Frozen serial transverse sections (20 [micro]m) of all brains and spinal cords were collected on gelatinized glass slides. The tissue sections were stored at -80 [degrees]C until use. The sections were washed with phosphate buffer saline (PBS, 10 mM) 5 times for 5 min and incubated with 0.01 M glycine in PBS for 10 min. Non-specific protein binding was blocked by the incubation of the sections for 30 min in a solution containing 2% BSA. Then, the sections were incubated overnight with rabbit anti-Fos as primary antibodies (k-25; 1:2000). Afterwards, the sections were incubated for 2 h with donkey anti-rabbit Alexa Fluor 555 as secondary antibodies (1:2000). The cover slip was mounted with glycerol solution. As an immunofluorescence control for non-specific labeling, sections were incubated without primary antibody. After each stage, slides were washed with PBS 5 times for 5 min.

2.4.6. Acquisition and analyses of images

Pictures from Fos-positive areas were acquired for each animal with an Olympus IX2-ICB (Tokyo, Japan). The brain regions were classified according to Paxinos and Franklin Atlas (Paxinos and Franklin, 2012). Neurons were counted by the free software Image J (National Institute of Health) using a plug-in that uses the same level of label intensity to select and count the Fos-positive cells (Brito et al., 2013).

2.4.7. Molecular docking studies

Docking calculations for the structure of GluR2-SlS2J ligand-binding core receptor (PDB code: 1FTJ), an ionotropic glutamate receptor, were performed. To use AutoDock Vina, to calculate ligand-receptor binding modes, the 1FTJ protein complexed with glutamate (natural agonist ligand) was selected as standard structure. The hydrogen atoms and implicit solvation were added initially for the geometry of protein structure minimization using the chimera package. The randomized conformation and the structure file were converted to the PDBQT format using AutoDockTools 1.5.6.

Then for all ligands gasteiger charges were calculated. Essential hydrogen atoms, Kollman united atom type charges and solvation parameters were added to the receptor using AutoDock tools provided by the server. Grid maps of 20x20x20 [Angstrom] grid points with 0.375 A spacing centered at the known ligand binding site were generated using the Autogrid program (Morris et al., 2009, 1996). Different antagonist drugs for ionotropic glutamate receptor were searched using the Solis and Wets local search method with a Lamarckian genetic algorithm (Solis and Wets, 1981). Initial position, orientation and torsions of the ligand molecules were set randomly.

2.5. Statistical analysis

Results were expressed as mean [+ or -] S.E.M. Differences between two groups were analyzed using Student's t-test. Differences among multiple groups were analyzed using one-way or two-way analyses of variance (ANOVA) followed by Tukey's test or Bonferroni's test, respectively. In all cases, differences were considered significant if p < 0.05. The statistical analyses were assessed using the GraphPad Prism 5.0 software (GraphPad Prism Software Inc., San Diego, CA, USA).

3. Results

Through the differential scanning calorimetry (DSC) curves (Fig. 1(A)), the following endothermic events could be observed: one event of CT in the temperature range of 30-115 [degrees]C, four events in the curve of the [beta]CD in the following temperature ranges: 30-132 [degrees]C, 120-270 [degrees]C, 300-360 [degrees]C and >360 [degrees]C. Four events were observed in the Physical Mixture (PM) like the [beta]CD curve and two events in the Co-evaporation (CE) curve (30-176 [degrees]C and 288-345 [degrees]C).

CT has considerable mass loss to 160 [degrees]C (99.11 [+ or -] 0.12), while the [beta]CD presented so in the 1st stage (13.17% [+ or -] 1.14) and, in most part, in the 3rd stage (78.66% [+ or -] 1.33). In a similar way to the [beta]CD, PM and CE presented higher weight loss in the 3rd stage (66.53% [+ or -] 1.78 and 69.90% [+ or -] 3.33, respectively), indicating complexation by both methods observed through thermogravimetry/derivative thermogravimetry (TG/DTG) (Fig. 1(B) and Table 1).

The moisture determined by Karl Fisher (Table 2) showed the lowest percentage of water for CT (0.22% [+ or -] 0.02) and 13.75% [+ or -] 0.39 for the [beta]CD. The PM and the CE showed similar results to the [beta]CD (11.20% [+ or -] 0.40 and 8.17% [+ or -] 0.33, respectively).

Fig. 1(C) shows the spectra of [beta]CD, citronellal and inclusion complexes, which were obtained with the methods described above, by Fourier transform infrared spectroscopy (FTIR). CT allowed the observation of four bands (2985-2716 [cm.sup.-1], 1720 [cm.sup.-1], 1449 [cm.sup.-1] and 1381 [cm.sup.-1]). The spectrum of pure [beta]CD showed a broad band with a maximum absorption between 3571-3220 [cm.sup.-1] and another clearly visible band at 1642 [cm.sup.-1]. Other bands were also observed for the [beta]CD (2926, 1413, 1366, 1331, 1155, 1085, 1026 and 1000-700 [cm.sup.-1]). In the analysis of the spectra representing the inclusion complexes, it was observed that the spectrum that represents the PM was shown to be similar to the [beta]CD spectrum, demonstrating that the band at 1720 [cm.sup.-1], present in CT, was slightly prominent. The spectrum, which is the displacement of CE, showed bands at 1720 to 1717 [cm.sup.-1] and at 1416 [cm.sup.-1] to 1419 [cm.sup.-1].

The photomicrographs obtained with Scanning Electron Microscopy (SEM) (Fig. 2) in increases of 750 and 1,500x, elucidated the morphological difference of [beta]CD in relation to obtaining methods.

Animals treated with CT-[beta]CD and CT had anti-hyperalgesic effect (p <0.001). CT-[beta]CD showed a significant longer-lasting effect compared to CT alone from the 6th hour after treatment (p <0.001), keeping for more 3 h the anti-hyperalgesic activity (Fig. 3(A)).

In the daily assessment of mechanical hyperalgesia (Fig. 3(B)), mice treated with CT-[beta]CD demonstrated a significant reduction of hyperalgesia until the end of the trial (12th day) compared to the vehicle group (p < 0.05, p < 0.01 or p < 0.001). The TRAM group, similarly, had a significant effect on all days of the experiment (p < 0.05, p < 0.01 or p < 0.001). There was not a statistical difference among CT-[beta]CD group and TRAM group, which shows that the CT-[beta]CD presented a similar anti-hyperalgesic effect to that observed in animals treated with the standard drug (tramadol).

In the evaluation of muscle strength, there was no statistical difference (p > 0.05) on any day of trial in mice treated with CT-[beta]CD (50 mg/kg) or TRAM (4 mg/kg), when compared to control (Fig. 3(C)).

Additionally, in the assessment by immunofluorescence for Fos protein on animal brains, we demonstrated that there was an increase of Fos protein expression, when compared to control, in the ventrolateral area of periaqueductal gray (PAG) (p <0.01) (Fig. 4) and rostroventromedular area (RVM) (p < 0.05) (Fig. 5), by the action of the substance tested. However, in the spinal cord areas assessed (Fig. 6), a significant reduction of the expression of Fos protein was observed in mice treated with CT-[beta]CD (p<0.001) when compared to control group.

The energy of affinity of CT and other ligand compounds to the GluR2-SlS2J was estimated using the Vina docking software. All modeled compounds showed favorable energy binding based in the docking score function. Compounds as felbamate, topiramate and memantine are important glutamatergic modulators that are clinically used in therapeutics as anticonvulsant compound, in pain or neurological disorders treatment (Ahmad-Sabry and Shareghi, 2015; Mishal et al., 2015; Olivan-Blazquez et al., 2014; Spritzer et al., 2016). CT presented better score docking for GluR2-SlS2J than felbamate and memantine, beyond score docking look like the topiramate (Table 2), showing that the interaction of the CT with glutamatergic receptors is equal or better than products available in the clinic.

The superimposed CT is accommodated in the binding pocket and overlaps with the co-crystallized glutamate agonist ligand. The docked glutamate ligand formed with two carbonyl groups mimic the [alpha]-carboxyl group common to agonists, forming hydrogen bonds to Arg96 and the hydroxyl and backbone amide of Thr91; amide nitrogen makes a hydrogen bond to the backbone carbonyl of Pro89 and Glul93 (Fig. 7(A)). The oxygen atom of carbonyl group of CT forms hydrogen bonds to Arg96, Serl42 and Thrl43 (Fig. 7(B)).

4. Discussion

CT is an important monoterpene with appreciable biological properties already described (de Santana et al., 2013; Lenardao et al., 2007; Melo et al., 2010; L. Quintans-Junior et al., 2011). However, its poor solubility in water, short half-life and controversial effectiveness have all limited its use in chronic approaches, such as in models of chronic pain. On the other hand, cyclodextrins (CDs), such as [beta]CD, have been a useful tool to improve several chemical and pharmacological properties of non-polar compounds as terpenes (Brito et al., 2015; Oliveira et al., 2015). Thus, we complexed CT with [beta]CD assessing whether it could improve the analgesic profile of CT, and also assessed its possible interactions with CNS areas related to pain control, aided with the docking study.

First of all, we demonstrated that the CT-[beta]CD complex was already prepared and subjected to the characterization analysis. Hence, four endothermic events shown in the DSC curve indicated oil volatilization, release of water molecules present in the [beta]CD structure, a little pronounced event observed as phase transition and fusion of [beta]CD, followed by decomposition and removal of carbonaceous material present in the sample, respectively (Menezes et al., 2013; Wang et al., 2014). The curve of the physical mixture (PM) was shown to be very similar to the [beta]CD curve. Since the co-evaporation curve (CE) was different from that of the individual components and showed only two stages of endothermic events, it is the first characteristic of the released water and adsorbed oil, once the first event comprises the temperature range of citronellal as well as [beta]CD. The second was attributed to the fusion of [beta]CD, followed by decomposition, suggesting the complexation of citronellal by the method of CE.

Data from the study of TG/DTG confirmed the DSC results, where the temperatures of higher weight loss of CT and [beta]CD coincided with thermal events of volatilization of the oil and dehydration of [beta]CD as described in the moisture content determined by Karl Fischer. Regarding the methods of obtaining the inclusion complexes, it may be inferred that the first stage is attributed to water release and oil surface, since the TG/DTG technique does not distinguish between dehydration and volatilization oil adsorbed on the surface of [beta]CD (Hadaruga et al., 2012). In the second stage, it was noted that the weight loss for citronellal and [beta]CD was significantly lower when compared to MP and CE. Thus, both methods are capable of forming inclusion complexes of CT in [beta]CD, once the complexed active principle required a higher temperature to volatilize.

In FTIR, the corresponding spectrum to citronellal allowed us to observe a band corresponding to the OH stretching vibration mode and another observed in the region attributed to the stretch mode of the H-C = 0, aldehyde characteristic. C[H.sub.2] angular deformation and C[H.sub.3] deformation have also been observed. The spectrum of pure [beta]CD showed a broad band corresponding to stretching vibration of the different OH groups present in the [beta]CD, clearly visible in the band at 1642 [cm.sup.-1]. The spectrum showed other bands, such as CH vibrations and stretching of CH and C[H.sub.2] groups and CH stretching vibrations, and of CO stretching vibrations, of the links between ether and hydroxyl groups. Finally, the vibrations of the CH links and the CC skeleton vibrations in the glucopyranose ring have also been presented, as noted in the literature (Menezes et al., 2013; Sambasevam et al., 2013).

In the analysis of the spectra representing the inclusion complexes, one can observe that the spectrum representing the PM was shown to be similar to the [beta]CD spectrum, demonstrating that the band at 1720 [cm.sup.-1] relating to the aldehyde, which characterizes the citronellal, was little prominent, suggesting its low complexing efficiency. Since the spectra representing the CE showed a displacement of the bands at 1720 to 1717 [cm.sup.-1] and 1416 to 1419 [cm.sup.-1], what indicates the interaction of citronellal with the [beta]CD, showing that this was the best method to form inclusion complex.

In the photomicrographs obtained with SEM, the [beta]CD presented crystal and rhomboid form as previously described in the literature (Hadaruga, 2012). This profile was kept in the PM images, showing the lack of interaction of citronellal with [beta]CD through this method. In the pictures that refer to CE was observed a change in the form, in which there was a greater reduction in particle size and apparent interaction, indicating that this method was more efficient in complexing the citronellal in [beta]CD.

After the characterization of CT-[beta]CD complex, we performed a time-effect assessment to double-check whether the [beta]CD complex formation is able to improve CT-like analgesic profile in animal model of FM. Hence, the time-effect evaluation showed that the complexes containing CT in [beta]CD were able to reduce the mechanical hyperalgesia of mice induced with CMP for 8 h when compared with vehicle, while pure CT reduced the mechanical hyperalgesia for 6 h. On the other hand, when the inclusion complex was compared with the free drug, one can see that the CT-[beta]CD had a better and significant effect between the 6th and 8th hour after the treatment. All these data together demonstrated that CT was efficiently complexed in [beta]CD and that the inclusion complex formed has a higher analgesic effect than does the pure form.

Additionally, we also evaluated the analgesic effect of the chronic treatment with CT-[beta]CD in animals induced with non inflammatory chronic muscle pain, which is suggested to be an animal model of FM, for 7 days through the digital analgesimeter, once, in this model, there is a significant decrease in mechanical withdrawal threshold of the paw, being interpreted as secondary hyperalgesia (Da Silva et al., 2010; Sluka et al., 2001).

Our results demonstrated that the daily treatment with CT-[beta]CD was able to increase the threshold sensitivity of mechanical hyperalgesia with no indication of tolerance or addiction. This effect is probably the result of the CNS activation, since Melo et al. (2010) previously demonstrated that CT had a central analgesic effect through the hot-plate test with a possible involvement of the opioid system. Besides, Quintans-Junior et al. (2010) demonstrated that CT produced significantly antinociceptive effect in the orofacial pain induced by formalin-, capsaicin- or glutamate-tests. Glutamate receptors seem to be a promising target for CT-like analgesic action (Quintans-Junior et al., 2010; 2011). Hence, the hypothesis of the glutamate system involvement was corroborated again by molecular docking study suggested above in this paper.

Given that non-specific muscle relaxation effect can reduce the response of motor coordination and might invalidate the behavioral tests (Le Bars et al., 2001; Passos et al., 2009; Raboisson and Dallel, 2004), we also assessed the muscular force for 7 days, just after the mechanical hyperalgesia evaluation, and the results showed that the chronic treatment with CT-[beta]CD did not produce any significant alteration in force, which validates the action of CT-[beta]CD on the mechanical hyperalgesia observed in this study.

Aiming to confirm the hypothesis that CT-[beta]CD is able to activate the CNS areas which can manage pain, we proceeded with the evaluation of cell activation in the ventrolateral area of periaque-ductal grey (PAG), rostroventromedial area (RVM) and cell inhibition in the laminae I-IV of spinal cord. For this purpose, we used the immunofluorescence for Fos protein protocol, once that in order to study the central pathways of pain, the Fos expression has been used to examine these neural circuitries (Harris, 1998; Snowball et al., 2000). Additionally, the Fos protein has been used as a marker for neuronal activity after noxious stimulation in the rodents (Brito et al., 2013; Bullitt, 1990; Gama et al., 2013). Our results showed that the treatment with CT-[beta]CD activated CNS areas involved in the descending pain pathway, specifically the PAG and RVM, and it inhibited the superficial dorsal horn of the lumbar spinal cord. These effects are compatible with the hypothesis that CT-[beta]CD could act modulating the descending pain inhibitory pathways (M.S. Melo et al., 2011, 2010).

In addition, the PAG, the central area that receives signals from the thalamus, hypothalamus, cortex and the spinothalamic tract, exciting the nuclei of rostroventral medulla (RVM), exerts antinociceptive effect and inhibits the responses of the spinal cord (Perl, 2011; Steeds, 2009; Zubrzycka et al., 2011). The antinociception evoked from the PAG is opioid receptor-mediated and it can be attenuated by the concurrent administration of an opioid antagonist such as naloxone (Akil et al., 1976; Jensen and Yaksh, 1986). Therefore, the CT-[beta]CD is able to activate CNS areas, specifically those related to pain inhibition. The opioid and glutamate systems are probably involved in the anti-hyperalgesic effect observed in the present study, as suggested previously by M.S. Melo et al (2011, 2010).

Therefore, Skyba and Sluka (2002) used NMDA and non-NMDA receptor antagonists and noted that both are involved in the maintenance of hyperalgesia in chronic muscle pain. Since the CT-[beta]CD was able to produce anti-hyperalgesic effect in this model, we decided to investigate the possibility of interaction between the CT and ionotropic glutamate receptor (GluR2-SlS2J).

Trying to better understand these hypotheses, we seek to make a consistent docking study for the possible interaction between CT and glutamate receptor, since the opioid participation in the analgesic profile of CT has already been demonstrated by our group (M.S. Melo et al., 2011, 2010; De Santana et al., 2013). Thus, glutamate, like most glutamate receptor agonists, is an amino acid; this way, the [alpha]-carboxyl and [alpha]-amino groups directly interact with the ligand-binding core through 7 ion pair and hydrogen bonding interactions to domains SI and S2. Overlapping of the glutamate structure shows the fundamental interactions with Arg-485, Glu-705, Thr-480, Ser-654 and Pro-478 (Armstrong and Gouaux, 2000; Armstrong et al., 1998).

The carbonyl group citronellal fragment is located just before the three key residues Arg96, Thrl43 and Serl42, which are involved in ligand interactions in all x-ray structures of GluR2S1S2J and docking molecules, showed that the fundamental interactions between the receptor and both the [alpha]-carboxyl group and the [alpha]-amino group of the agonists are conserved (Armstrong and Gouaux, 2000; Armstrong et al., 1998; Frandsen et al., 2005; Jin, 2005; Kasper et al., 2002; Koller et al., 2011).

In this context, it is possible to propose that the CT-[beta]CD has an anti-hyperalgesic effect on chronic widespread non-inflammatory muscle pain in mice, having a greater effect than the free form (CT alone). This analgesic profile is probably mediated by the activation of the descending inhibitory pathway, especially the PAG and RVM, and the inhibition of the superficial dorsal horn spinal cord, which seems to be associated, at least in part, with interaction of glutamate receptors and CT. These findings have created the prospect of CT pharmacological use, complexed with CDs, for the management of some dysfunctional pain, such as FM or other types of chronic muscle pain.


Article history:

Received 7 December 2015

Revised 4 June 2016

Accepted 9 June 2016

Conflict of interest

Authors declare no conflict of interest.


We thank Mr. Osvaldo A. Santos for the technical support. This work was supported by grants from CNPq, CAPES, FAPITEC/SE, FUNCAP/CE and FINEP, all from Brazil. PL Santos, RG Brito, MA Oliveira, PP Menezes are carrying out the master or PhD degrees in the Graduate Program in Health Sciences (PPGCS/UFS). We also thank teacher Abilio Borghi for the grammar review on the manuscript.


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Abbreviations: 1FTJ, PBD code of GluR2 ligand binding core receptor; Arg, Arginine; BSA, Bovine serum albumin; CDs, Cyclodextrins; CE, Co-evaporation; CMP, Chronic muscle pain; CNS, Central nervous system; CT, Citronellal; CT-[beta]CD, Citronellal complexed in [beta]-cyclodextrin; DSC, Differential and scanning calorimetry; FM, Fibromyalgia; FT1R, Fourier transform infrared spectroscopy; Glu, Glutamic acid; GluR2, ionotropic glutamate receptor; GluR2-SlS2J, GluR2 ligand-binding core; NP, Natural products; PAG, Periaqueductal grey; PBS, Phosphate buffered saline; PDB, Protein data bank; PDBTQ, Protein data bank, partial charge (Q). & atom type (T); PM, Physical mixture; Pro, Proline; RVM, Rostroventromedial area; S.E.M, Standard error of the mean; SI, S2, Protein domain; SEM, Scanning electron microscopy; Ser, Serine; TG/DTG, Thermogravimetry/ derivate thermogravimetry; Thr, Threonine; TRAM, Tramadol; Tyr, Tyrosine; UFS, Universidade Federal de Sergipe (Federal University of Sergipe); [beta]CD, [beta]-cyclodextrin.

Priscila L. Santos (a), Renan G. Brito (a), Marlange A. Oliveira (a), Jullyana S.S. Quintans (a), Adriana G. Guimaraes (a), Marcio R.V. Santos (a), Paula P. Menezes (b), Mairim R. Serafini (b), Irwin R.A. Menezes (c), Henrique D.M. Coutinho (c), Adriano A.S. Araujo (b), *, Lucindo J. Quintans-Junior (a),*

(a) Department of Physiology, Federal University of Sergipe, Sao Cristovao, SE, Brazil.

(b) Department of Pharmacy, Federal University of Sergipe, Sao Cristovao, SE, Brazil

(c) Department of Biological Chemistry, Regional University of Cariri, Crato, CE, Brazil.

* Corresponding authors: Fax: +55-79-3212-6640.

E-mail addresses: (A.A.S. Araujo), lucindojr@gmail. com,, (L.J. Quintans-Junior).

Table 1
Mass loss percentages obtained from thermogravimetry-derivate
thermogravimetry (TG-DTG) curves and respective percentages of water
determined by Karl Fischer of citronellal pure (CT), [beta]-
cyclodextrin ([beta]CD), physical mixture (PM) and co-evaporation

Sample        1st stage/% 32-160    2nd stage/% 160-307
              [degrees]C            [degrees]C

Citronellal   99.11 [+ or -] 0.12   0.27 [+ or -] 0.23
[beta]CD      13.17 [+ or -] 1.14   1.50 [+ or -] 0.10
PM             8.59 [+ or -] 1.65   8.52 [+ or -] 0.25
CE             4.67 [+ or -] 0.02   9.58 [+ or -] 0.19

Sample        3th stage/% 307-500   4th stage/% 500-900
              [degrees]C            [degrees]C

Citronellal    0.82 [+ or -] 1.17    1.19 [+ or -] 0.22
[beta]CD      78.66 [+ or -] 1.33    6.67 [+ or -] 0.12
PM            66.53 [+ or -] 1.78   11.18 [+ or -] 0.65
CE            69.90 [+ or -] 3.33    8.74 [+ or -] 0.11

Sample        % [H.sub.2]0

Citronellal    0.22 [+ or -] 0.02
[beta]CD      13.75 [+ or -] 0.39
PM            11.20 [+ or -] 0.40
CE             8.17 [+ or -] 0.33

Note: All determinations were made in triplicate. [beta]CD: [beta]-
Cyclodextrin; PM: Physical mixture; CE: Co-evaporation.

Table 2
Modeled compounds according to the binding energy based in docking
score function.

Ligand            Docking score   interacting residues with receptor
                  (kcal/mol)      H- bond

CLU (1FTJ)        -6.3            Arg96, Glul93, Pro89, Serl42, Tht91,
Felbamate         -4.5            Arg96, Glul93, Leul38. Pro89, Serl42,
Memantine         -0.3            Glul93, Tyr61
Topiramate        -6.4            Arg96, Serl42, Thr91, Thrl43
(+) Citronellal   -5.6            Arg96, Serl42, Thrl43
(-) Citronellal   -6.1            Arg96, Serl42, Thrl43

Note: Arg: Arginine; Glu: Glutamic acid; Leu: Leucine; Pro: Proline;
Ser: Serine; Thr: Threonine; Tyr: Tyrosine.


Please note: Some tables or figures were omitted from this article.
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Author:Santos, Priscila L.; Brito, Renan G.; Oliveira, Marlange A.; Quintans, Jullyana S.S.; Guimaraes, Adr
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Aug 15, 2016
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