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Anti-hyperalgesic properties of ethanolic crude extract from the peels of Citrus reticulata (Rutaceae)

Abstract

The therapeutic effects from Citrus reticulata on painful inflammatory ailments are associated to its flavonoids constituent and phytochemical studies with Citrus genus affirm that the peels have important amounts of it. These bioactive compounds have been a considerable therapeutic source and evaluate potential application of the peel extract is significant. This research aims to investigate the influence of ethanolic crude extract from the peels of Citrus reticulata and its possible mechanism of action in different animal models of pain. The extract reduced hyperalgesia in the second phase of formalin test (vehicle: 501.5 ± 40.0 s; C. reticulata extract 300 mg/kg: 161.8 ± 41.1 s), in the carrageenan model (vehicle at 4th h: 82.5 ± 9.6 %; C. reticulata extract 300 mg/kg at 4th h: 47.5 ± 6.5 %) and in Complete Freund’s Adjuvant model (vehicle: 501.5 ± 40.0 s; C. reticulata extract 300 mg/kg: 161.8 ± 41.1 s). The possible contribution of opioidergic and adenosinergic systems in the anti-hyperalgesic effect of C. reticulata extract was observed after treatment, with non-selective antagonists for both systems, which produced reversal effects. In conclusion, these properties of C. reticulata extract suggest a potential therapeutic benefit in treating painful conditions.

Key words
Citrus reticulata; medicinal plants; pain; opioids; adenosine

INTRODUCTION

For many populations, relations based on herbal medicines is being groundwork for treating many inflammatory and painful disorders, given that conventional medicines are associated with many adverse effects, toxicity and drug interactions (ChanCHAN HCS, MCCARHTY D, JIANING L, PALCZEWSKI K & YUAN S. 2017. Designing Safer Analgesics via μ-Opioid Receptor Pathways. Trends Pharmacol Sci 11: 1016-1037. et al. 2017). Development of safe alternatives for treating such diseases from compounds derived from plants have been studied for medicinal purposes and some of them have shown anti-inflammatory and analgesic properties, including Achillea millefolium L., Arnica montana L., Curcuma longa L., Salix alba L., Citrullus colocynthis (L.) Schrad, Conium maculatum L. and Cyperus rotundus L. amonh others from Citrus species (Al-SnafiAL-SNAFI AE. 2016. Medicinal plants possessed anti-inflammatory antipyretic and analgesic activities (part 2) – plant based review. Sch Acad J Pharm 5: 142-158. 2016).

These properties are associated with naturally occurring bioactive compounds that have been a considerable therapeutic source. Within these, flavonoids are unquestionably a class of agents with potential applications (BrodowskaBRODOWSKA KM. 2017. Natural flavonoids: classification, potential role, and application of flavonoid analogues. Eur J Biol Res 2: 108-23. 2017). Flavonones are the preponderance flavonoids in Citrus species (Waheed et al. 2009) and around the world, citrus plants like tangerines are widely consumed by the population, mainly as food supplements and nutraceuticals for many physiological, pharmacological and medicinal activities (YeYE X. 2017. Phytochemicals in Citrus: Applications in Functional Foods, Boca Raton: CRC Press, 520 p. 2017). China, Spain and Brazil account for 60.3% of world tangerine production, while Brazil was the second world leading citrus producer in 2014 (19 million tons) after China (33 million tons) (FAOFAO. 2016. Food and Agriculture Organization of the United Nations Production. Available from: <http://www. fao.org/faostat/en/#compare>. Accessed: 21 Feburary, 2018.
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2016).

For Brazilian folk medicine, Citrus aurantium L., Citrus sinensis (L.) Osbeck, Citrus limon (L.) Osbeck, Citrus aurantiifolia (Christm.) Swingle and Citrus reticulata Blanco, have been commonly used in the treatment of different conditions that feature inflammatory components such as bronchitis, stomach problems including gastritis, throat pain, among others (DeDE MEDEIROS PM, LADIO AH & ALBUQUERQUE UP. 2013. Patterns of medicinal plant use by inhabitants of Brazilian urban and rural areas: A macro scale investigation based on available literature. J Ethnopharmacol 150: 729-746. Medeiros et al. 2013).

These therapeutic effects from Citrus reticulata on painful inflammatory ailments are associated to its large spectrum of flavonoids constituent (Zhang et al. 2014, BarrecaBARRECA D, GATTUSO G, BELLOCO E, CALDERARO A, TROMBETTA D, SMERIGLIO A, LAGANÀ G, DAGLIA M, MENGHINI S & NABAVI SM. 2017. Flavonones: Citrus phytochemical with health-promoting properties. Biofactors: 1-12. et al. 2017) and phenolic acids, both with recognized anti-inflammatory and antinociceptive actions, as mentioned by Ambriz-PérezAMBRIZ-PÉREZ DL, LEYVA-LÓPEZ N, GUTIERREZ-GRIJALVE EP & HEREDIA JB. 2016. Phenolic compounds: Natural alternative in inflammation treatment. A Review. Cogent Food Agric 2: 1-14. et al. (2016). Additionally, there are phytochemical studies with Citrus genus that affirm that is in the peels that are found important amounts of flavonoids (NogataNOGATA Y, SAKAMOTO K, SHIRATSUCHI H, ISHII T, YANO M & OHTA H. 2006. Flavonoid composition of fruit tissues of citrus species. Biosci Biotechnol Biochem 70: 178-192. et al. 2006, BermejoBERMEJO A, LLOSÁ MJ & CANO A. 2011. Analysis of bioactive compounds in seven citrus cultivars. Food Sci Technol Int 1: 55-62. et al. 2011). Nevertheless, the consumption of Citrus occurs mainly in the form of juice, and the peels become unusable products. Even though it has been characterized for its valuable organic compounds, it is still a major environmental problem and product waste derived from the industrial processing of Citrus fruits (MahatoMAHATO N, SINHA M, SHARMA K & CHO MH. 2017. Citrus waste derived nutra-/pharmaceuticals for health benefits: current trends and future perspectives. J Funct Foods 40: 307-316. et al. 2017).

The constituents of the peels of Citrus reticulata emerge as a possible alternative to conventional medicines because of their effectiveness and low cytotoxicity (LimLIM TK. 2012. Edible Medicinal and Non-Medicinal Plants, Netherlands: Springer Netherlands, p. 695-715. 2012) thus it seems especially significant to evaluate new possibilities for clinical use of the peel extract as phytomedicine. Moreover, some properties related to the compounds present in it have already been studied and they suggest important anti-inflammatory and anti-oxidant role (AmorimAMORIM JL, SIMAS DL, PINHEIRO MM, MORENO DS, ALVIANO CS, DA SILVA AJ & FERNANDES PD. 2016. Anti-inflammatory properties and chemical characterization of the essential oils of four Citrus species. PLoS One 4: 153643. et al. 2016, ZhangZHANG Y, SUN Y, XI W, SHEN Y, QIAO L, ZHONG L, YE X & ZHOU Z. 2014. Phenolic compositions and antioxidant capacities of Chinese wild mandarin (Citrus reticulata Blanco) fruits. Food Chemistry 145: 674-680. et al. 2018). Despite this, no register was found about the potential antinociceptive and/or anti-hyperalgesic activities obtained with substances derived from the peels of Citrus reticulata fruits.

The present study is justified because this plant was selected for being widely cultivated in the southern region of Santa Catarina, as well as knowing that the peels are currently a by-product of the plant and that Citrus reticulata specie reveal higher content of flavonoid in their peels when compared to other Citrus fruits (Nogata et al. 2006, HuangHUANG Y & HO S. 2010. Polymethoxy flavones are responsible for the anti-inflammatory activity of citrus fruit peel. Food Chem 119: 868-873. & Ho 2010). Taking the above discussed into account, the aim of this study was to investigate antinociceptive and anti-hyperalgesic properties of ethanolic crude extract from peels of Citrus reticulata (ECE-CR) in different animal models of neurogenic and inflammatory pain (Santos et al. 1998) and preliminarily explore possible contribution of adenosinergic and opioidergic systems for this effect.

MATERIALS AND METHODS

Animals

In this preclinical study, experiments were conducted using male Swiss mice (n= 8/group) weighting 25-30 g. They were maintained under standardized conditions of 12 h light/12 h dark and room temperature at 22 ± 2 oC, with access to food and water ad libitum. After acclimatization of the animals to the Laboratory of Experimental Neuroscience (Lanex), at UNISUL/Brazil-SC, the animals where homogenously distributed between different treatment groups and experiments were conducted from 8:00 a.m. to 5:00 p.m. Animal care and the experimental protocols were approved by the Ethics Committee for Animal Use (CEUA)-UNISUL, under protocol number 13.039.4.03.IV. The number of animals and intensity of noxious stimuli used were the minimum necessary to demonstrate the consistent effects of treatments.

Preparation of the ethanolic crude extract of Citrus reticulata (ECE-CR)

The plant was collected at a cultivar in Araranguá, state of Santa Catarina, Brazil (latitude 28º56’05” south and longitude 49º29’09” west). Professor Jasper Zanco, faculty of the Agronomy course at University Southern Santa Catarina (UNISUL), identified the plant as belonging to the Rutaceae family, by comparing it directly with a voucher specimen (SRS 00050047) deposited at the Laelia Purpurata (SRS), herbarium of UNISUL.

Peels of the plant were placed to dry in an oven at 40°C for 48 h and the dried material was milled and standardized in particle sizes ranging from 250 µm to 850 µm.

The extract was produced at a ratio of 1:8 of peels/solvent to ethanol under dynamic maceration, the fluid remaining in contact with the peels for 10 days, under constant stirring in a closed vessel at room temperature. At the end, the ECE-CR was concentrated in rotavapor under reduced pressure to evaporate the solvent, in a water bath at a temperature of 35°C to 40°C until the total evaporation of the ethanol.

Evaluation of anti-nociceptive and anti-hyperalgesic activities of ECE-CR

Formalin test

Swiss mice were orally treated with sterile saline (0.1 ml/10 g weight) or different doses (30, 100 and 300 mg/kg) of ECE-CR, based on previous works performed in our laboratory in which this range of doses were antinociceptive for hydroalcoolic crudeDE MATTOS ES, FREDERICO MJ, COLLE TD, DE PIERI DV, PETERS RR & PIOVEZAN AP. 2007. Evaluation of antinociceptive activity of Casearia sylvestris and possible mechanism of action. J Ethnopharmacol 112: 1-6. extraxt of other plant (De Mattos et al. 2007, PiovezanPIOVEZAN AP ET AL. 2017. Hydroalcoholic crude extract of Casearia sylvestris Sw. reduces chronic post-ischemic pain by activation of pro-resolving pathways. J Ethnopharmacol 204: 179-188. et al. 2017). One hour after this treatment, the animals received an intraplantar (i.pl.) injection of 20 μL of 2.5% formalin. Nociceptive responses were determined by the time the animal spent licking the treated paw during the periods between 0-5 min (acute phase; phase I) and 15-30 min (inflammatory phase; phase II) after the injection of this agent (MartinsMARTINS DF, MAZZARDO-MARTINS L, SOLDI F, STRAMOSK J, PIOVEZAN AP & SANTOS AR. 2013. High-intensity swimming exercise reduces neuropathic pain in an model of complex regional pain syndrome type I: evidence for a role of the adenosinergic system. Neuroscience 234: 69-76. et al. 2013, López-CanoLÓPEZ-CANO M, FERNANDÉZ-DUEÑAS V, LLEBARIA A & CIRUELA F. 2017. Formalin murine model of pain. Bio-protocol 23: 1-8. et al. 2017).

Glutamate test

Acute nociception was induced by glutamate in mice that were initially orally treated with vehicle (saline, 0.1 ml/10 g weight) or different doses (30, 100 and 300 mg/kg) of ECE-CR. One hour after this treatment, the animals received an i.pl. injection of 20 µL of glutamate (10 µmol/paw) dissolved in a vehicle. The injection of glutamate in the paw of the animal induces direct stimulation of nociceptive neurons, as well as the release of various inflammatory mediators and neuropeptides involved in nociception, promoting immediately nociceptive activity, which was determined by the behavior of biting or licking the treated paw (BeirithBEIRITH A, SANTOS AR & CALIXTO JB. 2002. Mechanisms underlying the nociception and paw edema caused by injection of glutamate into the mouse paw. Brain Research 924: 219-228. et al. 2002, MartinsMARTINS DF, ROSA AO, GADOTTI VM, MAZZARDO-MARTINS L, NASCIMENTO FP, EGEA J, LÓPEZ MG & SANTOS ARS. 2011. The antinociceptive effects of AR-A014418, a selective inhibitor of glycogen synthase kinase-3 beta, in mice. J Pain 12: 315-322. et al. 2011). This index was recorded within 15 min after the administration of the compound (Sousa et al. 2017).

Carrageenan-induced mechanical hyperalgesia

The protocol was developed according to previously use by AlbanoALBANO MN, SILVEIRA MR, DANIELSKI LG, FLORENTINO D, PETRONILHO F & PIOVEZAN AP. 2013. Anti-inflammatory and antioxidant properties of hydroalcoholic crude extract from Casearia sylvestris Sw. (Salicaceae). J Ethnopharmacol 147: 612-617. et al. (2013), for the induction of mechanical hyperalgesia by i.pl. administration of carrageenan (300 µg/paw) in the right hind paw of mice. The mechanical hyperalgesia induced by sensibilization of paw was evaluated using von Frey monofilaments (VFH, Stoelting, Chicago, USA). The percentage values for paw withdrawal frequency to 10 applications of von Frey filament of 0.6 g for each animal was used as an indicator of response. This pressure was selected to evaluate mechanical hyperalgesia from previous study (Martins et al. 2011). The test was performed using a platform measuring 70 x 40 cm, which consists of a wire screen with a mesh size of 6 mm to facilitate the application of the filament on the ventral surface of the hind paw. Mice were placed individually in an observation acrylic bottomless chamber (9 x 7 x 11 cm) and covered with a lid. The criteria for the application of mechanical stimulus were: a) application made perpendicularly to the plantar surface, to provide enough pressure to bend the filament, thereby obtaining total pressure; b) animals were evaluated when all four paws were accommodated on the screen; c) the withdrawal response was considered positive when the animal completely removed the paw from the surface of the support screen.

Anti-hyperalgesic effect for ECE-CR was investigated in different groups of animals treated orally with vehicle (sterile saline, 0.1 ml/10 g weight) or different doses of ECE-CR (30, 100 and 300 mg/kg) at intervals of 1 h before (prophylactic treatment) or 1 h after the administration of carrageenan (therapeutic treatment). Basal values were registered before carrageenan injection.

CFA-induced hyperalgesia and possible mechanism of action

Since CFA model is adequate for study of artrithis, a higly prevalent painfull disease in the world in which the pathophysiology relays on inflammatory mechanisms. Confirmation of the effects of ECE-CR and preliminary exploration of its mechanism of action were evaluated with different groups of animals injected i.pl. with 20 µL of the vehicle (sterile saline, 0.1 ml/10 g weight, negative control) or a 30% solution of CFA for the induction of inflammatory pain, as used by Nascimento et al. (2010). To confirm the anti-hyperalgesic effect of ECE-CR, different groups of mice were orally treated with ECE-CR (30, 100 and 300 mg/kg), 24 h after the administration of CFA. The development of mechanical hyperalgesia was assessed as described in the previous section. For the dose with greater activity in these first 24 h (30 mg/kg), the anti-hyperalgesic activity of the plant was also investigated up to 5 days by its long-term effect (always 1h after daily treatment, once a day).

To explore the possible contribution of adenosinergic and opioidergic systems upon the anti-hyperalgesic effect of ECE-CR in a CFA-induced model, in the 3rd day after induction, different groups of animals were treated with vehicle (sterile saline, 0.1 ml/10 g weight, via i.pl.) or CFA (same conditions); prior to this (15 min before), these two groups were subdivided into two groups treated as follows: a) vehicle group: naloxone (non-selective opioidergic receptor’s antagonist, 1 mg/kg, subcutaneously [s.c.]) or caffeine (non-selective adenosinergic receptor’s antagonist, 10 mg/kg, intraperitoneally [i.p.]); b) CFA group: naloxone (non-selective opioidergic receptor’s antagonist, 1 mg/kg, s.c.) or caffeine (non-selective adenosinergic receptor’s antagonist, 10 mg/kg, i.p.). One hour after i.pl. treatment, mice were evaluated for mechanical hyperalgesia, as previously described.

Rotarod for evaluation of the influence of ECE-CR on motor coordination

To evaluate the possible occurrence of non-specific effects of ECE-CR on locomotor performance, the mice were submitted to the rotarod test (De Mattos et al. 2007). For that purpose, only animals which remained successfully on the revolving bar of the apparatus for two consecutive periods of 60 s were selected to receive orally either ECE-CR (30–300 mg/kg) or vehicle (2% Tween 80) on the following day. At 30, 60, 90 and 120 min after treatment, the animals were placed on the apparatus for up to 90 s at a time, and the amount of time that each animal remained on the revolving bar during each trial was recorded (in s).

Chemicals and drugs

Morphine hydrochloride was purchased from Merck A.G. (Darmstadt, Germany). Formalin was prepared by dilution of formaldehyde (LAFAN Química Fina, SP, Brasil) in saline. Glutamate, carrageenan, CFA, naloxone hydrochloride and caffeine were purchased from Sigma Chemical Co. (Porto Alegre, Brasil). Ethanol was acquired from Vetec Quimica Fina (Duque de Caxias, RJ, Brasil).

Statistical analysis

Results are expressed as media ± standard error of the mean (S.E.M) standard deviation and p< 0.05 value was considered statistically significant. The comparison between groups was assessed by using one-way analysis of variance (ANOVA) followed by Tukey’s test or by two-way ANOVA followed by Bonferroni’s test when appropriated. The GraphPad InStat® software was used for data analysis.

RESULTS

As it can be seen in Figure 1, the ECE-CR effect was more pronounced in pain of inflammatory origin, according to the results presented in the second phase of formalin-induced nociception and mechanical hyperalgesia on the carrageenan and CFA models. Moreover, a change in nociceptive response of neurogenic origin was not observed, neither in the 1st phase of the formalin test nor in the glutamate test.

Figure 1
Influence of ECE-CR on neurogenic (First phase) or inflammatory (Second Phase) nociception in the formalin test in mice. Animals were evaluated 1 h after treatment with the extract. Data are presented as mean ± S.E.M. for 8 animals. (*)p ≤ 0.05; One-way ANOVA followed by Tukey’s test.

Concerning to the anti-hyperalgesic effect of ECE-CR in the carrageenan model, different results were obtained depending on the type of treatment, whether it was prophylactic or therapeutic. Whereas the prophylactic treatment failed to alter this response in any of the evaluated doses, the therapeutic treatment of the animals at doses ranging from 100 to 300 mg/kg reduced hyperalgesic response, even in the 1st hour after treatment (data not shown).

In the evaluation of anti-hyperalgesic activity of ECE-CR significant change was observed in mechanical hyperalgesia in the hind paw of mice, 24 h after i.pl. injection of CFA (Figure 2). The evaluation on the time course of action of this extract demonstrated greater effectiveness in the first hour and lasted up to 3 h after its administration (Fig. 1a). Moreover, the effect on mechanical hyperalgesia was maintained until the 5th day after the induction of CFA model when the animals received daily treatment (Fig. 1b), even though no cumulative effect of the extract was registered.

Figure 2
Influence of treatment with ECE-CR on mechanical allodynia in the CFA-induced arthritis model in mice. a) Treatment with ECE-CR (30, 100 and 300 mg/kg) was performed 24 h after induction of the inflammation by the i.pl. administration of CFA (30% solution, 20 µl). b Treatment with HECR (30 mg/kg) was performed on days 1, 3 and 5 after induction of the inflammation by the i.pl. administration of the agent. Data are presented as mean ± S.E.M. for 8 animals. (*)p ≤ 0.05; Two-way ANOVA followed by Bonferroni test.

Regarding the involvement of the opioidergic and adenosinergic systems on the effects of ECE-CR, on the 5th day after the i.pl. injection of CFA in the paw of mice, pre-treatment (1 h earlier) with naloxone or caffeine reversed the anti-hyperalgesic effect caused by ECE-CR (vehicle: 87.5 ± 3.7%; ECE-CR 30 mg/kg orally: 27.5 ± 5.2%), 55.0% ± 6.3 and 65.0 ± 3.3%, respectively (Figure 3).

Figure 3
Contribution of opioidergic and adenosinergic systems on the effects of ECE-CR on mechanical allodynia in the CFA-induced arthritis model in mice. On the 5th day, after i.pl. treatment of the animals with vehicle (white bars) or CFA (30% solution, 20 µl grey bars), the mice received naloxone (1 mg/kg, s.c.) or caffeine (10 mg/kg, i.p.) and 15 min later they received ECE-CR (30 mg/kg; orally). One hour after this latest treatment, the animals were evaluated for mechanical allodynia. Data are presented as mean ± S.E.M. for 8 animals. (*)p ≤ 0.05; One-way ANOVA followed by Tukey’s test.

The results of the rotarod, for evaluation of the influence of ECE-CR on motor coordination, presented in Table I showed that the treatment of the animals with the extract (30 mg/kg, orally), selected as the most effective dose in this study, did not alter their resistance time on rotarod (in s), used as an index of evaluation for motor coordination of the animals at 22 r.p.m. (De Mattos et al. 2007) in any of the time frames chosen for observation in this study.

Table I
Influence of ECE-CR on locomotor activity of mice.

DISCUSSION

The present study evidenced anti-hyperalgesic properties of the ethanolic crude extract based on the fruit peels of Citrus reticulata in different animal models of nociception and hyperalgesia and provides some evidence on the mechanisms implicated in these effects.

Part of the results obtained in this study highlights that the antinociceptive activity of ECE-CR seem to be more efficient for inflammatory pain, because it has not changed the nociceptive response in animal models of neurogenic pain like in the glutamate test and the 1st phase of the formalin test. Therefore, the anti-hyperalgesic activity from the extract might occur without influence of central nervous system and possibly inhibits peripheral mechanisms of inflammatory mediators. In addition, this result could explain the primordial popular use of Citrus species as anti-inflammatory analgesic and antipyretic medication (AriasARIAS BA & RAMÓN-LACA L. 2005. Pharmacological properties of citrus and their ancient and medieval uses in the Mediterranean region. J Ethnopharmacol 97: 89-95. & Ramón-Laca 2005).

The activity of ECE-CR in models of inflammatory nociception as the 2nd phase of the formalin test and the carrageenan model reinforce the findings observed in the reduction of pain induced by CFA model, which comprises pivotal inflammatory components. This fact suggests that the phytochemicals present in ECE-CR may have important activity on relief of pain symptoms by peripheral mediated analgesic activity, possibly interfering with factors that cause peripheral and/or spinal cord sensitization observed in these models, such as histamine, serotonin, bradykinin, substance P, CGRP or PGE2 (Granados-SotoGRANADOS-SOTO V, ALONSO-LÓPEZ R, ASOMOZA-ESPINOSA R, RUFINO MO, GOMES-LOPES LD & FERREIRA SH. 2001. Participation of COX, IL-1 beta and TNF alpha in formalin-induced inflammatory pain. Proc West Pharmacol Soc 44: 15-17. et al. 2001, Morris 2003).

Another possible mechanism of action for ECE-CR on mechanical hyperalgesia observed in the CFA model, is that it’s possibly affecting T cell-derived mediators, such as cytokines, as these are also activated by mycobacteria, that leads to an increased immune response (HuangHUANG S, LIU HF, QUAN X, JIN Y, XUAN G, AN R, DIKYE T & LI B. 2016. Inflammatory responses in LPS-induced murine macrophages and complete Freund’s adjuvant-induced arthritis rats. Am J Chin Med 7: 1-14. et al. 2016). According to this, hesperidin, another Citrus flavanone compound has been the subject of various studies in the CFA-induced model, it’s efficacy was specified by Sakr (2017), who characterized reduction in the infiltration of inflammatory cells by inhibition of pro-inflammatory cytokines production (IL-1, IL-6 and TNF-α) secreted by macrophages, suppression of T lymphocyte proliferation, as well as increased anti-inflammatory cytokines (IL-4 and IL-10).

Besides this, the possible activity of ECE-CR on neural nociceptive pathways cannot be completely ruled out, since the present analysis of the mechanism of action has demonstrated that its anti-hyperalgesic activity is affected by antagonists of opioid and adenosinergic systems, which exert modulatory role on the transmission of painful information by nociceptive ascendant or descendant pathways. The contribution of a peripheral activity of the opioid system in the reversal process of mechanical hyperalgesia in the CFA model should be considered, since the inflammatory process that occurs after CFA injection in the mouse hind paw triggers the recruitment of monocytes and macrophages, which are responsible for an endogenous analgesic activity in other models (Sauer et al. 2014).

Regarding the participation of the adenosinergic system in the activity of ECE-CR, it is known that opioids induce the release of adenosine in the spinal cord and that this mediator can act as a neuromodulator in promoting anti-hyperalgesic and anti-inflammatory effects, possibly mediated by the activation of the descending pain control pathway. The adenosinergic system can also be effective in the control of peripheral inflammation, by helping reduce the harm caused by uncontrolled immune response (SawynokSAWYNOK J & LIU XJ. 2003. Adenosine in the spinal cord and periphery: release and regulation of pain. Prog Neurobiol 69: 313-340. & Liu 2003, Varani et al. 2017, JacobsonJACOBSON KA ET AL. 2017. A3 adenosine receptors as modulators of inflammation: from medicinal chemistry to therapy. Med Res Rev 2017: 1-42. et al. 2017).

The rotarod test showed that the selected dose (30 mg/kg) for most of the experiments due to its high efficacy, did not affect the duration of time in which the animals stayed on the revolving bar, at the different time frames chosen as the representative of acute (1 or 2 h) or prolonged pre-treatment (24 h). Because of this, it seems not to be the case that a possible unspecific effect of ECE-CR on motor coordination might be responsible for the inhibition of painful behaviors registered in this study.

CONCLUSIONS

The results of the present study have demonstrated that systemic treatment with ECE-CR of Citrus Reticulata peel presents anti-hyperalgesic effects on inflammatory pain models in mice. The investigation of the participation of the adenosinergic and opioidergic systems, as possible mechanisms of action, is a preliminary achievement requiring more studies to identify new cellular targets such as transient receptor potential (TRP) channels, acid sensing ion channels (ASICs) and adenosinergic receptor subtypes (A1/A2), as well as their influence on intracellular mechanisms of nociceptive/hyperalgesic stimulus signaling such as protein kinase C, adenylyl cyclase and protein kinase C (BaggioBAGGIO CH, FREITAS CS, MARCON R, WERNER MF, RAE GA, SMIDERLE FR, SASSAKI GL, IACOMINI M, MARQUES MC & SANTOS AR. 2012. Antinociception of β-D-glucan from Pleurotus pulmonarius is possibly related to protein kinase C inhibition. Int J Biol Macromol 50: 872-877. et al. 2012, Nascimento et al. 2010, MarconMARCON R, LUIZ AP, WERNER MF, FREITAS CS, BAGGIO CH, NASCIMENTO FP, SOLDI C, PIZZOLATTI MG & SANTOS AR. 2009. Evidence of TRPV1 receptor and PKC signaling pathway in the antinociceptive effect of amyrin octanoate. Brain Res 1295: 76-88. et al. 2009, review in: White et al. 2010,). These findings can also stimulate further investigation on the use of ECE-CR as a food supplement to people who suffer from different inflammatory disorders with painful symptoms.

ACKNOWLEGMENTS

This study was supported by Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC-CNPq). Adriele C. Schneider received a scholarship from CAPES, Brazil. Maria E.M. Lisbôa received a scholarship from PUIC-UNISUL.

REFERENCES

  • ALBANO MN, SILVEIRA MR, DANIELSKI LG, FLORENTINO D, PETRONILHO F & PIOVEZAN AP. 2013. Anti-inflammatory and antioxidant properties of hydroalcoholic crude extract from Casearia sylvestris Sw. (Salicaceae). J Ethnopharmacol 147: 612-617.
  • AL-SNAFI AE. 2016. Medicinal plants possessed anti-inflammatory antipyretic and analgesic activities (part 2) – plant based review. Sch Acad J Pharm 5: 142-158.
  • AMBRIZ-PÉREZ DL, LEYVA-LÓPEZ N, GUTIERREZ-GRIJALVE EP & HEREDIA JB. 2016. Phenolic compounds: Natural alternative in inflammation treatment. A Review. Cogent Food Agric 2: 1-14.
  • AMORIM JL, SIMAS DL, PINHEIRO MM, MORENO DS, ALVIANO CS, DA SILVA AJ & FERNANDES PD. 2016. Anti-inflammatory properties and chemical characterization of the essential oils of four Citrus species. PLoS One 4: 153643.
  • ARIAS BA & RAMÓN-LACA L. 2005. Pharmacological properties of citrus and their ancient and medieval uses in the Mediterranean region. J Ethnopharmacol 97: 89-95.
  • AZUMA T, SHIGESHIRO M, KODAMA M, TANABE S & SUZUKI T. 2013. Supplemental naringenin prevents intestinal barrier defects and inflammation in colitic mice. J Nutr 143: 827-834.
  • BAGGIO CH, FREITAS CS, MARCON R, WERNER MF, RAE GA, SMIDERLE FR, SASSAKI GL, IACOMINI M, MARQUES MC & SANTOS AR. 2012. Antinociception of β-D-glucan from Pleurotus pulmonarius is possibly related to protein kinase C inhibition. Int J Biol Macromol 50: 872-877.
  • BARRECA D, GATTUSO G, BELLOCO E, CALDERARO A, TROMBETTA D, SMERIGLIO A, LAGANÀ G, DAGLIA M, MENGHINI S & NABAVI SM. 2017. Flavonones: Citrus phytochemical with health-promoting properties. Biofactors: 1-12.
  • BEIRITH A, SANTOS AR & CALIXTO JB. 2002. Mechanisms underlying the nociception and paw edema caused by injection of glutamate into the mouse paw. Brain Research 924: 219-228.
  • BERMEJO A, LLOSÁ MJ & CANO A. 2011. Analysis of bioactive compounds in seven citrus cultivars. Food Sci Technol Int 1: 55-62.
  • BRODOWSKA KM. 2017. Natural flavonoids: classification, potential role, and application of flavonoid analogues. Eur J Biol Res 2: 108-23.
  • CHAN HCS, MCCARHTY D, JIANING L, PALCZEWSKI K & YUAN S. 2017. Designing Safer Analgesics via μ-Opioid Receptor Pathways. Trends Pharmacol Sci 11: 1016-1037.
  • DE MATTOS ES, FREDERICO MJ, COLLE TD, DE PIERI DV, PETERS RR & PIOVEZAN AP. 2007. Evaluation of antinociceptive activity of Casearia sylvestris and possible mechanism of action. J Ethnopharmacol 112: 1-6.
  • DE MEDEIROS PM, LADIO AH & ALBUQUERQUE UP. 2013. Patterns of medicinal plant use by inhabitants of Brazilian urban and rural areas: A macro scale investigation based on available literature. J Ethnopharmacol 150: 729-746.
  • FAO. 2016. Food and Agriculture Organization of the United Nations Production. Available from: <http://www. fao.org/faostat/en/#compare>. Accessed: 21 Feburary, 2018.
    » http://www.
  • GRANADOS-SOTO V, ALONSO-LÓPEZ R, ASOMOZA-ESPINOSA R, RUFINO MO, GOMES-LOPES LD & FERREIRA SH. 2001. Participation of COX, IL-1 beta and TNF alpha in formalin-induced inflammatory pain. Proc West Pharmacol Soc 44: 15-17.
  • HUANG Y & HO S. 2010. Polymethoxy flavones are responsible for the anti-inflammatory activity of citrus fruit peel. Food Chem 119: 868-873.
  • HUANG S, LIU HF, QUAN X, JIN Y, XUAN G, AN R, DIKYE T & LI B. 2016. Inflammatory responses in LPS-induced murine macrophages and complete Freund’s adjuvant-induced arthritis rats. Am J Chin Med 7: 1-14.
  • JACOBSON KA ET AL. 2017. A3 adenosine receptors as modulators of inflammation: from medicinal chemistry to therapy. Med Res Rev 2017: 1-42.
  • LIM TK. 2012. Edible Medicinal and Non-Medicinal Plants, Netherlands: Springer Netherlands, p. 695-715.
  • LÓPEZ-CANO M, FERNANDÉZ-DUEÑAS V, LLEBARIA A & CIRUELA F. 2017. Formalin murine model of pain. Bio-protocol 23: 1-8.
  • MARCON R, LUIZ AP, WERNER MF, FREITAS CS, BAGGIO CH, NASCIMENTO FP, SOLDI C, PIZZOLATTI MG & SANTOS AR. 2009. Evidence of TRPV1 receptor and PKC signaling pathway in the antinociceptive effect of amyrin octanoate. Brain Res 1295: 76-88.
  • MARTINS DF, ROSA AO, GADOTTI VM, MAZZARDO-MARTINS L, NASCIMENTO FP, EGEA J, LÓPEZ MG & SANTOS ARS. 2011. The antinociceptive effects of AR-A014418, a selective inhibitor of glycogen synthase kinase-3 beta, in mice. J Pain 12: 315-322.
  • MAHATO N, SINHA M, SHARMA K & CHO MH. 2017. Citrus waste derived nutra-/pharmaceuticals for health benefits: current trends and future perspectives. J Funct Foods 40: 307-316.
  • MARTINS DF, MAZZARDO-MARTINS L, SOLDI F, STRAMOSK J, PIOVEZAN AP & SANTOS AR. 2013. High-intensity swimming exercise reduces neuropathic pain in an model of complex regional pain syndrome type I: evidence for a role of the adenosinergic system. Neuroscience 234: 69-76.
  • MORRIS CJ 2003. Carrageenan-induced paw edema in the rat and mouse. Methods in Molecular Biology 225: 115-121.
  • NASCIMENTO FP, FIGUEREDO SM, MARCON R, MARTINS DF, MACEDO SJ JR, LIMA DA, ALMEIDA RC, OSTROSKI RM, RODRIGUES AL & SANTOS AR. 2010. Inosine reduces pain-related behavior in mice: involvement of adenosine A1 and A2A receptor subtypes and protein kinase C pathways. J Pharmacol Exp Ther 334: 590-598.
  • NOGATA Y, SAKAMOTO K, SHIRATSUCHI H, ISHII T, YANO M & OHTA H. 2006. Flavonoid composition of fruit tissues of citrus species. Biosci Biotechnol Biochem 70: 178-192.
  • PIOVEZAN AP ET AL. 2017. Hydroalcoholic crude extract of Casearia sylvestris Sw. reduces chronic post-ischemic pain by activation of pro-resolving pathways. J Ethnopharmacol 204: 179-188.
  • SAKR HI, KHOWAILED AA, BASTAWY NA, GABER SS, AHMED OM & GABER AS. 2017. Hesperidin and experimentally-induced arthritis in male rats. J Med Clin Res 10: 29567-29585.
  • SANTOS AR, VEDANA EM & DE FREITAS GA. 1998. Antinociceptive effect of meloxicam, in neurogenic and inflammatory nociceptive models in mice. Inflamm Res 47: 302-307.
  • SAUER RS, HACKEL D, MORSCHEL L, SAHLBACH H, WANG Y, MOUSA SA, ROEWER N, BRACK A & RITTNER HL. 2014. Toll like receptor (TLR)-4 as a regulator of peripheral endogenous opioid-mediated analgesia in inflammation. Molecular Pain 10: 1-10.
  • SAWYNOK J & LIU XJ. 2003. Adenosine in the spinal cord and periphery: release and regulation of pain. Prog Neurobiol 69: 313-340.
  • SOUSA FSS, ANVERSA RG, BIRMANN PT, SOUZA MN, BALAGUEZ R, ALVES D, LUCHESE C, WILHELM EA & SAVEGNAGO L. 2017. Contribution of dopaminergic and noradrenergic systems in the antinociceptive effect of alfa-phenylalanyl acetophenone. Pharmacol Rep 69: 871-877.
  • WAHEED A, MAHMUD S, SALEEM M & AHMAD T. 2009. Fatty acid composition of neutral lipid: Classes of Citrus seed oil. J Saudi Chem Soc 13: 269-272.
  • WHITE JP, CIBELLI M, REI FIDALGO A, PAULE CC, NOORMOHAMED F, URBAN L, MAZE M & NAGY I. 2010. Role of transient receptor potential and acid-sensing ion channels in peripheral inflammatory pain. Anesthesiol 112: 729-741.
  • YE X. 2017. Phytochemicals in Citrus: Applications in Functional Foods, Boca Raton: CRC Press, 520 p.
  • VARANI K, VINCENZI F, RAVANI A, PASQUINI S, MERIGHI S, GESSI S, SETTI S, CADOSSI M, BOREA PA & CADOSSI R. 2017. Adenosine receptors as biological pathway for the anti-inflammatory and beneficial effect of low frequency low energy pulsed electromagnetic fields. Mediators Inflamm 1-11.
  • ZHANG Y, SUN Y, XI W, SHEN Y, QIAO L, ZHONG L, YE X & ZHOU Z. 2014. Phenolic compositions and antioxidant capacities of Chinese wild mandarin (Citrus reticulata Blanco) fruits. Food Chemistry 145: 674-680.
  • ZHANG H, YAN Y & ZHOU Z. 2018. Phenolic and flavonoid contents of mandarin (Citrus reticulata Blanco) fruit tissues and their antioxidant capacity as evaluated by DPPH and ABTS methods. Journal Integr Agric 1: 256-263.

Publication Dates

  • Publication in this collection
    11 May 2020
  • Date of issue
    2020

History

  • Received
    21 Sept 2018
  • Accepted
    3 May 2019
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