molecules Article
Antinociceptive Effect of the Essential Oil from Croton conduplicatus Kunth (Euphorbiaceae) Raimundo Gonçalves de Oliveira Júnior 1 , Christiane Adrielly Alves Ferraz 1 , Juliane Cabral Silva 1 , Ana Paula de Oliveira 1 , Tâmara Coimbra Diniz 1 , Mariana Gama e Silva 1 , Lucindo José Quintans Júnior 2 , Ana Valéria Vieira de Souza 3 , Uiliane Soares dos Santos 3 , Izabel Cristina Casanova Turatti 4 , Norberto Peporine Lopes 4 , Vitor Prates Lorenzo 5 and Jackson Roberto Guedes da Silva Almeida 1, * 1
2 3 4 5
*
Center for Studies and Research of Medicinal Plants, Federal University of San Francisco Valley, 56306-385 Petrolina-PE, Brazil;
[email protected] (R.G.d.O.J.);
[email protected] (C.A.A.F.);
[email protected] (J.C.S.);
[email protected] (A.P.d.O.);
[email protected] (T.C.D.);
[email protected] (M.G.e.S.) Departament of Physiology, Federal University of Sergipe, 49100-000 Aracaju-SE, Brazil;
[email protected] Embrapa-Semiárido, 56302-970 Petrolina-PE, Brazil;
[email protected] (A.V.V.d.S.);
[email protected] (U.S.d.S.) Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 14040-903 Ribeirão Preto-SP, Brazil;
[email protected] (I.C.C.T.);
[email protected] (N.P.L.) Federal Institute of Education, Science and Technology Sertão Pernambucano, 56300-000 Petrolina-PE, Brazil;
[email protected] Correspondence:
[email protected]; Tel.: +55-87-2101-6862
Academic Editor: Olga Tzakou Received: 18 April 2017; Accepted: 26 May 2017; Published: 30 May 2017
Abstract: Medicinal plants have been widely used in the treatment of chronic pain. In this study, we describe the antinociceptive effect of the essential oil from Croton conduplicatus (the EO 25, 50, and 100 mg/kg, i.p.), a medicinal plant native to Brazil. Antinociceptive activity was investigated by measuring the nociception induced by acetic acid, formalin, hot plate and carrageenan. A docking study was performed with the major constituents of the EO (E-caryophyllene, caryophyllene oxide, and camphor). The EO reduced nociceptive behavior at all doses tested in the acetic acid-induced nociception test (p < 0.05). The same was observed in both phases (neurogenic and inflammatory) of the formalin test. When the hot-plate test was conducted, the EO (50 mg/kg) extended the latency time after 60 min of treatment. The EO also reduced leukocyte migration at all doses, suggesting that its antinociceptive effect involves both central and peripheral mechanisms. Pretreatment with glibenclamide and atropine reversed the antinociceptive effect of the EO on the formalin test, suggesting the involvement of KATP channels and muscarinic receptors. The docking study revealed a satisfactory interaction profile between the major components of the EO and the different muscarinic receptor subtypes (M2, M3, and M4). These results corroborate the medicinal use of C. conduplicatus in folk medicine. Keywords: pain; medicinal plants; essential oil; terpenoids
1. Introduction Pain is a complex experience involving noxious stimuli and emotional processing. It is a pathological consequence of various disorders, but which has a protective role representing, in many cases, the only symptom for the diagnosis of diseases [1]. In contrast, when persistent, pain causes negative emotional Molecules 2017, 22, 900; doi:10.3390/molecules22060900
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and physiological reactions, leaving the patient in a debilitating state. Epidemiological studies suggest that the world prevalence of chronic pain is approximately 40% [2–5] and its severity usually correlates with a decreasing in physical and mental health [6]. Pain commonly involves central and peripheral mechanisms, which makes it very difficult to choose an appropriated pharmacological therapy. Although highly effective, central analgesics are usually not disassociated with important adverse effects, such as dependence, respiratory depression, and constipation [7,8]. Furthermore, peripheral analgesics also have undesirable effects, such as gastrointestinal and renal lesions [9,10]. In this context, research groups have been dedicated to discover new molecules with analgesic potential and low toxicity. In fact, several pharmacological studies involving medicinal plants and their essential oils have been performed for this purpose [11–14]. Croton conduplicatus Kunth. (Euphorbiaceae) is a Brazilian medicinal plant, endemic of the Caatinga biome and popularly known as “quebra-faca” [15]. Its leaves and stem-bark have a strong and characteristic odor, being considered an aromatic plant. In folk medicine, this plant is used as a natural analgesic for the treatment of headache and stomach disorders [16]. However, to date, there are no reports in the literature that can scientifically validate the popular use of this species. Therefore, this paper describes the antinociceptive effect of the essential oil from C. conduplicatus (the EO) and possible mechanisms involved, aided by a docking study of its chemical constituents. 2. Results 2.1. Chemical Composition of the EO Croton conduplicatus produced a colorless essential oil with characteristic odor. GC-MS analysis showed the presence of 48 distinct peaks, of which 38 were identified, corresponding to 90.5% of its entire chemical composition. The sesquiterpenes (E)-caryophyllene (13.72%), and caryophyllene oxide (13.15%) and the monoterpene camphor (8.25%) were considered as the major constituents of the EO (Table 1). Table 1. Chemical constituents of the essential oil from stem bark of Croton conduplicatus Kunth. (Euphorbiaceae). Peak
RT (min)
RI
Compound
% GC-MS
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
8.44 8.91 9.45 10.56 12.76 14.54 15.02 15.13 15.74 15.81 16.04 16.97 17.09 17.28 17.84 17.99 18.48 19.08 19.11 20.55 24.79 25.17 25.91
914 926 941 969 1026 1072 1085 1088 1104 1106 1112 1137 1140 1145 1161 1165 1178 1194 1195 1236 1362 1373 1396
α-Thujene α-Pinene Camphene β-Pinene 1,8-Cineole cis-Linalool oxide NI trans-Linalool oxide Linalool NI exo-Fenchol cis-Pinocarveol Camphor Camphene hydrate Pinocarvone Borneol Terpinen-4-ol α-Terpineol Myrtenal Thymol, methyl ether Cyclosativene α-Copaene α-Gurjunene
0.04 5.57 0.97 4.88 2.56 0.31 0.22 1.20 0.74 0.72 0.56 0.51 8.25 0.36 0.16 1.29 1.05 0.62 0.89 0.34 0.33 0.97 0.39
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Table 1. Cont. Peak Molecules 2017, 22, 900
RT (min)
24 26.55 25 26.87 24 26 27.62 25 27 27.84 26 28 28.07 27 29 28.36 28 30 28.64 29 31 29.10 30 32 29.38 31 33 29.50 32 34 29.79 33 35 30.23 34 36 30.61 35 37 31.52 36 38 31.99 37 39 32.10 38 40 32.28 39 41 32.44 40 42 32.78 41 43 33.01 42 44 33.11 43 45 33.48 44 46 33.74 45 47 33.90 46 48 40.62 47 Total identified 48
RI 1417 1427 1417 1451 1427 1458 1451 1465 1458 1475 1465 1484 1475 1498 1484 1508 1498 1512 1508 1522 1512 1537 1522 1550 1537 1581 1550 1597 1581 1601 1597 1607 1601 1613 1607 1625 1613 1633 1625 1637 1633 1650 1637 1660 1650 1665 1660 1926 1665
Compound (E)-Caryophyllene NI (E)-Caryophyllene 13.72 α-Humulene 0.43 NI Allo-aromadendrene6.05 α-Humulene 9-epi-(E)-Caryophyllene Allo-aromadendrene 0.23 Germacrene D 0.19 9-epi-(E)-Caryophyllene NI Germacrene D 4.01 α-Muurolene NI 0.31 β-Bisabolene 3.66 α-Muurolene γ-Cadinene β-Bisabolene 0.26 δ-Cadinene γ-Cadinene 0.17 δ-CadineneNI 0.65 NI Elemol 0.39 Caryophyllene oxide0.81 Elemol CaryophylleneGuaiol oxide 13.15 Rosifoliol Guaiol 2.51 Humulene epoxide II0.83 Rosifoliol Eudesmol Humulene epoxide II 4.19 Muurolol Eudesmol 1.94 Muurolol NI 3.17 NI NI 3.04 NIAgarospirol 1.83 AgarospirolNI 3.01 NI NI 1.19 NI NI 0.97
% GC-MS 13.72 0.43 6.05 0.23 0.19 4.01 0.31 3.66 0.26 0.17 0.65 0.39 0.81 13.15 2.51 0.83 4.19 1.94 3.17 3.04 1.83 3.01 1.19 0.97 0.35 90.55
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26.55 26.87 27.62 27.84 28.07 28.36 28.64 29.10 29.38 29.50 29.79 30.23 30.61 31.52 31.99 32.10 32.28 32.44 32.78 33.01 33.11 33.48 33.74 33.90 40.62 1926 NI 0.35 Total identified 90.55 RT: retention time of compounds. RI: retention indices on DB-5MS column (relative to n-alkanes). NI: not identified compound. RT: retention time of compounds. RI: retention indices on DB-5MS column (relative to n-alkanes). NI: not identified compound.
2.2. Acetic-Acid-Writhing-Induced Nociception 2.2. Acetic-Acid-Writhing-Induced Nociception
Figure 1 shows that the EO produced a significant (p < 0.05) the antinociceptive effect in mice Figure 1 shows that the EO produced a significant (p < 0.05) the antinociceptive effect in mice during acetic acid writhing induced nociception test. The EO at doses of 25, 50, and 100 mg/kg during acetic acid writhing induced nociception test. The EO at doses of 25, 50, and 100 mg/kg (i.p.) (i.p.) presented 68, 84, and 91% of antinociceptive activity, respectively. Morphine and indomethacin presented 68, 84, and 91% of antinociceptive activity, respectively. Morphine and indomethacin reduced the number of writhings respectively. reduced the number of writhingsby by100 100and and 98%, 98%, respectively.
Figure 1. The effect of the essential oil of C. conduplicatus (the EO 25, 50, and 100 mg/kg, i.p.), morphine
Figure 1. The effect of the essential oil of C. conduplicatus (the EO 25, 50, and 100 mg/kg, i.p.), morphine (MOR 10 mg/kg, i.p.) and indomethacine (20 mg/kg, i.p.) in the acetic-acid-writhing-induced (MOR 10 mg/kg, and (20 mg/kg, i.p.) as in mean the acetic-acid-writhing-induced nociception test,i.p.) in mice (n indomethacine = 6, per group). Values are expressed ± SEM, where a indicates nociception test, in mice (n = 6,from perthe group). are expressed as mean ± SEM, wheretest. a indicates p < 0.05, significantly different controlValues group, according to ANOVA, followed by Tukey′s p < 0.05, significantly different from the control group, according to ANOVA, followed by Tukey0 s test.
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2.3. Formalin-Induced Nociception 2.3. Formalin-Induced Nociception model, the the EO treatment significantly reduced the paw licking In the theformalin-induced formalin-inducednociception nociception model, EO treatment significantly reduced the paw In the formalin-induced nociception model, the EO treatment significantly reduced the paw time compared to the control group, at all doses and in both the test 0.05). the first licking time compared to the control group, at allused, doses used, and phases in bothof phases of (p the< test (p In < 0.05). In licking time compared to the control group, at all doses used, and in both phases of the test (p < 0.05). In phase, EO (25, and 100 showed 57 and 52, 66%57ofand antinociceptive activity, respectively, the firstthe phase, the50, EO (25, 50,mg/kg) and 100 mg/kg)52,showed 66% of antinociceptive activity, the first phase, the EO (25, 50, and 100 mg/kg) showed 52, 57 and 66% of antinociceptive activity, whereas in the secondinphase, the effect varied 71 and 79%. In respectively, whereas the second phase, the between effect varied between 71addition, and 79%.indomethacin In addition, respectively, whereas in the second phase, the effect varied between 71 and 79%. In addition, was effective only in the second phase of the phase test, whereas morphine demonstrated satisfactory indomethacin was effective only in the second of the test, whereas morphine demonstrated indomethacin was effective only in the second phase of the test, whereas morphine demonstrated antinociceptive activity in both phases (Figure 2). (Figure 2). satisfactory antinociceptive activity in both phases satisfactory antinociceptive activity in both phases (Figure 2).
Figure 2. The effect of the essential essential oil oil of of C. conduplicatus conduplicatus (the EO EO 25, 50, 50, and100 100 mg/kg, i.p.), i.p.), morphine morphine Figure 2. 2. The The effect effect of of the the Figure essential oil of C. C. conduplicatus (the (the EO 25, 25, 50, and and 100mg/kg, mg/kg, i.p.), morphine (MOR 10 mg/kg, mg/kg, i.p.) and indomethacine (20 mg/kg, i.p.) in the formalin-induced nociception test, in (MOR 10 10 i.p.) and andindomethacine indomethacine (20 (20 mg/kg, mg/kg,i.p.) i.p.)in inthe theformalin-induced formalin-induced nociception nociception test, test, in in (MOR mg/kg, i.p.) mice (n = 6, per group). Values are expressed as mean ± SEM, where a indicates p < 0.05, significantly mice (n (n == 6, 6, per Values are SEM, where where aa indicates mice per group). group). Values are expressed expressed as as mean mean ± ± SEM, indicates pp < < 0.05, 0.05, significantly significantly 0 s test. different from the the control control group, group, according to to ANOVA,followed followed byTukey Tukey′s test. different from from different the control group, according according to ANOVA, ANOVA, followedby by Tukey′s test.
2.3.1. Involvement of Muscarinic Receptors Involvement of of Muscarinic Muscarinic Receptors 2.3.1. Involvement To evaluate the involvement of muscarinic receptors in the antinociceptive effect of the EO, involvement of of muscarinic muscarinic receptors receptors in in the the antinociceptive antinociceptive effect effect of of the the EO, EO, To evaluate the involvement animals were pretreated with atropine (ATP 0.1 mg/kg, i.p.) during the formalin test. Figure 3 (ATP 0.1 mg/kg, i.p.)i.p.) during the formalin test. Figure 3 shows animals were were pretreated pretreatedwith withatropine atropine (ATP 0.1 mg/kg, during the formalin test. Figure 3 shows that atropine pretreatment reversed the antinociceptive effect of the EO (25 mg/kg, i.p.) in that atropine pretreatment reversed reversed the antinociceptive effect of the EO (25 i.p.)mg/kg, in both i.p.) phases shows that atropine pretreatment the antinociceptive effect of mg/kg, the EO (25 in both phases of the test (p < 0.05), suggesting that the cholinergic system may be involved. of thephases test (p of < 0.05), suggesting the cholinergic may system be involved. both the test (p < 0.05),that suggesting that thesystem cholinergic may be involved.
Figure 3. The effect of the essential oil of C. conduplicatus (the EO 25 mg/kg, i.p.) and the EO + ATP Figure of the the essential essential oil oil of of C. C. conduplicatus conduplicatus (the (the EO EO 25 25 mg/kg, mg/kg, i.p.) Figure 3. 3. The The effect effect of i.p.) and and the the EO EO ++ ATP ATP (25 and 0.1 mg/kg, respectively, i.p.) in the first and second phase of formalin-induced nociception (25 and 0.1 mg/kg, respectively, i.p.) in the first and second phase of formalin-induced nociception (25 and 0.1 mg/kg, respectively, i.p.) in the first and second phase of formalin-induced nociception test test (n = 6, per group). Values are expressed as mean ± SEM, where a and b indicate p < 0.05, test = 6,group). per group). are expressed mean where ± SEM, where a and pb
