PHYTOTHERAPY RESEARCH Phytother. Res. (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5177
Antihyperalgesic Effects of an Aqueous Stem Bark Extract of Mangifera indica L.: Role of Mangiferin Isolated from the Extract Bárbara B. Garrido-Suárez,1 Gabino Garrido,2* Mary Elena García1 and René Delgado-Hernández1 1
Laboratorio de Farmacología Molecular, Centro de Investigación y Desarrollo de Medicamentos, Ave. 26 No. 1605, Nuevo Vedado, La Habana, Cuba 2 Departamento de Ciencias Farmacéuticas, Facultad de Ciencias, Edificio Ñ3, Universidad Católica del Norte, Angamos 0610, Antofagasta, Chile
This study aimed to assess the effects of a Mangifera indica stem bark extract (MSBE) and mangiferin (MG) on pain-related acute behaviors in the formalin 5% test. Rats received repeated oral MSBE (125–500 mg/kg) once daily for 7 days before formalin injection. Other four groups with the same treatments were performed in order to study the effect of MSBE on the formalin-induced long-term secondary mechano-hyperalgesia at 7 days after the injury by means of the pin-prick method. Additional groups received a single oral MSBE dose (250 mg/kg) plus ascorbic acid (1 mg/kg, i.p.). Also, repeated oral MG doses (12.5–50 mg/kg) during 7 days were administered. MSBE decreased licking/biting and flinching behaviors only in phase II and reduced the long-term formalin injury-induced secondary chronic mechano-hyperalgesia. The combination of MSBE plus ascorbic acid produced a reinforcement of this effect for flinching behavior, advising that antioxidant mechanisms are involved, at least in part, in these actions. Chronic administration of MG reproduced the effects of MSBE. For the first time, the antihyperalgesic effects of MSBE and MG in formalin 5% test, a recommended concentration for studying the antinociceptive activity of nitric oxide-related and N-methyl-D-aspartate-related compounds, were reported. These results could represent an important contribution to explain the analgesic ethnobotanical effects recognized to M. indica and other species containing MG. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: formalin test; hyperalgesia; Mangifera indica L.; mangiferin.
Abbreviations: AA, ascorbic acid; COX-2, cyclooxygenase-2; MG, mangiferin; MSBE, Mangifera indica L. (mango) stem bark extract; NF-κB, transcription nuclear factor kappa B; NMDA, N-methyl-D-aspartate; NO, nitric oxide; PGE2, prostaglandins E2; pNR1, phosphorylated NMDA receptor subunit 1; ROS, reactive oxygen species; SDH, spinal dorsal horn; TNFα, necrosis factor alpha; TRPV1, transient receptor potential vanilloid type 1 receptor.
INTRODUCTION Mango (Mangifera indica L., Anacardiaceae) stem bark has been traditionally used in many tropical and subtropical countries for medicinal purposes using an aqueous extract obtained by decoction (Núñez-Sellés et al., 2007). The ethnomedical use of MSBE in Cuba has been documented on more than 7000 patients in the last years, mainly on patients with malignant tumors (Tamayo et al., 2001). It is of particular interest that more than 95% of cancer patients treated with MSBE (2286 patients) evidenced pain relief and consequently an improvement in terms of their quality of life (Núñez-Sellés et al., 2002b). Also significant was that 87% of patients with chronic painful diseases such as Lupus erythematosus (675 patients) improved their quality of life, and 118 patients with painful peripheral neuropathy due to several causes remitted, after the first and three months of MSBE treatment (oral * Correspondence to: Gabino Garrido, Departamento de Ciencias Farmacéuticas, Facultad de Ciencias, Edificio Ñ3, Universidad Católica del Norte, Angamos 0610, Antofagasta, Chile. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
and topical administration), respectively (NúñezSellés et al., 2002b, 2007). The MSBE contains a definite mixture of components including polyphenols, triterpenes, flavonoids, phytosterols, fatty acids, and microelements (NúñezSellés et al., 2002a). Previous experiments with the extract have shown that it has antioxidant (Garrido et al., 2008; Martínez et al., 2000), antiinflammatory, analgesic (Garrido et al., 2001; 2004a; 2004b), and immunomodulatory (García et al., 2002; 2003) properties. This extract and MG, its major component (about 15–20% in the extract), prevent TNFα-induced IκB degradation and the binding of NF-κB to the DNA (Garrido et al., 2005; Leiro et al., 2004). NF-κB induces the transcription of genes implicated in the expression of some mediators and enzymes involved in inflammation, pain, oxidative stress, and synaptic plasticity (Romano et al., 2006). On the other hand, MSBE and MG shown neuroprotective effects in the glutamate-induced neuronal, glial injury model and antioxidant activity related to its iron-chelating properties in addition to scavenging activity of free radicals (Campos-Esparza et al., 2009; Gottlied et al., 2006; Lemus-Molina et al., 2009; Received 14 January 2014 Revised 28 March 2014 Accepted 29 April 2014
B. B. GARRIDO-SUÁREZ ET AL.
Pardo-Andreu et al., 2008). They were able to limit microglial activation in vitro models (Bhatia et al., 2008; Garrido et al., 2004b). These evidences suggest the potentiality of both MSBE and MG to modulate some of the molecular targets implicated in peripheral and central persistent pain mechanisms, especially central sensitization, a pivotal mechanism in neuropathic pain (Salter, 2006). The formalin test is widely used as a model of acute and tonic inflammatory pain to study pain mechanisms and to evaluate the analgesic action of several substances (Coderre et al., 1990; Hacimuftuoglu et al., 2006; Okuda et al., 2001; Sawynok and Reid, 2001; 2002). It is considered an experimental surrogate model of neuropathic pain; generally, it uses concentrations ranging from 0.5% to 5%, and while pain behaviors are dose-related over this range, different mechanisms are involved at low and high concentrations (Coderre et al., 1993). Accordingly, at low concentration, there is a predominant activity of capsaicin-sensitive neurogenic components, whereas at high concentration (5%), there is an additional involvement of a more complex inflammatory mechanism (Sawynok and Reid, 2002). Exclusively, at high concentration of formalin, spinal microglial cells are activated, and long-term secondary hyperalgesia is produced (Fu et al., 2000). Equally, formalin at 5%, but not 2% releases substantial glutamate from the rat spinal cord in addition to peripheral tissue (Okuda et al., 2001). This concentration into the plantar paw evokes an optimal behavioral response, and it should be used when studying the antinociceptive activity of NO-related and NMDA-related compounds (Okuda et al., 2001). It has also been established that ROS play a role in this model (Hacimuftuoglu et al., 2006). Previously, our group reported the exclusive inhibition of tonic phase (phase II) in the formalin test at low (1%, unpublicized data) and middle (2.5%) concentrations by MSBE (Garrido et al., 2001). The effects of MG have not been assessed in this model at high concentration. The purpose of this study was to examine the effect of chronic administration of MSBE on the three phases of formalin 5% test and its combination with a sub-effective dose of ascorbic acid to investigate any possible contribution of the antioxidant mechanisms. Therefore, the participation of MG in the analgesic activity of MSBE was assessed. MATERIAL AND METHODS Plant material. The MSBE was collected from a cultivated field located in the region of Pinar del Rio, Cuba. Voucher specimens of the plant (Code 41722) were deposited at the Herbarium of the Academy of Sciences, guarded by the Institute of Ecology and Systematics from the Ministry of Science, Technology and Environment, Havana, Cuba, and authenticated by MSc Ramona Prieto, curator, and MSc Isora Baró, Director of the Herbarium. Stem bark extract from M. indica was prepared by decoction in water for 1 h, and then, it was concentrated by evaporation and spray-dried in a Niro Atomizer Standard Spray Drying (Soeborg, Denmark) to obtain a fine homogeneous brown powder with a particle size of 30–60 mm (AcostaEsquijarosa et al., 2009). The chemical composition of this extract has been characterized by chromatographic (planar, liquid, and gas) methods, mass spectrometry, and UV–Vis spectrophotometry (García Rivera et al., 2011; Copyright © 2014 John Wiley & Sons, Ltd.
Núñez-Sellés, 2005; 2002a). The batch used in this study was analyzed in the Quality Department of the Pharmaceutical Chemistry Center (Havana, Cuba) and was found to have the following content: moisture 50%, total phenol (in anhydrous base) >30%, and MG >10%, according to the quality specifications established. The solid extract was dissolved in distilled water for pharmacological studies. Drugs. The MG (1,3,6,7-tetrahydroxy xanthone-C2-β-Dglucoside) was isolated from the MSBE by extraction with methanol according to the described standard method (Garcia Rivera et al., 2011; Nuñez-Sellés et al., 2002a). It was supplied by the Laboratory of Analytical Chemistry, Center of Pharmaceutical Chemistry (Havana, Cuba) with 90% purity assessed with a validated high-performance liquid chromatography-based method (García Rivera et al., 2011 Núñez-Sellés et al., 2002a). MG was suspended in carboxymethyl cellulose 0.05% for the tests. AA was purchased from Sigma-Aldrich (Saint Louis, MO, USA), and morphine sulfate was kindly provided by Laboratorios LIORAD (La Habana, Cuba). Animals. Experimental procedures were carried out in accordance with European regulations on animal protection (Directive 86/609), the Declaration of Helsinki, and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institute of Health (Publication № 85–23, revised 1996). All experimental protocols were approved by the Institutional Animal Care and Ethical Committee from the Drugs Research and Development Center. Male Sprague-Dawley rats (8–10 weeks) weighing 200–250 g were obtained from the Center for Experimental Animals Production (CENPALAB, La Habana, Cuba). They were kept in controlled conditions (22 ± 0.5°C, relative humidity 40–60%, a 7AM to 7PM alternate light– dark cycle, food and water ad libitum). The experiments took place during the light period, and animals belonging to the various treatment groups (n = 7–11 for each group) were tested in randomized order. Formalin test. Acute spontaneous nociceptive behavior. To perform the formalin test, the rats were placed individually in an open glass cylindrical chamber (34 × 30 × 28 cm). The animals were habituated to the chamber for 20 min prior to the injection and returned to the chamber after the injection for their observation. Formalin (50 μl) was injected s.c. into the plantar surface of the right hindpaw of the rat using a microsyringe with a 26-gage needle (Dubuisson and Dennis, 1977). The total number of flinches of the hindpaw and/or the hindquarters was recorded by visual observation for 5-min periods, for a total observation time of 45 min following injection of formalin. Also, licking/biting of the injected paw was recorded using a digital chronometer as the total licking/ biting time (s) per 5-min observation periods for 45 min after formalin injection, too. The response curves for both formalin-induced behaviors were generated by recording early (0–5 min), latency (5–15 min), and late (15–45 min) phase behaviors. Secondary chronic mechanical hyperalgesia. Formalin (50 μl) was injected s.c. into the dorsal surface of the right hindpaw of the rat using a microsyringe with a Phytother. Res. (2014)
ANTIHYPERALGESIC EFFECTS OF MANGIFERA INDICA AND MANGIFERIN
26-gage needle (Fu et al., 2000). Mechano-hyperalgesia of the opposite surface of the foot (plantar) was assessed using a modification of the pin-prick method (Tal and Bennett, 1994). With the rats standing on the wire mesh floor and confined beneath an inverted plastic box, the point of a blunted 23-gage needle was applied to the skin of the heel (touching but not penetrating). Normal rats respond with a very small and brief withdrawal. The injured rats respond with a withdrawal that is exaggerated in amplitude and duration. Behavioral responses to the pin prick were rated according to the following scale (Coderre et al., 2004): 0 = no response; 1 = rapid paw flicking, stamping, or shaking (less than 1 s); 2 = repeated paw stamping, shaking, or paw lifting less than 3 s; 3 = above behaviors or hindpaw licking for more than 3 s; and 4 = above behaviors for more than 3 s and hindpaw licking for more than 3 s. An additional point was added if any vocalizations occurred. Sedative behavioral test. To assess whether MSBE, MG, AA, induced sedation, or anesthesia interferes with posture and righting reflexes, a five-point scale was utilized by the observer (Devor and Zalkind, 2001). Scale for posture: 0 = normal posture, rearing, and grooming; 1 = moderate atonia and ataxia, weight support, but not rearing; 2 = support but severe ataxia; 3 = muscle tone but not weight support and only small purposive movements; and 4 = flaccid atonia, fully immobilized with no attempts at movements. Scale for righting reflexes: 0 = the rat struggles when placed on its side followed by rapid forceful righting; 1 = moderate resistance when the rat is placed on its side, with rapid but not forceful righting; 2 = no resistance to the rat being placed on its side, with effortful but ultimately successful righting; 3 = unsuccessful righting; and 4 = no movements. Medication protocols. Repeated oral MSBE administration on formalin-induced nociceptive behaviors. Given the possibility that some clinical effect of MSBE can appear only after its chronic administration, we firstly designed a long-term medication protocol to evaluate the effect of MSBE on acute spontaneous behaviors in formalin test at 5%. Rats received (dose volume 10 ml/kg, p.o.) pretreatment with MSBE (125, 250, and 500 mg/kg of body weight) or distilled water once daily for 7 consecutive days (n = 11 for each group) previous to test, the last dose was administered 1 h before formalin injection. An additional positive control group was acutely treated with morphine (10 mg/kg) by oral route (Holtman et al., 2010). The MSBE doses were selected according to previous reports (Garrido et al., 2001) and on pilot experiments in our laboratory. In order to investigate the possible effect of MSBE on the formalin-induced long-term secondary mechanohyperalgesia, others four groups (n = 7 for each group) with the same treatments were performed. Baseline nociceptive score was assessed before dorsal formalin injection and at 7 days post-injury, using a modification of the pin-prick method. Effects of a combination of MSBE plus AA on formalin-induced nociceptive behaviors. To assess the effect of single oral MSBE and its combination with AA on formalin test (250 mg/kg b.w., dose volume Copyright © 2014 John Wiley & Sons, Ltd.
10 ml/kg, p.o.) or distilled water (n = 10) per group, sub-effective dose of AA (1 mg/kg b.w., dose volume 5 ml/kg, i.p.) (n = 8) or its combination were administered to animals 1 h before formalin injection (n = 10). Repeated oral MG administration on formalin-induced nociceptive behaviors. To explore the possible contribution of MG to the MSBE analgesic mechanism on formalin test, rats were divided into four groups. The animals received (dose volume 10 ml/kg, p.o.) pretreatment with MG (12.5, 25, and 50 mg/kg b.w.) or carboxymethyl cellulose 0.05% once daily for 7 consecutive days each group (n = 11). The MG doses were selected on MSBE doses utilized previously (Garrido et al., 2001) considering that MG is about 15–20% in the extract and according to previous reports (Pardo-Andreu et al., 2010; Lopes et al., 2013). Statistical analyses. Data were analyzed using the statistical program Graph Pad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). The results are presented as mean ± S.E.M. Statistical analysis were carried out using one-way analysis of variance test followed by Bonfferoni’s multiple comparison test for evaluating statistically significant differences (p < 0.05) of each treated group from control.
RESULTS Effect of repeated oral dose of MSBE on formalin-induced nociceptive behaviors Formalin 5% injected into plantar surface of the right hindpaw produced a typical pattern of licking/biting and flinching behavior characterized by a biphasic time course (Fig. 1). Oral pretreatment with MSBE (125–500 mg/kg b.w.) once a day for 7 days reduced dose-related formalin-induced pain only during phase II, expressed as mean of time of licking/biting (Fig. 1A) and as number of flinching (Fig. 1B). These effects were significant in the peak pain-related behaviors in this test about 30 min (p < 0.01 and p < 0.001). Increasing doses of MSBE produced 16.8%, 33.8%, and 49.6% of reduction of the licking/biting response in the tonic phase compared with vehicle pretreated controls. Only the high dose (MSBE 500 mg/kg) was similar to morphine, which produced 67.4% of reduction of the licking/biting response in this phase (vehicle = 476 ± 45, MSBE 125 mg = 396 ± 28, MSBE 250 mg = 315 ± 31, p < 0.001, MSBE 500 mg = 240 ± 26, p < 0.001, morphine 10 mg = 155 ± 10, p < 0.001) (Fig. 1C). However, the flinching nocifensive response was reduced independently from the MSBE dose. The proportion of inhibition with respect to the vehicle treated group was among 64.7%, 48.4%, and 44.5%. The low dose (125 mg/kg) was more effective for reducing the number of flinches in the tonic phase. Nevertheless, the three doses were similar to morphine, which produced 61.5% of reduction of the flinching behavior (vehicle = 252 ± 24, MSBE 125 mg = 89 ± 4, p < 0.001, MSBE 250 mg = 130 ± 28, p < 0.001, MSBE 500 mg = 140 ± 22, p < 0.001, morphine 10 mg = 97 ± 4, p < 0.001) (Fig. 1D). The period of latency was not affected by MSBE for neither of the behaviors. Baseline nociceptive responses to pin pricks showed similar scores in all groups. Pretreatment with Phytother. Res. (2014)
B. B. GARRIDO-SUÁREZ ET AL.
Figure 1. Time course of the effects of pretreatment with three doses of Mangifera indica L. stem bark extract (MSBE) (125–500 mg/kg b.w.) on the licking/biting and flinching following formalin at 5% injected into plantar surface of the right hindpaw. (A) The data are presented as mean time licking/biting/5 min ± S.E.M. during 45 min. (B) The data are presented as mean of number of flinches/5min ± S.E.M. during 45 min. (C) The data are presented as cumulative mean licking/biting time ± S.E.M. during the phase I (0–5 min), latency (5–15 min), and phase II (15–45 min) of the formalin test. (D) The data are presented as cumulative mean of number of flinches ± S.E.M. during the phase I, latency, and phase II of the same test. **p < 0.01 and ***p < 0.001 indicate significant differences with respect to control group (vehicle), and ###p 0.001 indicates differences with respect to positive control group (morphine). In (A) and (B), only the statistical differences in the peak painrelated behaviors (about 30 min) are showed.
MSBE decreased the secondary chronic mechanical hyperalgesia in the opposite surface of the affected hindpaw evaluated at 7 days after formalin injection with respect to the controls (vehicle = 2.62 ± 0.1, MSBE 125 mg = 1.83 ± 0.2, p < 0.0001, MSBE 250 mg = 0.72 ± 0.1, p < 0.0001, MSBE 500 mg = 0.56 ± 0.1, p < 0.0001) (Fig. 2). Effects of a combination of MSBE plus AA on formalininduced nociceptive behaviors
N o c ic e p tiv e s c o r e (p in p r ic k )
The combination of MSBE (250 mg/kg b.w., p.o.) with a sub-effective dose of AA (1 mg/kg b.w., i.p.) decreased the time of licking–biting exclusivity in phase II with respect to the control group such as MSBE alone 3 2.5
***
2 1.5 1
***
0.5
***
0 vehicle
MSBE 125
Baseline
MSBE 250
MSBE 500
Post-injection
Figure 2. The effects of the pretreatment with Mangifera indica L. stem bark extract (MSBE) (125–500 mg/kg b.w.) on secondary chronic mechanical hyperalgesia (phase III) in the opposite surface of the affected hindpaw evaluated at 7 days after formalin injection. The data are presented as mean ± S.E.M. ***p
Antihyperalgesic Effects of an Aqueous Stem Bark Extract of Mangifera indica L.: Role of Mangiferin Isolated from the Extract Bárbara B. Garrido-Suárez,1 Gabino Garrido,2* Mary Elena García1 and René Delgado-Hernández1 1
Laboratorio de Farmacología Molecular, Centro de Investigación y Desarrollo de Medicamentos, Ave. 26 No. 1605, Nuevo Vedado, La Habana, Cuba 2 Departamento de Ciencias Farmacéuticas, Facultad de Ciencias, Edificio Ñ3, Universidad Católica del Norte, Angamos 0610, Antofagasta, Chile
This study aimed to assess the effects of a Mangifera indica stem bark extract (MSBE) and mangiferin (MG) on pain-related acute behaviors in the formalin 5% test. Rats received repeated oral MSBE (125–500 mg/kg) once daily for 7 days before formalin injection. Other four groups with the same treatments were performed in order to study the effect of MSBE on the formalin-induced long-term secondary mechano-hyperalgesia at 7 days after the injury by means of the pin-prick method. Additional groups received a single oral MSBE dose (250 mg/kg) plus ascorbic acid (1 mg/kg, i.p.). Also, repeated oral MG doses (12.5–50 mg/kg) during 7 days were administered. MSBE decreased licking/biting and flinching behaviors only in phase II and reduced the long-term formalin injury-induced secondary chronic mechano-hyperalgesia. The combination of MSBE plus ascorbic acid produced a reinforcement of this effect for flinching behavior, advising that antioxidant mechanisms are involved, at least in part, in these actions. Chronic administration of MG reproduced the effects of MSBE. For the first time, the antihyperalgesic effects of MSBE and MG in formalin 5% test, a recommended concentration for studying the antinociceptive activity of nitric oxide-related and N-methyl-D-aspartate-related compounds, were reported. These results could represent an important contribution to explain the analgesic ethnobotanical effects recognized to M. indica and other species containing MG. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: formalin test; hyperalgesia; Mangifera indica L.; mangiferin.
Abbreviations: AA, ascorbic acid; COX-2, cyclooxygenase-2; MG, mangiferin; MSBE, Mangifera indica L. (mango) stem bark extract; NF-κB, transcription nuclear factor kappa B; NMDA, N-methyl-D-aspartate; NO, nitric oxide; PGE2, prostaglandins E2; pNR1, phosphorylated NMDA receptor subunit 1; ROS, reactive oxygen species; SDH, spinal dorsal horn; TNFα, necrosis factor alpha; TRPV1, transient receptor potential vanilloid type 1 receptor.
INTRODUCTION Mango (Mangifera indica L., Anacardiaceae) stem bark has been traditionally used in many tropical and subtropical countries for medicinal purposes using an aqueous extract obtained by decoction (Núñez-Sellés et al., 2007). The ethnomedical use of MSBE in Cuba has been documented on more than 7000 patients in the last years, mainly on patients with malignant tumors (Tamayo et al., 2001). It is of particular interest that more than 95% of cancer patients treated with MSBE (2286 patients) evidenced pain relief and consequently an improvement in terms of their quality of life (Núñez-Sellés et al., 2002b). Also significant was that 87% of patients with chronic painful diseases such as Lupus erythematosus (675 patients) improved their quality of life, and 118 patients with painful peripheral neuropathy due to several causes remitted, after the first and three months of MSBE treatment (oral * Correspondence to: Gabino Garrido, Departamento de Ciencias Farmacéuticas, Facultad de Ciencias, Edificio Ñ3, Universidad Católica del Norte, Angamos 0610, Antofagasta, Chile. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
and topical administration), respectively (NúñezSellés et al., 2002b, 2007). The MSBE contains a definite mixture of components including polyphenols, triterpenes, flavonoids, phytosterols, fatty acids, and microelements (NúñezSellés et al., 2002a). Previous experiments with the extract have shown that it has antioxidant (Garrido et al., 2008; Martínez et al., 2000), antiinflammatory, analgesic (Garrido et al., 2001; 2004a; 2004b), and immunomodulatory (García et al., 2002; 2003) properties. This extract and MG, its major component (about 15–20% in the extract), prevent TNFα-induced IκB degradation and the binding of NF-κB to the DNA (Garrido et al., 2005; Leiro et al., 2004). NF-κB induces the transcription of genes implicated in the expression of some mediators and enzymes involved in inflammation, pain, oxidative stress, and synaptic plasticity (Romano et al., 2006). On the other hand, MSBE and MG shown neuroprotective effects in the glutamate-induced neuronal, glial injury model and antioxidant activity related to its iron-chelating properties in addition to scavenging activity of free radicals (Campos-Esparza et al., 2009; Gottlied et al., 2006; Lemus-Molina et al., 2009; Received 14 January 2014 Revised 28 March 2014 Accepted 29 April 2014
B. B. GARRIDO-SUÁREZ ET AL.
Pardo-Andreu et al., 2008). They were able to limit microglial activation in vitro models (Bhatia et al., 2008; Garrido et al., 2004b). These evidences suggest the potentiality of both MSBE and MG to modulate some of the molecular targets implicated in peripheral and central persistent pain mechanisms, especially central sensitization, a pivotal mechanism in neuropathic pain (Salter, 2006). The formalin test is widely used as a model of acute and tonic inflammatory pain to study pain mechanisms and to evaluate the analgesic action of several substances (Coderre et al., 1990; Hacimuftuoglu et al., 2006; Okuda et al., 2001; Sawynok and Reid, 2001; 2002). It is considered an experimental surrogate model of neuropathic pain; generally, it uses concentrations ranging from 0.5% to 5%, and while pain behaviors are dose-related over this range, different mechanisms are involved at low and high concentrations (Coderre et al., 1993). Accordingly, at low concentration, there is a predominant activity of capsaicin-sensitive neurogenic components, whereas at high concentration (5%), there is an additional involvement of a more complex inflammatory mechanism (Sawynok and Reid, 2002). Exclusively, at high concentration of formalin, spinal microglial cells are activated, and long-term secondary hyperalgesia is produced (Fu et al., 2000). Equally, formalin at 5%, but not 2% releases substantial glutamate from the rat spinal cord in addition to peripheral tissue (Okuda et al., 2001). This concentration into the plantar paw evokes an optimal behavioral response, and it should be used when studying the antinociceptive activity of NO-related and NMDA-related compounds (Okuda et al., 2001). It has also been established that ROS play a role in this model (Hacimuftuoglu et al., 2006). Previously, our group reported the exclusive inhibition of tonic phase (phase II) in the formalin test at low (1%, unpublicized data) and middle (2.5%) concentrations by MSBE (Garrido et al., 2001). The effects of MG have not been assessed in this model at high concentration. The purpose of this study was to examine the effect of chronic administration of MSBE on the three phases of formalin 5% test and its combination with a sub-effective dose of ascorbic acid to investigate any possible contribution of the antioxidant mechanisms. Therefore, the participation of MG in the analgesic activity of MSBE was assessed. MATERIAL AND METHODS Plant material. The MSBE was collected from a cultivated field located in the region of Pinar del Rio, Cuba. Voucher specimens of the plant (Code 41722) were deposited at the Herbarium of the Academy of Sciences, guarded by the Institute of Ecology and Systematics from the Ministry of Science, Technology and Environment, Havana, Cuba, and authenticated by MSc Ramona Prieto, curator, and MSc Isora Baró, Director of the Herbarium. Stem bark extract from M. indica was prepared by decoction in water for 1 h, and then, it was concentrated by evaporation and spray-dried in a Niro Atomizer Standard Spray Drying (Soeborg, Denmark) to obtain a fine homogeneous brown powder with a particle size of 30–60 mm (AcostaEsquijarosa et al., 2009). The chemical composition of this extract has been characterized by chromatographic (planar, liquid, and gas) methods, mass spectrometry, and UV–Vis spectrophotometry (García Rivera et al., 2011; Copyright © 2014 John Wiley & Sons, Ltd.
Núñez-Sellés, 2005; 2002a). The batch used in this study was analyzed in the Quality Department of the Pharmaceutical Chemistry Center (Havana, Cuba) and was found to have the following content: moisture 50%, total phenol (in anhydrous base) >30%, and MG >10%, according to the quality specifications established. The solid extract was dissolved in distilled water for pharmacological studies. Drugs. The MG (1,3,6,7-tetrahydroxy xanthone-C2-β-Dglucoside) was isolated from the MSBE by extraction with methanol according to the described standard method (Garcia Rivera et al., 2011; Nuñez-Sellés et al., 2002a). It was supplied by the Laboratory of Analytical Chemistry, Center of Pharmaceutical Chemistry (Havana, Cuba) with 90% purity assessed with a validated high-performance liquid chromatography-based method (García Rivera et al., 2011 Núñez-Sellés et al., 2002a). MG was suspended in carboxymethyl cellulose 0.05% for the tests. AA was purchased from Sigma-Aldrich (Saint Louis, MO, USA), and morphine sulfate was kindly provided by Laboratorios LIORAD (La Habana, Cuba). Animals. Experimental procedures were carried out in accordance with European regulations on animal protection (Directive 86/609), the Declaration of Helsinki, and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institute of Health (Publication № 85–23, revised 1996). All experimental protocols were approved by the Institutional Animal Care and Ethical Committee from the Drugs Research and Development Center. Male Sprague-Dawley rats (8–10 weeks) weighing 200–250 g were obtained from the Center for Experimental Animals Production (CENPALAB, La Habana, Cuba). They were kept in controlled conditions (22 ± 0.5°C, relative humidity 40–60%, a 7AM to 7PM alternate light– dark cycle, food and water ad libitum). The experiments took place during the light period, and animals belonging to the various treatment groups (n = 7–11 for each group) were tested in randomized order. Formalin test. Acute spontaneous nociceptive behavior. To perform the formalin test, the rats were placed individually in an open glass cylindrical chamber (34 × 30 × 28 cm). The animals were habituated to the chamber for 20 min prior to the injection and returned to the chamber after the injection for their observation. Formalin (50 μl) was injected s.c. into the plantar surface of the right hindpaw of the rat using a microsyringe with a 26-gage needle (Dubuisson and Dennis, 1977). The total number of flinches of the hindpaw and/or the hindquarters was recorded by visual observation for 5-min periods, for a total observation time of 45 min following injection of formalin. Also, licking/biting of the injected paw was recorded using a digital chronometer as the total licking/ biting time (s) per 5-min observation periods for 45 min after formalin injection, too. The response curves for both formalin-induced behaviors were generated by recording early (0–5 min), latency (5–15 min), and late (15–45 min) phase behaviors. Secondary chronic mechanical hyperalgesia. Formalin (50 μl) was injected s.c. into the dorsal surface of the right hindpaw of the rat using a microsyringe with a Phytother. Res. (2014)
ANTIHYPERALGESIC EFFECTS OF MANGIFERA INDICA AND MANGIFERIN
26-gage needle (Fu et al., 2000). Mechano-hyperalgesia of the opposite surface of the foot (plantar) was assessed using a modification of the pin-prick method (Tal and Bennett, 1994). With the rats standing on the wire mesh floor and confined beneath an inverted plastic box, the point of a blunted 23-gage needle was applied to the skin of the heel (touching but not penetrating). Normal rats respond with a very small and brief withdrawal. The injured rats respond with a withdrawal that is exaggerated in amplitude and duration. Behavioral responses to the pin prick were rated according to the following scale (Coderre et al., 2004): 0 = no response; 1 = rapid paw flicking, stamping, or shaking (less than 1 s); 2 = repeated paw stamping, shaking, or paw lifting less than 3 s; 3 = above behaviors or hindpaw licking for more than 3 s; and 4 = above behaviors for more than 3 s and hindpaw licking for more than 3 s. An additional point was added if any vocalizations occurred. Sedative behavioral test. To assess whether MSBE, MG, AA, induced sedation, or anesthesia interferes with posture and righting reflexes, a five-point scale was utilized by the observer (Devor and Zalkind, 2001). Scale for posture: 0 = normal posture, rearing, and grooming; 1 = moderate atonia and ataxia, weight support, but not rearing; 2 = support but severe ataxia; 3 = muscle tone but not weight support and only small purposive movements; and 4 = flaccid atonia, fully immobilized with no attempts at movements. Scale for righting reflexes: 0 = the rat struggles when placed on its side followed by rapid forceful righting; 1 = moderate resistance when the rat is placed on its side, with rapid but not forceful righting; 2 = no resistance to the rat being placed on its side, with effortful but ultimately successful righting; 3 = unsuccessful righting; and 4 = no movements. Medication protocols. Repeated oral MSBE administration on formalin-induced nociceptive behaviors. Given the possibility that some clinical effect of MSBE can appear only after its chronic administration, we firstly designed a long-term medication protocol to evaluate the effect of MSBE on acute spontaneous behaviors in formalin test at 5%. Rats received (dose volume 10 ml/kg, p.o.) pretreatment with MSBE (125, 250, and 500 mg/kg of body weight) or distilled water once daily for 7 consecutive days (n = 11 for each group) previous to test, the last dose was administered 1 h before formalin injection. An additional positive control group was acutely treated with morphine (10 mg/kg) by oral route (Holtman et al., 2010). The MSBE doses were selected according to previous reports (Garrido et al., 2001) and on pilot experiments in our laboratory. In order to investigate the possible effect of MSBE on the formalin-induced long-term secondary mechanohyperalgesia, others four groups (n = 7 for each group) with the same treatments were performed. Baseline nociceptive score was assessed before dorsal formalin injection and at 7 days post-injury, using a modification of the pin-prick method. Effects of a combination of MSBE plus AA on formalin-induced nociceptive behaviors. To assess the effect of single oral MSBE and its combination with AA on formalin test (250 mg/kg b.w., dose volume Copyright © 2014 John Wiley & Sons, Ltd.
10 ml/kg, p.o.) or distilled water (n = 10) per group, sub-effective dose of AA (1 mg/kg b.w., dose volume 5 ml/kg, i.p.) (n = 8) or its combination were administered to animals 1 h before formalin injection (n = 10). Repeated oral MG administration on formalin-induced nociceptive behaviors. To explore the possible contribution of MG to the MSBE analgesic mechanism on formalin test, rats were divided into four groups. The animals received (dose volume 10 ml/kg, p.o.) pretreatment with MG (12.5, 25, and 50 mg/kg b.w.) or carboxymethyl cellulose 0.05% once daily for 7 consecutive days each group (n = 11). The MG doses were selected on MSBE doses utilized previously (Garrido et al., 2001) considering that MG is about 15–20% in the extract and according to previous reports (Pardo-Andreu et al., 2010; Lopes et al., 2013). Statistical analyses. Data were analyzed using the statistical program Graph Pad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). The results are presented as mean ± S.E.M. Statistical analysis were carried out using one-way analysis of variance test followed by Bonfferoni’s multiple comparison test for evaluating statistically significant differences (p < 0.05) of each treated group from control.
RESULTS Effect of repeated oral dose of MSBE on formalin-induced nociceptive behaviors Formalin 5% injected into plantar surface of the right hindpaw produced a typical pattern of licking/biting and flinching behavior characterized by a biphasic time course (Fig. 1). Oral pretreatment with MSBE (125–500 mg/kg b.w.) once a day for 7 days reduced dose-related formalin-induced pain only during phase II, expressed as mean of time of licking/biting (Fig. 1A) and as number of flinching (Fig. 1B). These effects were significant in the peak pain-related behaviors in this test about 30 min (p < 0.01 and p < 0.001). Increasing doses of MSBE produced 16.8%, 33.8%, and 49.6% of reduction of the licking/biting response in the tonic phase compared with vehicle pretreated controls. Only the high dose (MSBE 500 mg/kg) was similar to morphine, which produced 67.4% of reduction of the licking/biting response in this phase (vehicle = 476 ± 45, MSBE 125 mg = 396 ± 28, MSBE 250 mg = 315 ± 31, p < 0.001, MSBE 500 mg = 240 ± 26, p < 0.001, morphine 10 mg = 155 ± 10, p < 0.001) (Fig. 1C). However, the flinching nocifensive response was reduced independently from the MSBE dose. The proportion of inhibition with respect to the vehicle treated group was among 64.7%, 48.4%, and 44.5%. The low dose (125 mg/kg) was more effective for reducing the number of flinches in the tonic phase. Nevertheless, the three doses were similar to morphine, which produced 61.5% of reduction of the flinching behavior (vehicle = 252 ± 24, MSBE 125 mg = 89 ± 4, p < 0.001, MSBE 250 mg = 130 ± 28, p < 0.001, MSBE 500 mg = 140 ± 22, p < 0.001, morphine 10 mg = 97 ± 4, p < 0.001) (Fig. 1D). The period of latency was not affected by MSBE for neither of the behaviors. Baseline nociceptive responses to pin pricks showed similar scores in all groups. Pretreatment with Phytother. Res. (2014)
B. B. GARRIDO-SUÁREZ ET AL.
Figure 1. Time course of the effects of pretreatment with three doses of Mangifera indica L. stem bark extract (MSBE) (125–500 mg/kg b.w.) on the licking/biting and flinching following formalin at 5% injected into plantar surface of the right hindpaw. (A) The data are presented as mean time licking/biting/5 min ± S.E.M. during 45 min. (B) The data are presented as mean of number of flinches/5min ± S.E.M. during 45 min. (C) The data are presented as cumulative mean licking/biting time ± S.E.M. during the phase I (0–5 min), latency (5–15 min), and phase II (15–45 min) of the formalin test. (D) The data are presented as cumulative mean of number of flinches ± S.E.M. during the phase I, latency, and phase II of the same test. **p < 0.01 and ***p < 0.001 indicate significant differences with respect to control group (vehicle), and ###p 0.001 indicates differences with respect to positive control group (morphine). In (A) and (B), only the statistical differences in the peak painrelated behaviors (about 30 min) are showed.
MSBE decreased the secondary chronic mechanical hyperalgesia in the opposite surface of the affected hindpaw evaluated at 7 days after formalin injection with respect to the controls (vehicle = 2.62 ± 0.1, MSBE 125 mg = 1.83 ± 0.2, p < 0.0001, MSBE 250 mg = 0.72 ± 0.1, p < 0.0001, MSBE 500 mg = 0.56 ± 0.1, p < 0.0001) (Fig. 2). Effects of a combination of MSBE plus AA on formalininduced nociceptive behaviors
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The combination of MSBE (250 mg/kg b.w., p.o.) with a sub-effective dose of AA (1 mg/kg b.w., i.p.) decreased the time of licking–biting exclusivity in phase II with respect to the control group such as MSBE alone 3 2.5
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Figure 2. The effects of the pretreatment with Mangifera indica L. stem bark extract (MSBE) (125–500 mg/kg b.w.) on secondary chronic mechanical hyperalgesia (phase III) in the opposite surface of the affected hindpaw evaluated at 7 days after formalin injection. The data are presented as mean ± S.E.M. ***p
