Antinociceptive effect of hydroalcoholic extract and isoflavone isolated from Polygala molluginifolia in mice: evidence for the involvement of opioid receptors and TRPV1 and TRPA1 channels.
Purpose: The plants of the genus Polygala (Polygalaceae) have been used for a long time in folk medicine to treat pain and inflammation. The species Polygala molluginifolia is native to southern Brazil and is popularly known as "canfora". The presented study analyzes the antinociceptive effect of hydroalcoholic extract from Polygala molluginifolia (HEPm) and an isoflavone (ISO) isolated from the extract, in behavioral models of pain in mice, as well as the mechanism underlying this effect.
Materials and methods: The phytochemical analysis of HEPm was performed through a capillary electrophoresis analysis and colorimetric test. The antinociceptive effects of HEPm and ISO (10-1000 mg/kg, i.g.) were evaluated by applying the formalin test; mechanical and thermal hyperalgesia to postoperative pain in mice. The possible involvement of opioid receptors, TRPV1 and TRPA1 channels in the antinociceptive effect of HEPm and ISO were also evaluated. Finally, the nonspecific effects of HEPm and ISO were evaluated by measuring locomotor activity (Open-field Test) and corporal temperature.
Results: The 5,3',4'-trihydroxy-6",6"-dimethylpyrano[2",3":7,6] isoflavone (ISO) was identified in HEPm by capillary electrophoresis analysis and selected for the experimental tests. The oral administration of HEPm or of ISO significantly inhibited the neurogenic and inflammatory phases of formalin-induced pain, edema formation and local hyperemia, without causing any change to locomotor activity. Acute and repeated treatment of animals with HEPm reduced mechanical and thermal (heat and cold) hyperalgesia in the postoperative pain. In addition, administering HEPm or ISO markedly reduced nociceptive behavior induced by the peripheral and central injection of TRPV1 and TRPA1 channels activators. Finally, the antinociception provided by the administration of HEPm or ISO was reversed by the preadministration of naloxone.
Conclusions: Taken together, these results provide the first experimental evidence of the significant antinociceptive effect of HEPm and ISO in animal models of acute pain without causing sedation or locomotor dysfunction. This effect appears to be mediated, at least in part, by the activation of opioid receptors and/or by the inhibition of TRPV1 and TRPA1 channels. Moreover, this study adds new scientific evidence and highlights the therapeutic potential of the medicinal plant Polygala molluginifolia in the development of phytomedicines with analgesic properties.
Pain is a global proportions and postoperative pain represents one of the most common forms of acute pain. The National Center for Health Statistics (2010) estimates that 51.4 million surgical procedures are performed annually in the USA, representing a large economic impact. Furthermore, it is estimated that approximately 80% of patients have acute pain (moderate to extreme) after surgery (Apfelbaum et al. 2003). This acute pain is followed by persistent pain in 10-50% of cases (Kehlet et al. 2006), which substantially affects the recovery and quality of life of these individuals. Several approaches have been used to reduce postoperative pain, especially the administration of opioids and non-steroidal anti-inflammatory drugs (Pogatzki-Zahn et al. 2012), however, they have limited efficacy and pronounced side effects (Benyamin et al. 2008). Therefore, new strategies to prevent postoperative pain should be further investigated. In this regard, medicinal plants seem to be excellent therapeutic options as they have been widely used throughout history and have proved to be rich sources of new compounds that can be especially used to treat pain and inflammation (Sen and Samanta 2014).
Considerable milestones in the development of analgesic drugs from natural products include the discoveries of morphine, a natural alkaloid extracted from the plant Papaver somniferum and salicylic acid isolated from Salix alba (Calixto et al. 2000; Haas 1983). Researchers have explored the involvement of the endogenous opioid system in the antinociceptive effect of medicinal herbs (Calixto et al. 2000; Lapa et al. 2009). However, hypersensitivity to painful stimuli (mechanical, thermal and chemical), called hyperalgesia, during opioid withdrawal was reported in several studies (reviewed in Angst and Clark 2006). Other studies show that transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), heat and cold sensors, respectively (Andrade et al. 2012; Khairatkar-Joshi and Szallasi 2009), are critically involved in hyperalgesia during inflammation (Andrade et al. 2012; Julius 2013) and postoperative pain (Pogatzki-Zahn et al. 2005; Wei et al. 2012). Therefore, antagonists of these channels stand out as potential analgesic therapeutics (Andrade et al. 2012; Khairatkar-Joshi and Szallasi 2009).
The medicinal plants of the genus Polygala belong to the Polygaceae family and have significant ethnopharmacological value as they have been empirically used for a long time to treat several diseases, especially for pain (Lapa et al. 2009; Meotti et al. 2006; Ribas et al. 2008) and inflammation (Arruda-Silva et al. 2014; Borges et al. 2014) relief. The Polygala molluginifolia (A. St.-Hil.) species is native to the Atlantic Forest. It is a small herb found in the grassland formations of southern Brazil (Arruda-Silva et al. 2014; Venzke et al. 2013) and is popularly known as "canfora". Few scientific data have been published on the biological activities of this herb before the present study. A recent study demonstrated by using phytochemical analysis that this species is rich in isoflavones with antioxidant potential (Venzke et al. 2013) and has a significant anti-inflammatory effects in the mouse pleurisy model (Arruda-Silva et al. 2014).
Despite much evidence regarding the genus Polygala, there is no study to date reporting the effects of the Polygala molluginifolia species on pain responses, or analyzing the mechanism through which it achieves its beneficial effects. Hence, the present study was designed to investigate the effect of postoperative pain treatment with the hydroalcoholic extract of Polygala molluginifolia (HEPm) and 5,3',4'-trihydroxy-6",6"-dimethylpyrano [2",3":7,6]isoflavone (ISO), an isoflavone isolated from the extract, in animal models of pain, such as formalin and plantar incision-induced postoperative pain. In addition, we analyzed the involvement of opioid receptors and TRPV1 and TRPA1 channels in the analgesic effect of HEPm and ISO.
2. Material and methods
2.1 Plant material, extraction and isolation of vegetal material
Polygala molluginifolia A. St.-Hil and Moq. was collected in Sao Lourenco do Sul (31[degrees]21'46"S and 51[degrees]58'54"W), Rio Grande do Sul State, Brazil, in January 2013. A voucher specimen was identified by Prof. Dra. Raquel Ltidtke (UFPel) and deposited by Prof. Dr. Rafael Trevisan at the Herbarium of Federal University of Santa Catarina (UFSC) under the number FLOR-48690.
The dried and powered whole plant material (85.3 g) was extracted with 96% ethanol at room temperature (four times a day over seven days). The resultant extracts were filtered and the ethanol was removed under reduced pressure to obtain the hydroalcoholic extract (14.84 g). Then, the hydroalcoholic extract (10.87 g) was solid-liquid partitioned with hexane and EtOAc, and the solvents were removed under reduced pressure. The residue from the partition was called water-soluble fraction. This process yielded three distinct fractions: hexane-soluble (0.73 g), EtOAc soluble (3.05 g) and water-soluble (6.23 g) fractions.
The EtOAc-soluble fraction underwent chromatographic fractionation on a silica gel column and eluted with increasing amounts of EtOAc in hexane to yield 35 fractions. The fractions obtained were monitored by TLC (Thin-layer chromatography) and revealed with sulfuric-anisaldehyde followed by heating. The similar fractions 12 and 13 were combined and purified with methanol to yield 5,3',4,-trihydroxy-6",6"-dimethylpyrano [2"3";7,6]isoflavone (ISO) 111.0 mg, as a yellow solid with a melting point of 165-167 [degrees]C. The structure of ISO was confirmed by comparing the results obtained from [sup.1]H and [sup.13]C with previous data described by our group (Arruda-Silva et al. 2014; Venzke et al. 2013).
2.2. Phytochemical analysis of the extract from Polygala molluginifolia
2.2.1. Capillary electrophoresis analysis of HEPm
The analyses were conducted using a capillary electrophoresis system (Agilent Technologies, Palo Alto, CA USA) equipped with a diode array detector set at 254 nm and data acquisition and treatment software (HP ChemStation) (Micke et al. 2006).
The internal temperature of the cartridge was set at 25[degrees]C and a fused-silica capillary (Polymicro, Phoenix, AZ, USA) of 48.5 cm (40 cm effective length) x 50 [micro]m inner diameter x 375 [micro]m outer diameter was used. The samples (HEPm and the isolated compounds co-injected with HEPm) were injected hydrodynamically (50 mbar for 5 s). The compounds were separated by constant voltage conditions of +25 kV in the injection side. Reagent p-nitrophenol was used as the internal standard. The concentration of the isolated compounds in HEPm was expressed as a percentage considering the total mass of isolated compounds in relation to the HEPm (% m/m).
2.2.2. Determination of total phenol content
The total phenolic content of the samples was determined using the Folin-Ciocalteu reagent (Moresco et al. 2014). In this method, the test solution was composed of 0.5 ml of the sample (HEPm diluted in methanol at a concentration of 1.0 mg/ml), 5.0 ml of distilled water and 0.5 ml of the Folin-Ciocalteu reagent. After a period of 3 mins, 1.0 ml of saturated sodium carbonate solution was added. These mixtures were shaken and allowed to stand for 1 h. The absorbance was measured at 725 nm in spectrophotometer (Perkin Elmer Lambda S) against a blank (solution without sample). The mean of three readings was used and expressed in mg of gallic acid/ g of HEPm.
2.2.3. Determination of total flavonoid content
The flavonoid content was determined spectrophotometrically according to Lamaison and Carnat (Nabavi et al. 2008). Briefly, 0.5 ml of 2% aluminum chloride (Al[Cl.sub.3]) in ethanol was mixed with the same volume of the sample (1.0 mg/ml). Absorption readings at 415 nm were taken after 1 h against a blank (methanol). The mean of three readings was used and expressed as mg of quercetin/ g of dry extract of HEPm.
A total of 528 2-month-old (25-35 g) male Swiss mice obtained from the animal facility of the Federal University of de Santa Catarina (Florianopolis, SC, Brazil) were housed in groups of 5 per cage at 22 [+ or -] 2[degrees]C and 60-80% humidity under a 12 h light/dark cycle (lights on at 06:00 h), with ad libitum access to standard laboratory diet and water. The animals were habituated to laboratory conditions for at least 1 h before testing, and all experiments were performed during the light phase of the cycle. The animals were randomly distributed between the experimental groups (5-10 animals per group), and the experimental procedures and protocols reported in this study were previously approved by the Ethics Committee for Animal Research of the Federal University of Santa Catarina (protocol number PP00745), where the study was conducted. Furthermore, all experiments were conducted in accordance with the National Institute of Health Animal Care Guidelines (NIH publications No. 80-23) and the ethical guidelines for investigations of experimental pain in conscious animals. The number of animals and intensities of noxious stimuli used were the minimum necessary to demonstrate the consistent effects of drug treatments. The animals used were euthanized by cervical dislocation immediately after the completion of the experiment.
2.4. Drugs and reagents
The following substances were used: formalin (Merck, Darmstadt, Germany): morphine sulfate (Uniao Quimica, Brazil); naloxone hydrochloride, cinnamaldehyde, camphor (Sigma-Aldrich, St. Louis, MO, USA): capsaicin (Tocris Bioscience, Ellisville, Missouri, USA) and isoflurane (Cristalia, SP, Brazil). All the drugs were dissolved in isotonic saline solution (0.9% NaCl) with the exception of the extract and the isolated isoflavone, which was dissolved in saline plus Tween 80. The final concentration of Tween 80 did not exceed 5% and did not cause any effect per se. Also, control groups animals were treated with vehicle (5% tween 80 in saline, 10ml/kg, i.g.) 1 h before each evaluation.
2.5. Formalin test
Formalin-induced nociception was measured as previously described (Lapa et al. 2009). Animals were injected with 20 [micro]l of a 2.5% formalin solution (0.92% formaldehyde in saline) intraplantarly (i.pl.) in the ventral surface of the right hindpaw. Animals were observed for 30 mins; the time between 0 and 5 mins represented the neurogenic phase and the time between 15 and 30 mins represented the inflammatory phase. Time spent licking the injected paw was recorded as an indicator of nociception. The animals were treated with HEPm (10-1000 mg/kg, i.g.) or ISO (10-100mg/kg, i.g.) 1 h before formalin injection in the right hindpaw. After the formalin injection, animals were immediately placed in individual glass cylinders (20-cm diameter) and the nociception was evaluated by the time spent licking the paw.
In addition, edema, local and systemic temperatures of the animals were verified before and after the observation time of the nociceptive response induced by intraplantar injection of formalin. The thickness difference (mm) between the right paw before and after formalin injection, was considered as an index of edema, measured from the central region of the paw, using an electronic digital micrometer 0-25 mm. The temperature difference ([degrees]C) between the ventral surface of the right paw, before and after formalin injection, was regarded as an index of the local temperature of the animals. The temperature difference ([degrees]C) of the right ear, before and after formalin test, was taken as an index of the systemic temperature in animals, verified by measuring the external acoustic meatus of the animal ear. For both evaluations, a Mallory-Pro Thermosensor (10[degrees]C-50[degrees]C) was used.
2.6. Plantar incision surgery
The plantar incision surgery (PIS) was performed as previously described by Pogatzki and Raja (2003). Briefly, mice were anesthetized with 1-2% isoflurane delivered via a nose cone. After sterile preparation of the right hind paw, a 5-mm longitudinal incision was made through the skin and fascia of the plantar surface using a number 11 scalpel blade. The incision started 2 mm from the proximal edge of the heel and extended toward the toes. The underlying muscle was elevated with curved forceps, leaving the muscle origin and insertion intact. After wound homeostasis, the skin was apposed with a 6.0 mm nylon mattress suture, and the wound was covered with 10% povidone-iodine solution. Sham-operated animals were anesthetized, but no incision was made. Animals were allowed to recover in their cages, and sutures were removed on the second postoperative day.
2.6.1 Assessment of mechanical hyperalgesia
The mice were individually placed in clear Plexiglas[R] boxes (9 x 7 x 11 [cm.sup.3]) on an elevated wire mesh (6 mm) platform (70 x 40 cm) to allow access to the ventral surface of the right hind paw. The withdrawal frequency was measured by the number of times (out of 10) the animal withdrew the paw after the 0.4 g von Frey filament (VFF) (Stoelting, Chicago, USA) was applied (Nucci-Martins et al. 2015). The results are expressed as the percentage of frequency of response. The animals were acclimatized for at least 1 h before the behavioral test. The day before surgery, the animals were subjected to testing to characterize the baseline response. Only animals that showed a response rate around 20% were selected.
To check if HEPm inhibits the mechanical hyperalgesia we investigated the antinociceptive effect, 24 h after PIS, the time course until the 6th hour after treatment with HEPm (30, 100 and 300 mg/kg, i.g.). Control animals received a similar volume of vehicle (10 ml/kg, i.g.) while sham-operated animals received vehicle (10 ml/kg, i.g.) or HEPm (300 mg/kg, i.g.). To investigate the effects of daily treatment with HEPm (30 and 300 mg/kg, i.g.), the treatment was repeated for 6 consecutive days immediately after PIS.
2.6.2. Measurement of thermal hyperalgesia
To assess thermal hyperalgesia to hot and cold stimulus in mice, the Hot/Cold Plate Analgesia Meter (Hot-Cold Plate, AVS Projetos, Campinas, SP, Brazil) was used with a minor modification of the method described by Nucci-Martins et al. (2015). Mice were placed in clear plastic chambers on the surface of the apparatus and the time (s) between placement and the shaking or licking of paws or jumping was recorded as the index of response latency. To analyze heat thermal hyperalgesia mice were placed on the hot plate (48 [+ or -] 1[degrees]C) and to analyze cold thermal hyperalgesia mice were placed on the cold plate (10 [+ or -] 1[degrees]C). Mice were treated with HEPm (300 mg/kg, i.g.) or vehicle (10 ml/kg, i.g.) 1 h beforehand. The thermal hyperalgesia was evaluated by the right hind paw withdrawal latency. The cut-off latency was 60 s for the hot plate test and was 120 s for the cold plate test. The hyperalgesic behavior was tested during the 1st and 2nd day after surgery.
2.7. Pain behavior induced by the intraplantar injection of TRPV1 and TRPAl agonists in mice
To evaluate the possible involvement of TRPV1 and TRPA1 channels on the antinociceptive effect of HEPm and ISO, mice were submitted to a test using either capsaicin or cinnamaldehyde, both specific activators of these channels, as previously described by Lapa et al. (2009) and Cordova et al. (2011). Briefly, the mice were pretreated with HEPm (10-300 mg/kg, i.g.), ISO (30 and 100 mg/kg, i.g.), vehicle (10 ml/kg, i.g.), 1 h prior, or subcutaneous camphor (a TRPA1 antagonist used as positive control, 7.6 mg/kg, s.c.), 30 min prior to the injection of 20 [micro]l of capsaicin (TRPV1 activator, 5 nmol/site, i.pl.) or cinnamaldehyde (TRPA1 activator, 10 nmol/site, i.pl.) in the plantar surface of the right hindpaw. Immediately after the capsaicin or cinnamaldehyde injection, animals were placed into clear observation chambers (9 x 11 x 13 cm) and the nociceptive response was evaluated as the time spent licking the injected paw during 5 min.
2.8. Pain behavior induced by the intrathecal injection of TRPV1 and TRPA1 agonists in mice
To evaluate the possible involvement of TRPV1 channels on the antinociceptive effect of HEPm, mice were subjected to a test using capsaicin, a specific activator of these channels, as previously described by Nucci-Martins et al. (2015). Furthermore, to evaluate the possible involvement of TRPA1 channels on the HEPm antinociceptive effect, dose-response curve (3-3000 pmoi/site, i.t.) standardization for evaluation in mice was carried out. According to the results, the best dose (30 pmol/site, i.t.) and time (30 min) were chosen in order to highlight the nociceptive behavior.
The intrathecal injections were given to fully conscious mice as previously described (Nucci-Martins et al. 2015). The animals were restrained manually and a 30-gauge needle connected by polyethylene tubing to a 25 [micro]1 microsyringe (Hamilton, Birmingham, UK) was inserted through the skin and between the vertebrae into the subarachnoid space of the L5-L6 spinal segments. The injections were given over a period of 5 s and the tail reflex movement was considered to be indicative of successful administration. Biting behavior was defined as a single head movement directed at the flanks or hind limbs, resulting in the contact of the animal's snout with the target organ.
The amount of time (seconds) that the animal spent biting or licking the caudal region was taken as evidence of nociception and was evaluated following local injections of one of the following agonists: capsaicin (TRPV1 activator, 100 pmol/i.t.) (5 min) or cinnamaldehyde (TRPAl activator, 30 pmol/i.t) (30 min). The animals received vehicle (10 ml/kg, i.g.), HEPm (10, 100 or 300 mg/kg, i.g.) or ISO (30 or 100 mg/kg, i.g.) 1 h before the intrathecal injection of 5 [micro]l of each drug.
2.9. Involvement of opioid receptors in HEPm and ISO antinociceptive effect
The involvement of the opioid system in the antinociceptive effect of HEPm and ISO was performed as previously described (Lapa et al. 2009). The mice subjected to intraplantar injection of 20 [micro]l of cinnamaldehyde (10 nmol/site, i.pl.) were pretreated with either: a systemic (i.p.) injection of saline (10 ml/kg) or naloxone (a nonselective opioid receptor antagonist, 1 mg/kg) 20 min before HEPm (300 mg/kg, i.g.), ISO (100 mg/kg, i.g.) or morphine (2.5 mg/kg, s.c.) treatment. The nociceptive response was evaluated as the time spent licking the injected paw during 5 min, 1 h after the treatment with HEPm or ISO or 30 min after treatment with morphine.
2.10. Measurement of locomotor activity
In order to rule out the false positive results in analgesia, the effect of HEPm and ISO on spontaneous locomotor activity was assessed in the Open-field Test (Lapa et al. 2009). The open field apparatus consisted of a wooden box measuring 40 x 60 x 50 cm. The floor of the arena was divided into 12 equal squares, and the number of squares crossed by the animal with all its paws was counted during a 6-min session. In the evaluation, the mice were treated with HEPm (10-1000 mg/kg, i.g.) or vehicle (10 ml/kg, i.g.) 1 h before the test.
2.11. Statistical analysis
The results were presented as mean [+ or -] standard error of the mean (S.E.M.). The [ID.sub.50] value was determined from an experiment using nonlinear regression GraphPad software (GraphPad Software, Inc., San Diego, CA). The Formalin, Capsaicin, Cinnamaldehyde, Naloxone, Hot and Cold Plates and Open-field Tests were performed by one way ANOVA followed by the Student-Newman-Keuls multiple comparison test. The results regarding postoperative pain were obtained by using the two-way analysis of variance (ANOVA) followed by the Bonferroni test for multiple comparisons. In all analyses, P values were considered statistically significant only if they were less than 0.05. The statistical software GraphPad Prism[R] v5.01 (San Diego, CA, USA) was used for calculation.
3.1. Phytochemical analysis of HEPm
In prior works (Arruda-Silva et al. 2014; Venzke et al. 2013) we described the chemical profile of HEPm by HPLC analysis and the isolation of pyranoisoflavones of ethyl acetate fraction of the species Polygala molluginifolia. In the present study we analyzed the profile by capillary electrophoresis analysis of a new extract prepared from this species.
The electrophoretic profile of HEPm was obtained by analysis at 254 nm (Fig. 1A). The electropherogram showed 5 significant peaks, in which 3 are majorities. It was possible to identify the pyrano isoflavone 5,3,'4,-trihydroxy-6",6"-dimethylpyrano [2",3":7,6] isoflavone (2), referred to by ISO, representing 0.34% of the HEPm, used to conduct the experiments in this work (Fig. 1A and IB). The other two majority compounds found in HEPm are 3',4'-dihydroxy-6",6",6'",6",-tetramethylbis(pyrano[2",3":5,6::2'",3'":7,8]isoflavone (1) and flavonoid rutin (3), representing 0.32% and 0.41% of the HEPm, respectively (Fig. 1A). All 3 majority compounds found in this work were also identified in previous studies (Arruda-Silva et al. 2014; Venzke et al. 2013).
In addition, quantitative colorimetric analysis showed that 11.09% of the compounds found in HEPm were phenols and 67.18% of these phenols were flavonoids (Table 1).
3.2. Acute HEPm or ISO administration reduces formalin-induced nociception
The results depicted in Figs. 2 and 3 (panel A and B) show that HEPm and ISO administered intragastrically caused significant inhibition of both the neurogenic and the inflammatory phases of formalin-induced licking. The calculated [ID.sub.50] values of HEPm for the first and second phases were 210.1 (60.4-730.6) mg/kg and 49.9 (29.5-84.2) mg/kg, respectively. The HEPm inhibitions observed for the first phase were 34 [+ or -] 5%, 39 [+ or -] 7%, 57 [+ or -] 7% and 59 [+ or -] 10% at doses of 10, 100, 300 and 1000 mg/kg, respectively (Fig. 2A) and, for the second phase, 60 [+ or -] 5%, 81 [+ or -] 8% and 86 [+ or -] 6% at doses of 100, 300 and 1000 mg/kg, respectively (Fig. 2B). The ISO inhibitions observed for the first phase were 27 [+ or -] 5% and 42 [+ or -] 6% (Fig. 3A) and, for the second phase, 40 [+ or -] 8% and 71 [+ or -] 5% (Fig. 3B), both at doses of 30 and 100mg/kg, respectively. The calculated [ID.sub.50] value of ISO for the second phase was 46.4 (33.4-64.3) mg/kg.
Furthermore, the HEPm and ISO also produced an antiedematogenic effect, at all doses tested, when compared to the control group. The HEPm (10-1000 mg/kg, i.g.) reduced paw edema caused by intraplantar formalin injection, with inhibitions of 35 [+ or -] 6%, 35 [+ or -] 2%, 29 [+ or -] 5% and 23 [+ or -] 8%, respectively (Fig. 2C). The ISO (10-100 mg/kg, i.g.) reduced edema with inhibitions of 39 [+ or -] 7%, 30 [+ or -] 5% and 51 [+ or -] 7%, respectively (Fig. 3C).
Moreover, the HEPm and ISO were also able to reduce hyperemia (increased temperature) caused by intraplantar formalin injection, at all doses tested, when compared to the control group. The HEPm (10-1000 mg/kg, i.g.) reduced hyperemia with inhibitions of 43 [+ or -] 6%, 30 [+ or -] 7%, 35 [+ or -] 8% and 36 [+ or -] 7%, respectively (Fig. 2D) and the ISO (10-100 mg/kg, i.g.) reduced hyperemia with inhibitions of 68 [+ or -] 6%, 54 [+ or -] 11% and 67 [+ or -] 7%, respectively (Fig. 3D). However, the HEPm and ISO did not promote change in body temperature or nonspecific hypothermic effect, when compared to the control group (results not shown).
3.3. Acute and daily treatment with HEPm reduces mechanical and thermal hyperalgesia after hind paw incision
3.3.1 Mechanical hyperalgesia
The results of Fig. 4 show that HEPm (30, 100 and 300 mg/kg, i.g.) caused a significant inhibition of plantar incision-induced mechanical hyperalgesia when compared to the control group. The effect was maintained 3 and 4h after treatment with doses of 30 and 300 mg/kg, respectively. Nevertheless, the effect of the dose of 100mg/kg was kept for only 2h. The observed inhibitions 1 h after treatment were 56 [+ or -] 7%, 62 [+ or -] 12% and 74 [+ or -] 14% at the doses of 30, 100 and 300 mg/kg, respectively (Fig. 4A). When administered daily for 6 days, HEPm (30 and 300 mg/kg, i.g.) significantly reduced the mechanical hyperalgesia caused by plantar incision with inhibitions varying between 27 [+ or -] 17% and 56 [+ or -] 7% at the dose of 30 mg/kg and 56 [+ or -] 6% to 75 [+ or -] 15% at the dose of 300 mg/kg (Fig. 4B).
3.3.2. Thermal hyperalgesia
The plantar incision surgery induced a decrease in paw withdrawal latency during thermal stimulus (heat and cold) in comparison to non-injured mice (Fig. 5). Intragastric pretreatment with HEPm (30 and 300 mg/kg) reduced the thermal hyperalgesia induced by the incision, increasing paw withdrawal latency of 80 [+ or -] 14% and 44 [+ or -] 11% for the heat (Fig. 5A) and 74 [+ or -] 24% and 104 [+ or -] 20% for the cold (Fig. 5B) assessments, respectively.
3.4. HEPm and ISO reduce capsaicin and cinnamaldehyde-induced peripheral nociception
We investigated the peripheral involvement of thermo-TRPs (Fig. 6) in the antinociceptive action of HEPm and ISO. Both treatment with HEPm and treatment with ISO inhibited the nociceptive behavior (licking) responses induced by capsaicin or cinnamaldehyde intraplantar injection (TRPV1 and TRPA1 activators, respectively) when compared to the control group. The inhibition values for the paw capsaicin test were 28 [+ or -] 5%, 31 [+ or -] 6% and 39 [+ or -] 7% with HEPm treatment at doses of 30, 100 and 300 mg/kg, respectively (Fig. 6A) and of 36 [+ or -] 7% and 50 [+ or -] 4% with ISO treatment at doses of 30 and 100 mg/kg, respectively (Fig. 6C). Furthermore, the inhibition values for paw cinnamaldehyde test were 30 [+ or -] 7%, 41 [+ or -] 7% and 48 [+ or -] 4% with HEPm treatment at doses of 30, 100 and 300 mg/kg, respectively (Fig. 6B) and of 45 [+ or -] 3% and 53 [+ or -] 2% with ISO treatment at doses of 30 and 100 mg/kg, respectively (Fig. 6D).
3.5. HEPm and ISO reduce capsaicin and cinnamaldehyde-induced central nociception
We investigated the central involvement of thermo-TRPs (Fig. 7) in the antinociceptive action of HEPm and ISO. Both the treatment with HEPm and the treatment with ISO inhibited the nociceptive behavior (licking/biting) responses induced by capsaicin or cinnamaldehyde intrathecal injection (TRPV1 and TRPA1 activators, respectively) when compared to the control group. The inhibition values for spinal capsaicin test were 29 [+ or -] 10% and 36 [+ or -] 5% with HEPm treatment at doses of 100 and 300 mg/kg, respectively (Fig. 7A) and of 32 [+ or -] 11% and 35 [+ or -] 6% with ISO treatment at doses of 30 and 100 mg/kg, respectively (Fig. 7C). Furthermore, HEPm and ISO were able to completely inhibit the pain caused by the intrathecal injection of cinnamaldehyde, at all tested doses. These findings show a strong central involvement of TRPA1 channels in the analgesic effect of HEPm and ISO (Fig. 7B and 7D, respectively). Similarly, the camphor (7.6 mg/kg, s.c.) positive control produced significant inhibition of 92 [+ or -] 7% (results not shown).
3.6. Involvement of opioid receptors in the systemic antinociceptive action of HEPm and ISO
In order to identify the opioid receptors in the HEPm or ISO action, naloxone (a non-selective antagonist of opioid receptors) was used. The antinociception induced by morphine (nonselective opioid receptor agonist, used as positive control) was reversed by pretreatment with naloxone (Fig. 8A). Similarly, as shown in Fig. 8, administration of naloxone (1 mg/kg, i.p.) significantly antagonized the antinociceptive effects of morphine, HEPm and ISO (panel A, B and C) when compared to the control group, after intraplantar injection of cinnamaldehyde. In addition, the inhibitions observed in the cinnamaldehyde test for treatments with morphine (2.5 mg/kg, s.c.), HEPm (300 mg/kg, i.g.) and ISO (100 mg/kg, i.g.) were 59 [+ or -] 8%, 51 [+ or -] 3% and 47 [+ or -] 4% (Fig. 8 panel A, B and C, respectively).
3.7. Evaluation of locomotor activity
Intragastric administration of either HEPm or ISO, at all doses tested, did not alter locomotor activity of animals in the Open-field Test when compared with the vehicle (control group). In animals treated with HEPm, the mean [+ or -] SEM for crossing number was 135 [+ or -] 6 for the control group and 133 [+ or -] 5; 127 [+ or -] 6; 142 [+ or -] 8; 116 [+ or -] 12 for the groups treated with HEPm: 10, 100, 300 and 1000 mg/kg, respectively. In animals treated with ISO, the mean [+ or -] SEM for crossing number was 142 [+ or -] 10 for the control group and 127 [+ or -] 6; 127 [+ or -] 7; 132 [+ or -] 8 for the groups treated with ISO: 10, 30 and 100 mg/kg, respectively.
The results of the present study demonstrated, for the first time, that systemic (oral) administration of hydroalcoholic extract of Polygala molluginifolia (HEPm) and an isoflavone (ISO) isolated from this extract elicits a potent dose-dependent inhibition of the nociceptive behavioral response in animal models of pain without causing sedation or locomotor dysfunction. This antinociceptive effect may be mediated, at least in part, by the activation of endogenous opioid receptors and/or by the inhibition of TRPV1 and TRPA1 channels.
Phytochemical studies of the genus Polygala reveal that the presence of xanthones (Bashir et al. 1992) and flavonoids (Lapa et al. 2009) is quite common, followed by coumarins (Hamburger et al. 1985; Meotti et al. 2006; Ribas et al. 2008), saponins (Li et al. 2006), lignans (Dall'Acqua et al. 2002) and more rarely, isoflavones (Bashir et al. 1992). The presence of isoflavones and the flavonoid rutin, including the majority 5,3',4'-trihydroxy-6",6"-dimethylpyrano [2",3'":7,6] isoflavone (Arruda-Silva et al. 2014; Venzke et al. 2013) was recently identified in the Polygala molluginifolia (A.St-Hil.) species. Venzke et al. (2013) also identified this presence and conducted experiments in this work, referred to by ISO. Furthermore, Venzke et al. (2013) also demonstrated antioxidant activity, acetylcholinesterase inhibition and PAMPA (parallel artificial membrane permeability assay) permeability to the four isoflavones isolated and identified from Polygala molluginifolia. According to these data, we also determined, spectrophotometrically, phenols and flavonoids that are typical for this plant species. It is well described in the literature that phenolic compounds, such as flavonoids, exhibit good antioxidant activity (Pietta 2000) and may effectively contribute to reducing pain and inflammation (Lapa et al. 2009).
Arruda-Silva et al. (2014) demonstrated that the anti-inflammatory action of Polygala molluginifolia in the model of pleurisy in mice was due to p65 phosphorylation inhibition in the NF-[kappa]B pathway and decreased levels and expression of pro-inflammatory cytokines, such as TNF-[alpha] and lL-1[beta]. Considering the anti-inflammatory profile of this medicinal plant, we investigated a possible antinociceptive effect of Polygala molluginifolia using the formalin test.
The formalin test is a classic tonic pain animal model for the preliminary study of drugs with analgesic and anti-inflammatory effects (Tjolsen et al. 1992). Injection of formalin into the hind paw induces a biphasic pain response; the first phase is neurogenic (early phase--F1) and results from direct activation of primary afferent sensory neurons, predominantly by C-fiber activation, whereas the second phase is inflammatory (late phase--F2) and appears to be dependent on the combination of an inflammatory reaction in the peripheral tissue and central sensitization in the dorsal horn of the spinal cord. These functional changes seem to be initiated by the C-fiber barrage during the early phase (Tjolsen et al. 1992). Thus, the early phase can be suppressed by the administration of centrally acting analgesics, such as morphine, whereas the late phase responds to various drugs with established clinical analgesic, such as opioids, steroidal and non-steroidal anti-inflammatory (Tjolsen et al. 1992). Furthermore, studies have demonstrated that TRPV1 and TRPA1 antagonists are also able to inhibit pain responses induced by formalin (McNamara et al. 2007). In addition, Endres-Becker et al. (2007) demonstrated that pi-opioid receptor activation can inhibit TRPV1 activity via [G.sub.i/0] proteins and the cAMP pathway in inflammatory pain. Thus, our results clearly demonstrated the ability of HEPm and ISO to inhibit both phases of the formalin test, leading us to think of a possible involvement of the opioid, TRPV1 and TRPA1 receptors in the analgesic/anti-inflammatory effect of Polygala molluginifolia.
Opiates are currently the mainstay for the treatment of moderate to severe pain. However, the abrupt termination of prolonged opioid application induces a withdrawal syndrome, including hyperalgesia in animals and in humans (Angst and Clark 2006). The opioid hypothesis was preliminarily investigated by attempting to reverse analgesia after administration of naloxone (opioid antagonist) (Hill 1981). In this regard, our results show that pre-administration of naloxone was able to counteract the antinociception of HEPm and ISO, clearly demonstrating an important involvement of Polygala molluginifolia in the endogenous opioid receptors activation to produce analgesia. In addition, these results indicate a possible interaction between the endogenous opioid receptors and the TRPA1 channel, since the animal's treatment with morphine was able to inhibit nociception induced by intraplantar injection of cinnamaldehyde (TRPA1 activator) and the pretreatment with naloxone reversed this effect.
A particularly intense research focus is TRP thermo-channels, because the hypersensitivity to pain that occurs over several pathological conditions is often a result of an increase in the expression and/or activity of TRPV1 or TRPA1 channels (Julius 2013). These channels can also be activated by several natural compounds, such as capsaicin, a major ingredient of chili pepper that activates TRPV1 (Khairatkar-Joshi and Szallasi 2009) and cinnamaldehyde from cinnamon, which activates TRPA1 (Andrade et al. 2012). Our results demonstrate that oral administration of either HEPm or ISO decreases nociceptive behavior induced by intraplantar and intrathecal injections of capsaicin (TRPV1 activator) and cinnamaldehyde (TRPA1 activator), highlighting the total (100%) spinal TRPA1 channel inhibition. Therefore, we suggest an important peripheral and central involvement of TRPV1 and TRPA1 channels in the analgesia produced by Polygala molluginifolia.
Neurotransmitters released by noxious stimuli may contribute to enhanced excitability following surgical injury. Pogatzki and Raja (2003) emphasize that the postoperative pain in humans can be mimicked by paw incisions in mice. More interesting data resulting from the present study is the demonstration, for the first time, that acute or daily treatment with HEPm significantly reduced the mechanical hyperalgesia and thermal hyperalgesia (heat and cold) in postoperative pain induced by plantar incision surgery (PIS), in mice. Furthermore, the antihyperalgesic effect of HEPm lasted up to 4 h after acute treatment and was not susceptible to tolerance and/or produced no cumulative effect, because HEPm maintained its efficacy when administered repeatedly. Taken together, our results clearly show that Polygala molluginifolia has therapeutic potential for the treatment of postoperative pain.
The literature describes the hot plate test as a very sensitive model for centrally acting drugs, like morphine and its derivatives (Lapa et al. 2009), being a neurogenic-modulated model that produces, at constant temperature, two kinds of behavioral responses; paw licking and jumping (for review see Chapman et al. 1985). Both of these are considered to be supraspinally-integrated responses. In our results, the HEPm inhibited the thermal hyperalgesia (heat) in the hot plate test after incision surgery, indicating that Polygala molluginifolia may be activating central opioid receptors. Furthermore, is well described in the literature that TRPV1 and TRPAl channels are sensitive to temperature, being activated by heat ([greater than or equal to] 43[degrees]C) and cold ([less than or equal to] 17[degrees]C), respectively (Andrade et al. 2012; Khairatkar-Joshi and Szallasi 2009). Honore et al. (2005) showed that A-425619 (TRPV1 antagonist), effectively relieves acute and chronic inflammatory pain and postoperative pain. Additionally, a dose of A-425619 dependently reduces capsaicin-induced mechanical hyperalgesia. Recently, Wei et al. (2012) demonstrated that Chembridge-5861528 (TRPAl antagonist), administered intaperitoneally, intraplantarly and intrathecally suppresses mechanical hypersensitivity after incision. Taken together, our results demonstrate that oral treatment with HEPm was able to inhibit thermal hyperalgesia to both heat and cold, making values that approach baseline levels. Thus, these data also reinforce the findings of acute experiments and confirms the involvement of TRPV1 and TRPAl channels in the analgesic effect of Polygala molluginifolia.
In addition, we observed that acute treatment with HEPm and ISO (10-100 mg/kg) did not affect the spontaneous locomotor activity of mice. These results suggest that animal's sensory and motor capabilities remained normal, excluding the possibility of nonspecific effects, such as sedation or motor dysfunction, on the antinociceptive effect of Polygala molluginifolia.
Finally, Maione et al. (2009) suggested a functional interaction between TRPV1 and [micro]-opioid receptors in the descending antinociceptive pathway to produce analgesia. Studies indicate that removing TRPV1-expressing primary afferent neurons potentiates the spinal analgesic effect of delta-opioid agonists on mechanonociception (Chen and Pan 2008). Furthermore, Vetter et al. (2006) demonstrated that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. However, Forster et al. (2009) demonstrated that nociceptor activation and sensitization by morphine is conveyed by TRPV1 and TRPA1. Taken together, these data can provide insights into interactions among opioid, vanilloid 1 and ankyrin 1 receptors. Such information may lead to the discovery of analgesics with little or no side effects and an improving treatment of pain of various etiologies, including postoperative pain. Thus, we suggested that the straight action of Polygala molluginifolia on central and peripheral pathways, observed in our results, may be dependent on opioid system activation and TRPV1 and TRPA1 channels inhibition.
In summary, the present study demonstrated, for the first time, the analgesic properties of the medicinal plant Polygala molluginifolia in animal models of acute pain, highlighting an important antinociceptive action, similar to other Polygala genus species. We evidenced the potential of HEPm in inhibiting mechanical and thermal hyperalgesia (heat and cold) in postoperative pain in mice, without causing sedation or locomotor dysfunction. Even though the precise mechanism by which Polygala molluginifolia promotes its beneficial effects is not completely known, we suggest that this may be mediated, at least in part, by the activation of endogenous opioid receptors and/or by inhibition of TRPV1 and TRPA1 channels, peripherally and centrally. Thus, this study adds new scientific evidence and highlights the potential of the medicinal plant Polygala molluginifolia in the development of phytomedicines with analgesic properties.
Received 20 May 2015
Revised 21 January 2016
Accepted 2 February 2016
Abbreviations: HEPm, hydroalcoholic extract of Polygala molluginifolia; ISO, 5 3'4'-trihydroxy-6"6"-dimethylpyrano [2"3":7 6]isoflavone; PIS, plantar incision surgery; Sham Animals that were anesthetized, but no incision was made; TRPV1, transient receptor potential vanilloid 1; TRPA1, transient receptor potential ankyrin 1; UFPel, Universidade Federal de Pelotas.
Conflict of interest
The authors declare that there are no conflicts of interest.
This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Fundacao de Apoio a Pesquisa Cientifica e Tecnologica do Estado de Santa Catarina (FAPESC), Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES).
Andrade, E.L., Meotti, F.C., Calixto, J.B., 2012. TRPA1 antagonists as potential analgesic drugs. Pharmacol. Ther 133 (2), 189-204.
Angst. M.S., Clark, J.D., 2006. Opioid-induced hyperalgesia: a qualitative systematic review. Anesthesiology 104 (3), 570-587.
Apfelbaum, J.L., Chen, C., Mehta, S.S., Gan, T.J., 2003. Postoperative pain experience: results from a national survey suggest postoperative pain continues to be undermanaged. Anesth. Analg 97 (2), 534-540.
Arruda-Silva, F., Nascimento, M.V.P.S., Luz, A.B.G., Venzke, D., Queiroz, G.S., Frbde, T.S., Pizzolatti, M.G., Dalmarco, E.M., 2014. Polygala molluginifolia A. St.-Hil. and Moq. prevent inflammation in the mouse pleurisy model by inhibiting NF-[kappa]B activation, int. Immunopharmacol 19 (2), 334-341.
Bashir, A., Hamburger, M., Msonthi, J.D., Hostettmann, K., 1992. Isoflavones and xanthones from Polygala virgata. Phytochemistry 31 (1), 309-311.
Benyamin, R., Trescot, A.M., Datta, S., Buenaventura, R., Adlaka, R., Sehgal, N., Glaser, S.E., Vallejo, R., 2008. Opioid complications and side effects. Pain Phys. 11 (2), S105-S120.
Borges, F.R., Silva, M.D., Cordova, M.M., Schambach, T.R., Pizzolatti, M.G., Santos, A.R., 2014. Anti-inflammatory action of hydroalcoholic extract, dichloromethane fraction and steroid alpha-spinasterol from Polygala sabulosa in LPS-induced peritonitis in mice. J. Ethnopharmacol 151 (1), 144-150.
Calixto, J.B., Beirith, A., Ferreira, J., Santos, A.R.S., Filho, V.C., Yunes, R.A., 2000. Naturally occurring antinociceptive substances from plants. Phytother. Res 14 (6), 401-418.
Cordova, M.M., Werner, M.F., Silva, M.D., Ruani, A.P., Pizzolatti, M.G., Santos, A.R.S., 2011. Further antinociceptive effects of myricitrin in chemical models of overt nociception in mice. Neurosci. Lett 495 (3), 173-177.
Chapman, C.R., Casey, K.L., Dubner, R., Foley, D.M., Graceley, R.H., Reading, A.E., 1985. Pain measurement: an overview. Pain 22, 1-31.
Chen, S.R., Pan, H.L., 2008. Removing TRPVl-expressing primary afferent neurons potentiates the spinal analgesic effect of delta-opioid agonists on mechanonociception. Neuropharmacology 55 (2), 215-222.
Dall'Acqua, S., Innocenti, G., Viola, G., Piovan, A., Caniato, R., Cappelletti, E.M., 2002. Cytotoxic compounds from Polygala vulgaris. Chem. Pharm. Bull (Tokyo) 50 (11), 1499-1501.
Endres-Becker, J., Heppenstall, P.A., Mousa, S.A., Labuz, D., Oksche, A., Schafer, M., Stein, C., Zollner, C., 2007. Mu-opioid receptor activation modulates transient receptor potential vanilloid 1 (TRPV1) currents in sensory neurons in a model of inflammatory pain. Mol. Pharmacol 71 (1), 12-18.
Forster, A.B., Reeh, P.W., Messlinger, K., Fischer, M.J., 2009. High concentrations of morphine sensitize and activate mouse dorsal root ganglia via TRPV1 and TRPA1 receptors. Mol. Pain 5, 17.
Haas, H., 1983. History of antipyretic analgesic therapy. Am. J. Med 75 (5A), 1-3.
Hamburger, M., Gupta, M., Hostettmann, K., 1985. Coumarins from Polygala paniculata. Planta Med 3, 215-217.
Hill, R.G., 1981. The status of naloxone in the identification of pain control mechanisms operated by endogenous opioids. Neurosci. Lett 21 (2), 217-222.
Honore, P., Wismer, C.T., Mikusa, J., Zhu, C.Z., Zhong, C., Gauvin, D.M., Gomtsyan, A., El Kouhen, R., Lee, C.H., Marsh, K., Sullivan, J.P., Faltynek, C.R., Jarvis, M.F., 2005. A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel transient receptor potential type V1 receptor antagonist, relieves pathophysiological pain associated with inflammation and tissue injury in rats. J. Pharmacol. Exp. Ther 314(1), 410-421.
Julius, D., 2013. TRP channels and pain. Ann. Rev. Cell Dev. Biol 29, 355-384.
Kehlet, H., Jensen, T.S., Woolf, C.J., 2006. Persistent postsurgical pain: risk factors and prevention. Lancet 367 (9522), 1618-1625.
Khairatkar-Joshi, N., Szallasi, A., 2009. TRPV1 antagonists: the challenges for therapeutic targeting. Trends Mol. Med 15 (1), 14-22.
Lapa, F.R., Gadotti, V.M., Missau, F.C., Pizzolatti, M.G., Marques, M.C., Dafre, A.L., Farina, M., Rodrigues, A.L., Santos, A.R.S., 2009. Antinociceptive properties of the hydroalcoholic extract and the flavonoid rutin obtained from Polygala paniculata L. in mice. Basic Clin. Pharmacol. Toxicol 104 (4), 306-315.
Li, T.Z., Zhang, W.D., Yang, G.J., Liu, W.Y., Chen, H.S., Shen, Y.H., 2006. Saponins from Polygala japonica and their effects on a forced swimming test in mice. J. Nat. Prod 69 (4), 591-594.
Maione, S., Starowicz, K., Cristino. L., Guida, F., Palazzo, E., Luongo, L., Rossi, F., Marabese, I., de Novellis, V., Di Marzo, V., 2009. Functional interaction between TRPVl and mu-opioid receptors in the descending antinociceptive pathway activates glutamate transmission and induces analgesia. J. Neurophysiol 101 (5), 2411-2422.
McNamara, C.R., Mandel-Brehm, J., Bautista, D.M., Siemens, J., Deranian, K.L., Zhao, M., Hayward. N.J., Chong, J.A., Julius, D., Moran, M.M., Fanger, C.M., 2007. TRPA1 mediates formalin-induced pain. Proc. Natl. Acad. Sci. U S A 104 (33), 13525-13530.
Meotti, F.C., Ardenghi, J.V., Pretto, J.B., Souza, M.M., d' Avila Moura, J., Junior, A.C., Soldi, C., Pizzolatti, M.G., Santos, A.R., 2006. Antinociceptive properties of coumarins, steroid and dihydrostyryl-2-pyrones from Polygala sabulosa (Polygalaceae) in mice. J. Pharm. Pharmacol 58 (1), 107-112.
Micke, G.A., Fujiya, N.M., Tonin, F.G., de Oliveira Costa, A.C., Tavares, M.F.M., 2006. Method development and validation for isoflavones in soy germ pharmaceutical capsules using micellar electrokinetic chromatography. J. Pharm. Biomed. Anal 41 (5), 1625-1632.
Moresco, H.H., Pereira, M., Bretanha, L.C., Micke, G.A., Pizzolatti, M.G., Brighente, I.M.C., 2014. Myricitrin as the main constituent of two species Of Myrcia. J. Appl. Pharm. Sci 4 (2), 1-7.
Nabavi, S.M., Ebrahimzadeh, MA, Nabavi, S.F., Hamidinia, A., Bekhradnia, A.R., 2008. Determination of antioxidant activity, phenol and flavonoid content of Parrotia persica Mey. Pharmacologyonline 2, 560-567.
NationalCenterforHealth, Statistics, 2010. FastStats: inpatient surgery. Centers for Disease Control and Prevention--CDC. USA http://www.cdc.gov/nchs/fastats/ inpatient-surgety.htm.
Nucci-Martins, C., Martins. D.F., Nascimento, L.F., Venzked, D., Oliveira, A.S., Frederico, M.J.S., Silva, F.R.M.B., Brighented, I.M.C., Pizzolatti, M.G., Santos, A.R.S., 2015. Ameliorative potential of standardized fruit extract of Pterodon pubescens Benth on neuropathic pain in mice: evidence for the mechanisms of action. J. Ethnopharmacol. (in press).
Pietta, P.-G., 2000. Flavonoids as antioxidants. J. Nat. Prod 63 (7), 1035-1042.
Pogatzki-Zahn, E.M., Schnabel, A., Zahn, P.K., 2012. Room for improvement: unmet needs in postoperative pain management. Expert Rev. Neurother 12 (5), 587600.
Pogatzki-Zahn, E.M., Shimizu, I., Caterina, M., Raja, S.N., 2005. Heat hyperalgesia after incision requires TRPV1 and is distinct from pure inflammatory pain. Pain 115 (3), 296-307.
Pogatzki, E.M., Raja, S.N., 2003. A mouse model of incisional pain. Anesthesiology 99 (4), 1023-1027.
Ribas, C.M., Meotti, F.C., Nascimento, F.P., Jacques, A.V., Dafre, A.L., Rodrigues, A.L., Farina, M., Soldi, C., Mendes, B.G., Pizzolatti, M.G., Santos, A.R., 2008. Antinociceptive effect of the Polygala sabulosa hydroalcoholic extract in mice: evidence for the involvement of glutamatergic receptors and cytokine pathways. Basic Clin, Pharmacol. Toxicol 103 (1), 43-47.
Sen, T., Samanta, S., 2014. Medicinal Plants, Human Health and Biodiversity: A Broad Review. Springer,, Berlin, Heidelberg, pp. 1-52 chapter 273, pp.
Tjolsen, A., Berge, O.-G., Hunskaar, S., Rosland, J.H., Hole, K., 1992. The formalin test: an evaluation of the method. Pain 51 (1), 5-17.
Venzke, D., Carvalho, F.K., Ruani, A.P., Oliveira, A.S., Brighente, I., Micke, G.A., Barison, A., Pizzolatti, M.G., 2013. PAMPA permeability, acetylcholinesterase inhibition and antioxidant activity of pyranoisoflavones from Polygala molluginifolia (polygalaceae). J. Braz. Chem. Soc. 24 (12), 1991-1997.
Vetter, 1., Wyse, B.D., Monteith, G.R., Roberts-Thomson, S.J., Cabot, P.J., 2006. The mu opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway. Mol. Pain 2, 22.
Wei, H., Karimaa, M., Korjamo, T., Koivisto, A., Pertovaara, A., 2012. Transient receptor potential ankyrin 1 ion channel contributes to guarding pain and mechanical hypersensitivity in a rat model of postoperative pain. Anesthesiology 117 (1), 137-148.
Catharina Nucci-Martins (a,b), Leandro F. Nascimento (a,b), Dalila Venzke (c), Lizandra C. Brethanha (c), Alysson V.F. Sako (c), Aldo S. Oliveira (c), Ines M.C. Brighente (c), Gustavo A. Micke (c), Moacir G. Pizzolatti (c), Adair R.S. Santos (a,b),*
(a) Laboratory of Neurobiology of Pain and Inflammation, Department of Physiological Sciences, Center of Biological Sciences, Federal University of Santa Catarina, Trindade, Florianopolis, SC 88040-900, Brazil
(b) Graduate Program in Neuroscience, Center of Biological Sciences, Federal University of Santa Catarina, SC 88040- 900, Florianopolis, Brazil
(c) Department of Chemistry, Center of Physical and Mathematical Sciences, Federal University of Santa Catarina, Trindade, Floriandpolis, SC 88040-900, Brazil
* Corresponding author at; Department of Physiological Sciences, Center of Biological Sciences, Federal University of Santa Catarina, Trindade, Floriandpolis, SC 88040-900, Brazil. Tel:+55 48 3721 4685; fax:+55 48 3721 9672.
E-mail address: firstname.lastname@example.org, email@example.com (A.R.S. Santos).
Table 1 Total phenol and flavonoid content of extract of Polygala molluginifolia. Sample Total phenol Total flavonoid content (a) content (b) Extract of P. 110.86 [+ or -] 2.31 74.48 [+ or -] 0.12 molluginifolia (HEPm) (a) Results in mg of gallic acid/g of HEPm. (b) Results in mg of quercetin/g of HPEm. Each value is the mean [+ or -] SD of three independent measurements.
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|Author:||Nucci-Martins, Catharina; Nascimento, Leandro F.; Venzke, Dalila; Brethanha, Lizandra C.; Sako, Alys|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||May 15, 2016|
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