The transient receptor potential vanilloid 1 (TRPV1) receptor is relevant to the perception of noxious information and has been studied as a therapeutic target for the development of new analgesics. The goal of this study was to perform in vivo and in vitro screens to identify novel, efficacious, and safe TRPV1 antagonists isolated from leaves of the medicinal plant Vernonia tweedieana Baker. All of the fractions and the hydroalcoholic extract produced antinociception in mice during the capsaicin test, but the dichloromethane fraction also had antioedematogenic effect. Among the compounds isolated from the dichloromethane fraction, only α-spinasterol reduced the nociception and edema induced by capsaicin injection. Moreover, α-spinasterol demonstrated good oral absorption and high penetration into the brain and spinal cord of mice. α-Spinasterol was able to displace [3H]resiniferatoxin binding and diminish calcium influx mediated by capsaicin. Oral administration of the dichloromethane fraction and α-spinasterol also produced antinociceptive effect in the noxious heat-induced nociception test; however, they did not change the mechanical threshold of naive mice. The treatment with α-spinasterol did not produce antinociceptive effect in mice systemically pretreated with resiniferatoxin. In addition, α-spinasterol and the dichloromethane fraction reduced the edema, mechanical, and heat hyperalgesia elicited by complete Freund's adjuvant paw injection. The dichloromethane fraction and α-spinasterol did not affect body temperature or locomotor activity. In conclusion, α-spinasterol is a novel efficacious and safe antagonist of the TRPV1 receptor with antinociceptive effect.
The transient receptor potential vanilloid type 1 (TRPV1) receptor is a member of the TRP family (Caterina et al., 1997). It is composed of six transmembrane domains that form a nonselective cation channel (principally for calcium) (Schumacher, 2010). The TRPV1 receptor is expressed mainly in the nervous system and is particularly abundant in C and Aδ nociceptive fibers, where it plays a key role in the detection of noxious painful stimuli (Szallasi et al., 2007). It is activated by exogenous substances, such as capsaicin (the active component of chili peppers), resiniferatoxin (RTX; isolated from Euphorbia resinifera), endogenous inflammatory agents, extracellular protons, and noxious heat (>43°C) (Schumacher, 2010).
Because the TRPV1 receptor plays an important role in the detection and integration of noxious stimuli and is sensitized by different mechanisms, including various inflammatory mediators, it is possible to observe the participation of this receptor on diverse painful pathologies including inflammatory, visceral, and cancer pain (Adcock, 2009; Wong and Gavva, 2009). For these reasons, the TRPV1 has been studied as a relevant therapeutic target for the development of novel analgesics (Wong and Gavva, 2009). Antagonists of TRPV1 have been reported to produce antihyperalgesic effects in animal pain models and human diseases (Rami et al., 2006; Chizh et al., 2007; Lehto et al., 2008). However, some of these antagonists are implicated in the development of severe hyperthermia (Gavva et al., 2008). Thus, novel TRPV1 antagonists without a hyperthermic effect should be explored as new therapeutic agents for the treatment of pain (Wong and Gavva, 2009).
Natural products have been an important source for new drugs, including analgesic molecules (Calixto et al., 2000; Koehn and Carter, 2005). Products isolated from plants that are able to modulate the TRPV1 receptor, such as capsaicin, could be used to treat pain and symptoms of respiratory illness (Corson and Crews, 2007; Adcock, 2009; Schumacher, 2010). Vernonia species are herbaceous plants found all over the world and used as analgesics and antitussives (Iwalewa et al., 2003; Njan et al., 2008; Risso et al., 2010). Some studies have also reported the antinociceptive and anti-inflammatory effects of these species (Iwalewa et al., 2003; Njan et al., 2008; Risso et al., 2010). The species Vernonia tweedieana Baker is widely distributed on the plains of Paraguay, Argentina, and Southern Brazil, and its leaves are used in Brazilian folk medicine to treat cough (Zanon et al., 2008). However, there are no studies evaluating the analgesic potential of V. tweedieana or investigating the compounds and the mechanism responsible for its biological actions.
Because the expression, activation, and modulation of TRPV1 in sensory neurons seem to be an integral component of both pain and cough pathways (Adcock, 2009), and several Vernonia species are used as analgesics or antitussives and possess antinociceptive or anti-inflammatory effects, we postulated that V. tweedieana may be a potential source of TRPV1 antagonists with analgesic-like activity. Thus, as part of our ongoing research to identify phytochemicals isolated from natural products that can act as novel TRPV1 antagonists, we performed in vivo and in vitro screens to identify novel, efficacious, and safe TRPV1 antagonists contained in V. tweedieana.
Materials and Methods
Plant Material, Extraction, and Isolation Procedures
V. tweedieana Baker leaves were collected in March 2004 in Ijuí, Rio Grande do Sul, Southern Brazil. A voucher herbarium specimen was deposited at the herbarium of the Federal University of Santa Maria, Brazil under number SMDB 9536. The leaves were dried in a stove with circulating air at 40°C and pulverized in a mill. Dried and powdered leaves (1900 g) were extracted by maceration with 65% ethanol in water at room temperature for 7 days. The extract was filtered, and the ethanol was removed under reduced pressure. The hydroalcoholic extract (HE) was successively fractionated by using solvents with increasing polarity: dichloromethane (Dcm), ethyl acetate (Act), and n-butanol (But) (4 × 200 ml for each solvent). The extract and fractions were concentrated with a rotary evaporator (Buchi Laboratory Equipment, Flawil, Switzerland) at a temperature not exceeding 40°C to yield the Dcm (30.0 g; 1.58%), Act (25.62 g; 1.35%), and But (55.85 g; 2.94%) soluble fractions. A mixture of α- and β-amyrin, lupeol, stigmasterol, and α-spinasterol was isolated from the Dcm fraction at a concentration of 0.001% (w/w) each as described previously (Zanon et al., 2008).
Male albino Swiss mice (25–35 g) bred in-house were used. Animals were housed in a controlled environment (22 ± 2°C) with a 12-h light/dark cycle (lights on 6:00 AM to 6:00 PM) and fed standard laboratory chow and tap water ad libitum. The animals were acclimated to the experimental room for at least 1 h before the experiments. Each animal was used only once. All protocols were approved by Ethics Committee of the Federal University of Santa Maria (process number 087/2011) and in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). The number of animals and intensity of noxious stimuli used were the minimum necessary to demonstrate the consistent effects of the drug treatments in accordance with current ethical guidelines for the investigation of experimental pain in conscious animals (Zimmermann, 1983).
In Vitro Screening of TRPV1 Antagonists
[3H]RTX Binding Assay.
To determine whether the Dcm fraction or its individual components were capable of binding the TRPV1 receptor, we performed the [3H]RTX binding assay as described previously (Rossato et al., 2011). In brief, mouse spinal cords were homogenized in buffer A (5 mM KCl, 5.8 mM NaCl, 2 mM MgCl2, 0.75 mM CaCl2, and 137 mM sucrose, pH 7.4) with 10 mM HEPES and centrifuged for 10 min at 1000g at 4°C, and the supernatant was further centrifuged for 30 min at 35,000g at 4°C. The resulting pellets were resuspended in buffer A and frozen until analysis. The binding mixture containing buffer A [plus 0.25 mg/ml of bovine serum albumin (BSA)], membranes (0.5 mg/ml of protein), and [3H]RTX (2 nM) in the presence or absence of the isolated compounds (10 μM), Dcm fraction (0.1 mg/ml), or vehicle [0.1% dimethyl sulfoxide (DMSO)] with a final volume of 500 μl. The α-spinasterol was also tested in different concentrations (3–100 μM). For the measurement of nonspecific binding, 100 μM nonradioactive RTX was included in different tubes. The reaction was initiated by incubating the tubes at 37°C for 60 min and stopped by transferring the tubes to an ice bath and adding 100 μg of bovine α1-acid glycoprotein to allow the detection of specific binding. Finally, [3H]RTX in the bound and free membranes was separated by centrifugation for 30 min at 35,000g at 4°C. Radioactivity in the pellet was quantified by scintillation. The pellets were suspended in 1 ml of scintillation fluid, and radioactivity was counted in a scintillator apparatus (Tri-Carb 2100TR; PerkinElmer Life and Analytical Sciences, Waltham, MA). Specific binding was calculated as the difference of the total and nonspecific binding, and the results are reported as percentage of specific binding. Total protein was measured with Coomassie blue dye, and BSA was used as a standard (Bradford, 1976).
Calcium (Ca2+) Influx Assay.
After evaluating the ability of selected compounds to bind the TRPV1 receptor, we estimated their capacity to affect capsaicin-induced calcium influx in synaptosomes (Rossato et al., 2011). Mouse spinal cords were homogenized in assay buffer (50 mM phosphate buffer and 320 mM sucrose, pH 7.4) and centrifuged for 5 min at 1000g at 4°C. Then the supernatant was centrifuged for 20 min at 10,000g at 4°C. The final pellet was resuspended in Krebs-Ringer buffer (Ca2+-free) at a final protein concentration of 1 mg/ml and incubated with Fura-2/AM (10 μM) for 30 min at 37°C. The samples were centrifuged for 30 s at 12,000g, and the final pellet was resuspended in 1.5 ml of Krebs-Ringer medium (Ca2+-free). To start the reaction, 15 μl of 0.1 M CaCl2 was added to each sample, and after 10 min, different concentrations of α-spinasterol (10, 30, 100, and 300 μM), N-(3-methoxyphenyl)-4-chlorocinnamide (SB-366791) (1 μM, used as a positive control), or vehicle (0.1% DMSO) were added, followed after 1 min by the addition of 15 μl of capsaicin (20 μM). Ca2+ influx was measured by monitoring the fluorescence at 382 nm (excitation) and 505 nm (emission) in a spectrofluorimeter (RF-5301 PC; Shimadzu, Kyoto, Japan). We have also analyzed the possible Ca2+ influx induced only by α-spinasterol (100 μM), SB-366791 (1 μM; used as a positive control), or vehicle (0.1% DMSO). Background fluorescence was determined by using an equivalent sample of synaptosomes that were not loaded with Fura-2/AM. Calibration was performed by recording the maximal and minimal fluorescence values after adding 15 μl of 10% (w/s) Triton X-100 at the end of each experiment. The results are expressed as the percentage of the maximum response obtained with Triton X-100 and then were compared with the capsaicin influx (Rossato et al., 2011).
In Vivo Screening of TRPV1 Antagonists
First, to verify the possible antinociceptive and antioedematogenic effects of V. tweedieana hydroalcoholic extract and fractions, mice received oral administration of the HE (100 mg/kg), Dcm (100 mg/kg), Act (100 mg/kg), and But (100 mg/kg) fractions or vehicle (10 ml/kg) 1 h before the capsaicin test. The extract and fractions were dissolved in a solution of 5% Tween 80, 20% polyethylene glycol, and 75% saline (0.9% NaCl). We evaluated the possible antinociceptive and antioedematogenic effects of isolated compounds from the Dcm fraction (mixture of α- and β-amyrin, lupeol, stigmasterol, and α-spinasterol) in a separate experiment. Compounds (0.3 μmol/kg p.o.) or vehicle (10 ml/kg; 5% Tween 80 and 20% polyethylene glycol in saline) were administered 1 h before the capsaicin test. The doses were chosen based on their concentration (0.001%) in the Dcm fraction.
Afterward, we generated a dose-response curve for α-spinasterol in the capsaicin test. Animals were treated with α-spinasterol (0.03–1 μmol/kg p.o.) or vehicle 1 h before capsaicin injection. Control animals received a similar volume of vehicle solution (10 ml/kg p.o.). To determine the time-course curve, α-spinasterol (0.3 μmol/kg p.o.) was administered in separate groups of animals, and its antinociceptive effect was assessed at different times after administration (0.5, 1, 2, and 4 h). The selective TRPV1 receptor antagonist SB-366791 (3 μmol/kg p.o.) was used as a positive control, and its antinociceptive and antioedematogenic effects were evaluated 1 h after administration in dose-response curves or 0.5, 1, 2, and 4 h after treatment for the time-response curves. The isolated compounds and SB-366791 were dissolved in a solution of 5% Tween 80, 20% polyethylene glycol, and 75% saline (0.9% NaCl). The SB-366791 dose was based on a previous report (Niiyama et al., 2009).
In the other nociceptive tests, measures were assessed at 0.5, 1, 2, and 4 h after the administration of vehicle (10 ml/kg p.o.), Dcm fraction (100 mg/kg p.o), α-spinasterol (0.3 μmol/kg p.o.), or SB-366791 (3 μmol/kg p.o.; used as positive control). In the desensitization protocol of TRPV1-positive fibers we used morphine (10 mg/kg s.c.) as the positive control.
The motor performance (open-field and rotarod tests) and body temperature were evaluated 1 h after administration of Dcm fraction (100 mg/kg p.o.), α-spinasterol (0.3 μmol/kg p.o.), or vehicle (10 ml/kg p.o.).
Capsaicin-Induced Spontaneous Nociception and Edema Test.
The capsaicin test was used to screen new TRPV1 compounds because it is capable of inducing nociception in experimental animals and humans. Furthermore, it has the advantage of being a simple and short method to measure nociception, and nociception intensity (and animal discomfort) can be reduced by using submaximal doses of capsaicin (Simone et al., 1989; Sakurada et al., 1992; Walker et al., 1999). The intraplantar capsaicin test was carried out as described previously (Sakurada et al., 1992). After the acclimation period, 20 μl of capsaicin (1 nmol/paw) was subcutaneously injected under the dorsal surface of the right hind paw (intraplantarly). The animals were individually observed for 5 min after capsaicin injection. The amount of time spent licking or biting the injected paw was timed with a chronometer and used as a measure of nociception. Vehicle (0.15% ethanol in saline and 0.9% NaCl) was prepared and used as a control for the capsaicin experiments. Treatment with vehicle did not evoke nociception behavior (data not shown).
We also evaluated edema that developed 15 min after capsaicin injection (1 nmol/paw intraplantarly) as described previously (Milano et al., 2008b). Edema formation was described as Δ = test paw thickness − basal paw thickness. Capsaicin-induced edema and nociception were observed in the same group of animals.
Heat Stimulus-Induced Nociception.
The paw reaction test to a heat stimulus was performed as described previously (Hargreaves et al., 1988; Rossato et al., 2011). A radiant light beam from a 60-W light bulb was directed onto the right hind paw (99% of the total intensity was used). The time between the onset of the stimulus and paw withdrawal was measured and used as an index of the thermal nociceptive threshold. The baseline latency was determined before the test, and a maximum latency of 30 s was imposed to prevent tissue damage.
Mechanical Stimulus-Induced Nociception.
The mechanical threshold was measured by using von Frey filaments in the up-and-down paradigm as described previously (Dixon, 1980; Rossato et al., 2011). First, mice were acclimated (1 h) in individual clear Plexiglas boxes (9 × 7 × 11 cm) on an elevated wire mesh platform to allow access to the plantar surface of the hind paws. Then, the paw was touched with a series of seven von Frey hairs in logarithmic increments of force (0.02, 0.07, 0.16, 0.4, 1.4, 4.0, and 10.0g). The von Frey hairs were applied perpendicular to the plantar surface with sufficient force to cause slight buckling against the paw and held for approximately 2 to 4 s. Absence of paw lifting after 5 s led to the use of the next filament with increased weight, whereas paw lifting indicated a positive response and led to the use of the next weaker filament. This paradigm continued until six measurements were collected or four consecutive positive or negative responses occurred. The 50% mechanical paw withdrawal threshold (PWT; in g) response was then calculated from these scores as described previously (Dixon, 1980; Rossato et al., 2011).
Complete Freund's Adjuvant-Induced Inflammatory Nociception.
To induce the development of inflammatory heat and mechanical hyperalgesia and edema, mice were lightly anesthetized with halothane, and CFA (20 μl; 1 mg/ml of the heat-killed Mycobacterium tuberculosis) was injected intraplantarly (Rossato et al., 2011). The assessment of the mechanical threshold, paw withdrawal latency (s), to a heat stimulus and edema were performed as described above. Basal values were observed before the injection of CFA, and the development of heat hyperalgesia, mechanical hyperalgesia, and edema was observed 48 h after CFA injection.
Desensitization of TRPV1-Positive Fibers.
To further explore the role of TRPV1-positive fibers in the antinociceptive effect of α-spinasterol in the paw reaction test to a heat stimulus, animals were submitted to a systemic desensitization protocol with RTX as described previously (Pecze et al., 2009). Pecze et al. (2009) previously demonstrated that 50 μg/kg s.c. of RTX largely reduced the expression of TRPV1 as well as increased noxious heat latencies and abolished capsaicin-induced eye irritation 7 days after treatment in adult mice. Moreover, they also observed that greater doses of RTX caused mice mortality.
Animals were anesthetized by using a mixture of ketamine (90 mg/kg) and xylazine (3 mg/kg) before systemic administration of RTX (50 μg/kg s.c.) or vehicle (0.5% ethanol and 0.5% Tween 80 in saline). The baseline latency was determined before RTX injection, and 7 days after the test, a maximum latency of 30 s was imposed to prevent tissue damage. The time between the onset of the heat stimulus and the paw reaction latency was automatically measured and taken as an index of the thermal nociceptive threshold as described above. In addition, we performed the capsaicin nociceptive test (as described above) 7 days after the injection of RTX or vehicle to confirm the reduction of TRPV1 expression.
To determine the effect of systemic RTX treatment on TRPV1 receptor expression in sensory nerves, we performed a Western blot analysis on RTX- and vehicle-treated animals (Gewehr et al., 2011). The sciatic nerves were quickly isolated and homogenized in lysis buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM NaF, 10 μg/ml aprotinin, 10 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dl-dithiothreitol, and 2 mM sodium orthovanadate. After centrifugation (3000g for 30 min at 4°C), the supernatant was collected. The protein content was determined by using BSA as a standard (Bradford, 1976). Sciatic nerve protein (30 μg) was mixed with loading buffer (200 mM Tris, 10% glycerol, 2% SDS, 2.75 mM β-mercaptoethanol, and 0.04% bromophenol blue) and boiled for 10 min. The proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes, according to the manufacturer's instructions (PerkinElmer Life and Analytical Sciences). The proteins on the polyvinylidene difluoride membrane were stained with a solution of 0.5% Ponceau and 1% glacial acetic acid in water, and this served as the loading control (Romero-Calvo et al., 2010). After staining, the membranes were dried, scanned, and quantified. Membranes were processed by using the SNAP I.D. system (Millipore Corporation, Billerica, MA), blocked with 1% BSA and 0.05% Tween 20 in Tris-borate saline (TBS-T) and then incubated for 10 min with an anti-TRPV1 antibody at room temperature (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:150 in TBS-T. Blots were washed three times with TBS-T, followed by incubation with an alkaline phosphatase-coupled secondary antibody (1:3000, anti-rabbit IgG; Santa Cruz Biotechnology, Inc.) for 10 min at room temperature. Protein bands were visualized with a 5-bromo-4-chloro-3-indolyl phosphate/p-nitro blue tetrazolium system (Millipore Corporation). The membranes were dried, scanned, and quantified with the Scion Image version of National Institutes of Health Image (Scion Corporation, Frederick, MD).
Evaluation of Different Adverse Effects Possibly Associated with Dcm Fraction or α-Spinasterol Treatment
To evaluate possible nonspecific muscle-relaxant or sedative effects we examined spontaneous motor coordination at the open-field test (Rossato et al., 2011). The apparatus consisted of a wooden box measuring 40 × 60 × 50 cm. The floor of the arena was divided into 12 equal squares, and the number of squares crossed with all paws was counted in a 5-min session.
Forced motor activity was also evaluated by using the rotarod test (Rossato et al., 2011). In brief, 24 h before the experiments, all animals were trained on the rotarod (3.7 cm in diameter; 8 rpm) until they could remain in the apparatus for 60 s without falling. The number of falls and latency to first fall from the apparatus were recorded up to 240 s.
Severe hyperthermia is a previously reported side effect of TRPV1 receptor antagonists. The difference between the preinjection and postinjection values was calculated (Δ°C) as described previously (Rossato et al., 2011). The TRPV1 antagonist [(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide] (AMG-9810) (30 μmol /kg p.o.) was used as a positive control.
Determination of the α-Spinasterol Pharmacokinetic Profile.
For the study of the pharmacokinetic profile of α-spinasterol (0.3 μmol/kg p.o.), plasma, brain, and spinal cord samples were collected 0.5, 1, 2, 4, and 6 h after drug administration in mice (Zhao et al., 2010; Watabiki et al., 2011). The samples of brain and spinal cord were homogenized in 100 mM phosphate buffer, pH 7.4, and the protein samples were precipitated with acetone. After centrifugation (10,000g; 10 min; 4°C), the supernatant was separated and used for analysis. The mean plasma-to-brain and plasma-to-spinal cord ratios were calculated from drug concentrations in each tissue 1 h after drug administration.
High-performance liquid chromatography (HPLC-diode array detection) was performed with the Prominence Autosampler (SIL-20A) HPLC system (Shimadzu), equipped with Shimadzu LC-20AT reciprocating pumps connected to the DGU 20A5 degasser with the CBM 20A integrator, diode array detector (photodiode) SPD-M20A UV-VIS detector, and LC solution 1.22 SP1 software. Reverse-phase chromatographic analyses were carried out under isocratic conditions by using a C18 column (4.6 × 250 mm) packed with 5-μm diameter particles. The mobile phase was methanol/0.5% aqueous H3PO4 (88:12, v/v), following the method described by Zou and Chen (2008) with slight modifications. The chromatography peak was confirmed by comparing its retention time with that of a reference standard and by diode array detector spectra (190–400 nm). The flow rate was 0.8 ml/min, the injection volume was 40 μl, and the wavelength was 210 nm. All of the samples and the mobile phase were filtered through a 0.45-μm membrane filter (Millipore Corporation) and then degassed in an ultrasonic bath before use. A stock solution of spinasterol standard was prepared in the HPLC mobile phase at a concentration range of 0.018 to 0.606 nmol/ml. The calibration curve for spinasterol was Y = 461482x + 4284.1 (r = 0.9994). All chromatography operations were carried out in triplicate at ambient temperature.
Drugs and Reagents
Capsaicin was purchased from Sigma (St. Louis, MO), dissolved in 90% ethanol and 10% Tween 80, and diluted to the appropriate concentration in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate buffer). Methanol and phosphoric acid were purchased from Merck (Darmstadt, Germany). BSA, bovine α1-acid glycoprotein, CFA, Fura-2/AM, HEPES, Triton X-100, morphine, resiniferatoxin, SB-366791, and AMG-9810 were purchased from Sigma. [3H]RTX was purchased from PerkinElmer Life and Analytical Sciences and diluted in assay buffer immediately before use.
The results are presented as the mean ± S.E.M., except for the ID50 and IC50 values, which are reported as geometric means accompanied by their respective 95% confidence limits. The ID50 and IC50 values were determined by nonlinear regression analyses with a sigmoid dose-response equation using GraphPad 5.0 (GraphPad Software, Inc., San Diego, CA). The percentages of maximal inhibition (Imax) are reported as the mean ± S.E.M. of inhibition obtained in each individual experiment in relation to the control values (vehicle for the in vivo results, 100% specific binding for the [3H]RTX binding assay, and 100% response obtained with Triton X-100 for the Ca2+ influx assay). The significance level was set at p < 0.05. Data were analyzed by using Student's t test, one-way analysis of variance (ANOVA), or two-way ANOVA followed by Bonferroni's post hoc test.
Evaluation of Antinociceptive and Antioedematogenic Effects of the Hydroalcoholic Extract, Fractions, and Isolated Compounds from V. tweedieana in the Capsaicin Test.
The hydroalcoholic extract, fractions, and isolated compounds from V. tweedieana had antinociceptive and antioedematogenic effects in the capsaicin-induced nociceptive model. Treatment with the HE extract and the Dcm, Act, and But fractions (100 mg/kg) produced an antinociceptive response in this model with inhibition of 51 ± 4, 67 ± 5, 44 ± 4, and 33 ± 9%, respectively, compared with the vehicle-treated group (Fig. 1A). Statistical analysis of the nociception time after capsaicin injection (one-way ANOVA followed by Bonferroni's post hoc test) revealed that the Dcm fraction produced a greater antinociceptive effect compared with the other fractions or the HE extract. In addition, only the Dcm fraction showed an antioedematogenic effect, with 66 ± 9% inhibition (Fig. 1B).
Among the compounds isolated from the Dcm fraction (Table 1), α-spinasterol (0.3 μmol/kg p.o.) had the greatest effect on capsaicin-induced nociception 1 h after treatment (58 ± 4% inhibition; Fig. 1C). Stigmasterol (0.3 μmol/kg p.o.) also decreased capsaicin-induced nociception (40 ± 7% inhibition). However, it did not reduce the associated edema, whereas α-spinasterol (0.3 μmol/kg p.o.) produced an antioedematogenic effect (inhibition of 66 ± 10%; Fig. 1D). The mixture of α- and β-amyrin and lupeol had no effect on nociception or edema elicited by capsaicin injection at the dose tested (0.3 μmol/kg p.o.). The TRPV1 antagonist (SB-366791; 3 μmol/kg p.o.) showed antinociceptive and antioedematogenic effect in the capsaicin model as observed in Fig. 1.
The antinociceptive and antioedematogenic effects of α-spinasterol and the mechanisms related to these actions were then further characterized. The dose-response curve showed that α-spinasterol had maximal antinociceptive and antioedematogenic effects of 64 ± 6 and 70 ± 4%, respectively, at 1 μmol/kg p.o. 1 h after treatment. The ID50 values for the antinociceptive and antioedematogenic effects were 0.055 μmol/kg (with a range of 0.039–0.077 μmol/kg) and 0.11 μmol/kg (with a range of 0.074–0.16 μmol/kg), respectively (Fig. 2 A and B). The results presented in Fig. 2C show that α-spinasterol (0.3 μmol/kg p.o.) markedly inhibited the capsaicin-induced nociceptive response from 0.5 to 4 h after administration, with 60 ± 6% inhibition. The antioedematogenic effect of α-spinasterol occurred from 0.5 to 2 h after treatment, with 62 ± 11% inhibition (Fig. 2D). SB-366791 (3 μmol/kg p.o.; used as a positive control) produced an antinociceptive effect from 0.5 to 2 h after treatment, with 68 ± 5% inhibition (Fig. 2E). It also had an antioedematogenic effect during the capsaicin test (74 ± 6% inhibition) from 0.5 to 1 h after treatment (Fig. 2F).
Characterization of α-Spinasterol as a TRPV1 Antagonist.
We observed that the Dcm fraction (1 mg/ml) and α-spinasterol (10 μM) displaced the specific binding of [3H]RTX from spinal cord membranes by 49 ± 2 and 67 ± 11%, respectively (Fig. 3A). On the other hand, stigmasterol, the mixture of α- and β-amyrin, and lupeol (10 μM) isolated from the Dcm fraction did not alter the specific binding of [3H]RTX (Fig. 3A). The IC50 value for α-spinasterol was 1.4 μM (with a range of 0.7–2.6 μM), and the maximal inhibition was 64 ± 3% for 10 μM (Fig. 3B). Because α-spinasterol was able to bind to TRPV1 receptor, we investigated whether this molecule could alter capsaicin-mediated Ca2+ influx. In the presence of α-spinasterol, there was a substantial reduction in the response to capsaicin, with a maximal inhibition of 62 ± 10% and an IC50 of 40 μM (with a range of 23–68 μM) (Fig. 3C). In addition, the SB-366791 (1 μM) produced a large reduction in the Ca2+ influx induced by capsaicin (69 ± 5% inhibition). On the other hand, in the presence of α-spinasterol (100 μM), SB-366791 (1 μM), or vehicle, we have not observed any significant Ca2+ influx.
Dcm Fraction and α-Spinasterol Presented Antinociceptive Effects on Noxious Heat-Mediated Nociception Rather than Altered the Sensitivity to Mechanical Stimulus.
The administration of α-spinasterol (0.3 μmol/kg p.o.) produced antinociception in the noxious heat induced-nociception test from 0.5 to 2 h after administration (26 ± 6% inhibition; 1 h after administration). In addition, the treatment with the Dcm fraction (100 mg/kg p.o) showed an antinociceptive effect from 1 to 2 h (19 ± 2% inhibition), and SB-366791 (3 μmol/kg p.o.) produced an antinociceptive effect from 0.5 to 1 h (20 ± 6% inhibition) (Fig. 4A). On the other hand, the administration of the Dcm fraction, α-spinasterol, or SB-366791 did not affect the mechanical threshold of uninjured animals in any of the observed time points (Fig. 4B).
Participation of TRPV1-Positive Fibers in the Antinociceptive Effect of α-Spinasterol on Noxious Heat Stimulus-Induced Nociception.
Mice were pretreated with RTX to evaluate the role of positive TRPV1 fibers in the antinociceptive effect of α-spinasterol. Systemic pretreatment with RTX significantly reduced the immunoreactivity of the TRPV1 protein in the sciatic nerve 7 days after injection (72 ± 10% compared with the control group), confirming a reduction in TRPV1-positive sensory fibers (Fig. 5A).
Demonstrating the effectiveness of the TRPV1 desensitization, we have observed that the nociceptive response induced by capsaicin intraplantar injection (1 nmol/paw) in mice was abolished by pretreatment with RTX (nociception time of 130 ± 7 and 5 ± 2 s, for animals preadministered with vehicle or RTX, respectively; p < 0.001; Student's t test; n = 6–7).
As expected, RTX pretreatment also increased the paw withdrawal latency to noxious heat stimulus compared with basal latency values (before RTX pretreatment) (Fig. 5B). The treatment with α-spinasterol (0.3 μmol/kg p.o.) or SB-366791 (3 μmol/kg p.o.) was not capable of producing an additional increase on withdrawal latencies in RTX-pretreated animals (Fig. 5B). On the other hand, morphine treatment increased paw withdrawal latency to noxious heat stimulus (10 mg/kg s.c.; 1 h after treatment) in mice pretreated either with vehicle or RTX (Table 2).
Antihyperalgesic and Antioedematogenic Effects of the Dcm Fraction and α-Spinasterol in the CFA Model.
The antihyperalgesic effect of α-spinasterol was observed for CFA-induced heat hyperalgesia from 0.5 to 2 h after treatment (100% inhibition observed at 1 h after treatment); the Dcm fraction also had an antinociceptive effect from 0.5 to 1 h (100% inhibition; Fig. 6A). Antihyperalgesic effects of Dcm fraction and α-spinasterol were also observed for CFA-induced mechanical hyperalgesia from 0.5 to 2 h after treatment, with 82 ± 14 and 96 ± 15% of inhibition, respectively (Fig. 6B). The Dcm fraction and α-spinasterol also reduced CFA-induced edema; this effect was observed from 0.5 to 2 h after Dcm fraction treatment or 0.5 to 4 h after α-spinasterol treatment, with 38 ± 10 and 35 ± 6% inhibition observed 2 h after treatment, respectively (Fig. 6C). However, SB-366791 (3 μmol/kg p.o.) only reduced CFA-induced heat hyperalgesia from 0.5 to 1 h (100% inhibition), without affecting mechanical hyperalgesia or edema (Fig. 6).
The Administration of Dcm Fraction or α-Spinasterol Produced No Detectable Adverse Effects.
Dcm fraction (100 mg/kg p.o) or α-spinasterol (0.3 μmol/kg p.o.) did not alter forced or spontaneous locomotion evaluated by rotarod and open-field tests (Table 3). Dcm fraction and α-spinasterol did not change body temperature, whereas the TRPV1 antagonist AMG-9810 induced a significant increase in rectal temperature (Table 3).
The plasma concentration of α-spinasterol reached a maximal concentration (Cmax) of 0.25 ± 0.003 nmol/ml 1 h after oral administration of 0.3 μmol/kg (Fig. 7). We were able to detect α-spinasterol in plasma from 0.5 to 4 h after administration. α-Spinasterol was also detected in the brain and spinal cord with Cmax of 1.63 ± 0.12 and 5.8 ± 0.88 nmol/g, respectively, 1 h after oral administration (Fig. 7). The temporal profiles of drug levels in the brain and spinal cord were almost identical to that in plasma. The plasma-to-brain and plasma-to-spinal cord ratios were 6.5 and 23.4 for the Cmax at 1 h after treatment, respectively.
The TRPV1 receptor is a relevant target for the development of novel analgesic drugs, and its involvement has been studied in a wide range of diseases, including migraine, inflammatory, cancer, and osteoarthritic-related pain (Adcock, 2009; Wong and Gavva, 2009; Schumacher, 2010). The identification of novel TRPV1 ligands that possess antinociceptive activity without the development of severe hyperthermia is really important. In this study, we performed in vivo and in vitro screens to identify novel TRPV1 antagonists derived from a medicinal plant commonly used to treat cough and respiratory diseases (Zanon et al., 2008), which are pathologies related to TRPV1 participation (Adcock, 2009). Collectively, we observed that the hydroalcoholic extract and fractions of V. tweedieana have antinociceptive effects in the capsaicin test, where only the Dcm fraction possess antiodematogenic action. Furthermore, among the compounds isolated from the Dcm fraction, α-spinasterol and stigmasterol showed antinociceptive effects during the capsaicin test. However, only α-spinasterol had an antioedematogenic effect in the dose tested. This sterol also diminished capsaicin-induced Ca2+ influx in synaptosomes and displaced [3H]RTX binding in mice spinal cord membranes. In addition, α-spinasterol had antinociceptive effects in a heat-induced nociception test and an inflammatory pain model without causing motor alteration or hyperthermia.
The capsaicin test is a particularly relevant model for screening new TRPV1 compounds because capsaicin is a selective TRPV1 agonist capable of inducing an acute nociception in experimental animals and pain in humans (Simone et al., 1989; Sakurada et al., 1992; Walker et al., 1999). We have used this model to screen for antinociceptive and antioedematogenic effects of the newly studied compounds. As expected for SB-366791, TRPV1 receptor antagonism reduced both nociception and edema produced by intraplantar capsaicin injection. On the other hand, we demonstrated previously that nonsteroidal anti-inflammatory drugs inhibited nociception but not edema induced by capsaicin (Oliveira et al., 2009). Thus, we selected a fraction of V. tweedieana and its isolated compounds that could reduce both capsaicin-induced responses. Only the Dcm fraction and α-spinasterol reduced edema formation and spontaneous nociception. Our results are in accordance with previous findings that demonstrated that α-spinasterol exerted antinociceptive effects in different acute pain models, including glutamate-mediated spontaneous nociception and acetic acid-induced abdominal constriction (Meotti et al., 2006; Ribas et al., 2008; Freitas et al., 2009). However, the mechanisms underlying the antinociceptive effect of α-spinasterol remain to be elucidated.
To further characterize the α-spinasterol as a TRPV1 antagonist we have evaluated its capacity to displace [3H]RTX-specific binding and reduce capsaicin-induced calcium influx. Of the compounds tested only α-spinasterol displaced the specific binding of [3H]RTX from spinal cord membranes. Our results clearly indicate that α-spinasterol is a TRPV1 ligand. Furthermore, this compound inhibited capsaicin-induced Ca2+ influx in the same potency range as observed in the binding assay. This demonstrates that like the noted TRPV1 antagonist, SB-366791, the α-spinasterol acts as a TRPV1 receptor antagonist in this assay. The value of inhibition of capsaicin-induced calcium influx observed for SB-366791 (1 μM) was similar to that reported previously (Varga et al., 2005). The fact that the steroid α-spinasterol bound the TRPV1 receptor was not unexpected because other steroid compounds, such as pregnenolone sulfate and dehydroepiandrosterone, also act as TRPV1 antagonists (Chen and Wu, 2004; Chen et al., 2004). In addition, it has been observed as a cholesterol-binding motif in TRPV1, which might be modulated by cholesterol concentration (Picazo-Juárez et al., 2011). Moreover, the low micromolar concentration of α-spinasterol necessary to bind and inhibit TRPV1 receptors can be reached with oral administration because it is well distributed in the nervous system, where TRPV1 is expressed.
TRPV1 is a polymodal receptor expressed mainly in sensory neurons, and it is important for the perception of acute noxious information (Szallasi et al., 2007). Several reports indicated that mice lacking TRPV1 have diminished responses to acute thermal but not mechanical stimuli (Caterina et al., 1997; Davis et al., 2000). Accordingly, we observed that α-spinasterol inhibited nociceptive behavior induced by acute noxious heat, but it did not change the mechanical pain threshold of naive animals. In fact, a study showed that TRPV1 receptors are expressed mainly in sensory fibers that transduce heat pain and express μ-opioid receptor (Scherrer et al., 2009). However, α-spinasterol action in heat-evoked nociception is not opioid receptor-dependent because its antinociceptive action was not reversed by naloxone (data not shown). To confirm that the effect of α-spinasterol depends on TRPV1 in vivo, we depleted TRPV1-positive fibers by using a systemic RTX treatment. As expected, RTX-injected animals have reduced TRPV1 receptor expression in the sciatic nerve similar to what was observed previously in sensory ganglion by immunohistochemistry and Western blot analysis (Pecze et al., 2009). Confirming TRPV1 desensitization, an increase in the latency to noxious heat stimuli and an abolishment in the nociceptive response elicited by capsaicin intraplantar injection were also detected in RTX-treated mice. Unlike higher doses (up to 200 μg/kg) that produce large deficits in noxious heat stimulus detection (Chen and Pan, 2006), the lower dose of RTX used by us (50 μg/kg) seems, at least in part, to preserve the integrity of the thermal sensory fibers. In fact, RTX treatment in mice only partially increased paw withdrawal latency, and animals continued to respond to noxious heat stimulation.
The antinociceptive effect of both α-spinasterol and the selective TRPV1 antagonist SB-366791 in the thermal test was abolished in mice pretreated with RTX, indicating the participation of TRPV1-positive fibers in their antinociceptive effect. Next, we assessed whether drug-induced antinociception could still be detected in RTX-treated mice. We chose morphine because it may induce antinociception in TRPV1 knockout mice (Vardanyan et al., 2009) and RTX-treated rats (Chen and Pan, 2006). Unlike α-spinasterol and SB-366791, the antinociceptive effect produced by morphine was observed in both vehicle- and RTX-treated mice, indicating that ablation of positive TRPV1 fibers by RTX treatment is capable of decreasing only TRPV1-mediated antinociception. Collectively, these results suggest that the TRPV1 receptor is essential for the antinociceptive action of α-spinasterol in vivo.
The importance of the TRPV1 receptor in the pain related to inflammatory process has been recognized (Szallasi et al., 2007; Schumacher, 2010). In preclinical trials, TRPV1 antagonists reduced inflammatory related mechanical and thermal hyperalgesia, whereas animals lacking the TRPV1 protein were deficient in pain inflammatory signals and unable to develop thermal hyperalgesia (Walker et al., 2003; Gavva et al., 2005; Honore et al., 2005; Barton et al., 2006). In addition, pain related to inflammatory conditions is associated with increased TRPV1 receptor expression (Ji et al., 2002; Amaya et al., 2004; Zhang et al., 2005). Our study demonstrates that α-spinasterol may actively inhibit the edema, mechanical, and heat hyperalgesia observed in the CFA-induced inflammatory pain model. The fact that α-spinasterol did not alter mechanical nociception in naive animals but reduced mechanical hyperalgesia in inflamed mice may be explained by the increase of TRPV1 receptor expression in mechanically activated myelinated A-fibers after CFA injection (Amaya et al., 2004; McGaraughty et al., 2008). The antinociceptive effect of α-spinasterol in CFA-induced mechanical hyperalgesia could also be related to the capacity of this molecule to access the central nervous system (CNS). This is supported by evidence showing that TRPV1 receptor antagonists that access the CNS have better antihyperalgesic effects than peripheral antagonists, mainly against mechanical hyperalgesia (Cui et al., 2006). Indeed, α-spinasterol is well distributed in the CNS with plasma-to-brain and plasma-to-spinal cord ratios of 6.5 and 23.4, respectively. In addition, this sterol possesses antioedematogenic effect in the CFA model that was not observed for the TRPV1 antagonist SB-366791. This effect could be associated with different activities previously reported to α-spinasterol. In fact, α-spinasterol has potent antioxidant effects against several reactive oxygen species (Coballase-Urrutia et al., 2010), and antioxidants are able to reduce the CFA-induced paw edema (Wang et al., 2011; Sindhu et al., 2012). In addition, α-spinasterol suppressed lipopolysaccharide-induced expression of inducible nitric-oxide synthase and cycloxygenase-2 and also the release of nitric oxide, prostaglandin E2, tumor necrosis factor-α, and interleukin-1β in microglial cells (Jeong et al., 2010). Because all of these inflammatory mediators are able to modulate TRPV1 expression and/or activity (Szallasi et al., 2007; Adcock, 2009; Schumacher, 2010), this mechanism could also contribute indirectly to α-spinasterol inhibition of TRPV1 function.
Many drugs elicit false-positive responses in nociception tests because they cause sedation, motor activity impairment, or body temperature alteration (Negus et al., 2006). The induction of severe hyperthermia by some TRPV1 antagonists, such as N-(4-hydroxy-benzo[d]thiazol-2-yl)acetamide (N-acetyl benzothiazole) (AMG-517) (or its analog of AMG-9810), has hampered the development of these drugs as analgesics (Gavva et al., 2008). Neither the Dcm fraction nor α-spinasterol induced locomotor alteration or hyperthermia in mice. This indicates that α-spinasterol might have better features than other known TRPV1 antagonists. Finally, we found that orally administered α-spinasterol had good absorption with the peak concentrations in the plasma, brain, and spinal cord comparable with the time of maximal antinociception. These findings are relevant because oral administration is more clinically viable; it is low cost, safe, and easy to administer (Buxton, 2006).
In conclusion, α-spinasterol showed antinociceptive and antioedematogenic effects in distinct pain models, particularly hyperalgesia, a common complaint of chronic pain patients (Schaible et al., 2009). The plant steroid α-spinasterol has been identified as a novel, safe, and orally effective TRPV1 receptor antagonist, and it may be an attractive future target for pain therapy.
Participated in research design: Trevisan, Rossato, Walker, Klafke, Rosa, Oliveira, Tonello, Guerra, Boligon, Zanon, Athayde, and Ferreira.
Conducted experiments: Trevisan, Rossato, Walker, Klafke, Rosa, Oliveira, Tonello, Guerra, Boligon, and Zanon.
Performed data analysis: Trevisan, Rossato, Athayde, and Ferreira.
Wrote or contributed to the writing of the manuscript: Trevisan, Rossato, Athayde, and Ferreira.
This study was supported by the Conselho Nacional de Desenvolvimento Científico, Financiadora de Estudos e Projetos, Programa de Apoio aos Núcleos de Excelência, Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- transient receptor potential vanilloid 1
- analysis of variance
- bovine serum albumin
- central nervous system
- complete Freund's adjuvant
- dimethyl sulfoxide, Act, ethyl acetate
- hydroalcoholic extract
- paw withdrawal threshold
- Tween 20 in Tris-borate saline
- acetoxymethyl ester
- high-performance liquid chromatography
- [(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide]
- N-(4-hydroxy-benzo[d]thiazol-2-yl)acetamide (N-acetyl benzothiazole).
- Received April 30, 2012.
- Accepted July 25, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics