BACKGROUND
pterodon pubescens
benth.
, known as sucupira, is widespread throughout the brazilian states of goiás, minas gerais and são paulo.
sucupira seeds are commercially available in the brazilian medicinal plant market.
the crude alcoholic extracts of this plant are used in folk medicine as anti-inflammatory, analgesic and anti-rheumatic preparations  <cit> .
phytochemical studies of the pterodon genus have shown the presence of alkaloids, isoflavones and diterpenes.
furanditerpenes were identified and isolated from pterodon fruits  <cit> .
studies have suggested that furanditerpenes possessing the vouacapan skeleton contribute to the anti-inflammatory and antinociceptive properties of pterodon pubescens seed oil  <cit> .
diterpenes 6α-hydroxyvouacapan-7β-17β-lactone and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester, found in p. emarginatus and p. polygalaeflorus seeds were previously reported to be associated with the anti-inflammatory activity of these species  <cit> .
herein we report the antinociceptive activity of 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester and geranylgeraniol isolated from pterodon pubescens benth.
when evaluated in writhing, capsaicin, glutamate and hot-plate animal experimental models.
RESULTS
some authors have reported the antinociceptive activity of the crude extract and fractions obtained from p. pubescens and established a relationship with anti-inflammatory activity  <cit> .
this report evaluated for the first time the contribution of geranylgeraniol  and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester , isolated from p. pubescens , to the antinociceptive activity using various experimental models to evaluate a distinct pain modulation.
calixto et al  <cit>  demonstrated the antiplatelet activity of geranylgeraniol attributed to cyclooxygenase enzyme inhibition, but did not mention data on antinociceptive activity.
some vouacapan compounds have been suggested to have a relationship with p. pubescens' antinociceptive activity.
nevertheless pain modulation by this species has never been reported, being described herein for the first time.
the following screening results demonstrated the activity and general mechanisms involved in antinociception caused by geranylgeraniol  and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester .
the most relevant additional findings of the present work are that,  compounds c <dig> and c <dig> may present synergistic activity;  both intraperitoneal  and oral  treatment of compounds c <dig> and c <dig> reduced reactivity to the writhing test demonstrating differences in potency related to the route of administration;  both compounds c <dig> and c <dig> demonstrated possible activity related to vanilloid receptors and/or glutamate peripheral receptors, with c <dig> being more potent by the i.p.
route;  the antinociceptive activity of compounds c <dig> and c <dig>  do not appear to exert their antinociceptive effects in the hot-plate test via opioid receptors.
initially, the open field test was performed in order to exclude the possibility that the antinociceptive action of geranylgeraniol  and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester  could be related to non-specific disturbances in the locomotor activity of the animals.
treatment with compounds c <dig> and c <dig>  did not cause any significant change in the ambulation of mice when tested in the open field.
however, pentobarbital  significantly  reduced the locomotor activity of animals in this test.
the mean number of crossings was  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig> ±  <dig>  and  <dig> ±  <dig>  for vehicle, pentobarbital, c <dig> and c <dig>  respectively.
after these results, the acetic acid writhing test model was employed.
this is a convenient stimulus assay for screening, because the intensity of response depends on the interaction of several factors, neurotransmitters and neuromodulators that determine nociception, such as kinines, acetylcholine, substance p and prostaglandins.
therefore, this model is responsive to analgesic substances possessing with the most varied action mechanisms  <cit> , being sensitive to drugs with analgesic activity such as aspirin, kinin receptor antagonists  and opioid analgesics with central or peripheral action  <cit> .
this model permitted evaluation of antinociceptive activity caused by both neurogenic and/or inflammatory pain.
this assay was used during the initial studies with compounds c <dig> and c <dig> in order to establish differences in potency using different routes of administration.
the antinociceptive activity of compounds c <dig> and c <dig> were compared with the dichloromethane extract   in the writhing test using  <dig> mg.kg- <dig> doses.
the reductions in the number of abdominal constrictions were 62%, 64% and 66% for eb, c <dig> and c <dig> respectively  .
these results suggested a possible synergistic activity among compounds c <dig> and c <dig> .
accordingly, dose-response curves for i.p.
and p.o.
administered compounds were determined in the writhing test to calculate ed <dig> values and possible differences caused by administration routes.
the p.o.
administration of compound c <dig> showed dose-related activity reducing by 51%, 57%, and 75%  the abdominal constrictions with  <dig> mg.kg- <dig>   <dig> mg.kg- <dig>  and  <dig> mg.kg- <dig> doses respectively, presenting ed <dig> =  <dig>  mg.kg- <dig> .
the same treatment with compound c <dig> reduced constrictions by 43%  and 71%  with ed <dig> =  <dig>  mg.kg- <dig>  these results showed that compound c <dig> was more potent than c <dig> when given by p.o.
administration.
the dose-related activity for compounds administered i.p.
in the writhing test, demonstrated that compound c <dig> reduced constrictions by 58% and 98%  with  <dig> mg.kg- <dig> and  <dig> mg.kg- <dig> doses respectively  .
compound c <dig> reduced constrictions by 84%, 90%, and 98% with  <dig> mg.kg- <dig>   <dig> mg.kg- <dig>  and  <dig> mg.kg- <dig> doses respectively, showing ed <dig> =  <dig>  mg.kg- <dig>  more potent than c <dig>  in the i.p.
tests, the numbers of abdominal constrictions of the control group were fewer compared to the control group of the p.o.
route, maybe caused by injection-stress of the animals.
in order to evaluate the antinociceptive response of compounds c <dig> and c <dig> in neurogenic pain caused by direct chemical stimulation on nociceptors, the capsaicin test was evaluated.
the active principle, capsaicin, from capsicum genus peppers, is the painful substance used to determine if compounds possessing antinociceptive activity act by the vanilloid vr <dig> receptors.
the animal response is caused by release of neuropeptides, such as substance p, neurokinines, somastotastin, peptide related to the calcitonin gene  with participation of nociceptive afferent c fibers, and in part, a fibers  <cit> .
the induced nociceptive process with capsaicin is related to the activation of the tachykininergic system and seems to be mediated by activation of a specific receptor, whose presence has been confirmed with capsazepine, a competitive vanilloid antagonist.
evidence also suggests that bradykinin  acting through b <dig> receptors seems to be involved in the neurogenic nociception caused by capsaicin in mice  <cit> .
in this test, i.p.
administration of compound c <dig> reduced by 43%, 48%, 58%, and 67%  with  <dig> mg.kg- <dig>   <dig> mg.kg- <dig>   <dig> mg.kg- <dig>  and  <dig> mg.kg- <dig> doses respectively  .
compound c <dig> showed dose-related activity too, reducing by 51%, 56%, 66%, and 69%  the reaction time, with  <dig> mg.kg- <dig>   <dig> mg.kg- <dig>   <dig> mg.kg- <dig>  and  <dig> mg.kg- <dig> doses respectively .
these results showed that the antinociceptive activity of compounds c <dig> and c <dig> may be related to the tachykininergic system or vanilloid vr <dig> receptors.
the same experimental model was tested with p.o.
administration of compounds c <dig>  and c <dig>  employing morphine  as positive control, in order to evaluate the activity of compounds on this route .
compound c <dig> reduced reaction time by 82% whereas c <dig> reduced by 65%, with the positive control demonstrating reduction by 52% .
this assay demonstrated that compound c <dig> was more potent in neurogenic pain modulated by vanilloid receptors than compound c <dig>  corroborating the previous result with intraperitoneal administration.
once we had demonstrated the activity of compounds geranylgeraniol  and 6α,7β-dihydroxyvouacapan-17β-oate methyl ester  with the writhing and capsaicin tests, we considered the glutamate test.
this model is based on the activation of peripheral glutamate receptors responsible for several types of pain sensation.
the nociceptive response induced by glutamate appears to involve peripheral, spinal and supraspinal sites of action and is mediated by both nmda and non-nmda receptors as well as by the release of nitric oxide or by some nitric oxide-related substances.
nitric oxide inhibitors and both nmda and non-nmda receptor antagonists, and other drugs have been previously reported to inhibit the acetic-acid and capsaicin- induced nociceptive response  <cit> .
our results showed that i.p.
administration of compounds c <dig> and c <dig> produced a significant dose-related inhibition of the nociceptive response caused by intraplantar injection of glutamate into the mouse's hind paw.
in this test, the p.o.
route was not evaluated because of possible changes of pharmacokinetic, metabolic or distribution parameters, which could disrupt the interaction among compounds and receptors .
compound c <dig> , with  <dig> mg.kg- <dig>   <dig> mg.kg- <dig>   <dig> mg.kg- <dig>  and  <dig> mg.kg- <dig> doses reduced reaction time by 67%, 72%, 75%, and 80% respectively , corresponding to on ed <dig> =  <dig>  mg.kg- <dig> .
compound c <dig>  showed ed <dig> =  <dig>  mg.kg- <dig>  reducing by 62%, 83%, and 96% the reaction time with  <dig> mg.kg- <dig>   <dig> mg.kg- <dig>  and  <dig> mg.kg- <dig> doses  respectively , again more potent than c <dig> in peripheral neurogenic modulation.
thus, these previous findings and the present results suggested that the antinociceptive action of compounds c <dig> and c <dig> in the acetic acid, capsaicin, and glutamate tests could be the result of both the inhibition of nos and the blockade of glutamate receptors.
further studies are being undertaken to confirm receptors involved on this activity.
compounds c <dig> and c <dig> were evaluated in the hot-plate thermal nociception model by p.o.
route.
this route was chosen based on preliminary set of experiments which showed to induce less stress to animals.
the hot-plate test is a neurogenic-modulated model that produces, at constant temperature, two kinds of behavioral response, which are paw licking and jumping.
both of these are considered to be supraspinally- integrated responses  <cit> .
in order to evaluate the antinociceptive activity of compounds c <dig> and c <dig> mediated by opioid receptors, the hot plate test was carried out using the opioid antagonist naloxone hydrochloride.
the p.o.
doses for compounds c <dig> and c <dig> were chosen based on preliminary studies in our laboratory.
pre-treatment with naloxone , did not reverse the antinociceptive effect of both compounds when the animals were treated p.o.
with  <dig> mg.kg- <dig> doses , especially at  <dig> minutes after challenge, suggesting that these compounds do not possess similar action to morphine or derivatives when evaluated in this assay .
statistical analysis anova
duncan's test: *p <  <dig> ; **p <  <dig>
ns: no significant result
morphine, naloxone, c <dig>  c <dig> and vehicle given orally.
CONCLUSIONS
both i.p.
and p.o.
treatment with compounds c <dig> and c <dig> reduced response in the writhing, capsaicin, glutamate, and hot-plate tests demonstrating their contribution to the antinociceptive activity of crude pterodon pubescens benth extracts.
compounds, c <dig> and c <dig>  demonstrated activity in models demonstrative of vanilloid receptors and glutamate peripheral receptors.
in the hot-plate model, the antinociceptive activity was maintained when naloxone hydrochloride, an opioid antagonist, was administered prior to sample dosing suggesting that compounds c <dig> and c <dig> do not exert their antinociceptive effects in the hot-plate test via opioid receptors.
the findings presented herein also suggested that crude pterodon pubescens benth extract probably is a mixture of substances with synergistic interactive effect, since the crude extract  with lower concentrations of compounds c <dig>  and c <dig>  presented statistically analogous effects to those of the pure compounds when tested separately  with p.o.
administration in the writhing experimental model.
further studies will be undertaken to establish the mechanisms of action for compounds c <dig> and c <dig>  the synergistic interactions will be evaluated employing the isobole method  <cit> .

 pterodon pubescens benth., known as sucupira, is widespread throughout the brazilian states of goiás, minas gerais and são paulo. sucupira seeds are commercially available in the brazilian medicinal plant market. the crude alcoholic extracts of this plant are used in folk medicine as anti-inflammatory, analgesic and anti-rheumatic preparations. phytochemical studies of the pterodon genus have shown the presence of alkaloids, isoflavones and diterpenes. furanditerpenes were identified and isolated from pterodon fruits. studies have suggested that furanditerpenes possessing the vouacapan skeleton contribute to the anti-inflammatory and antinociceptive properties of pterodon pubescens seed oil. diterpenes 6α-hydroxyvouacapan-7β-17β-lactone and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester, found in p. emarginatus and p. polygalaeflorus seeds were previously reported to be associated with the anti-inflammatory activity of these species. herein we report the antinociceptive activity of 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester and geranylgeraniol isolated from pterodon pubescens benth. when evaluated in writhing, capsaicin, glutamate and hot-plate animal experimental models.  some authors have reported the antinociceptive activity of the crude extract and fractions obtained from p. pubescens and established a relationship with anti-inflammatory activity. this report evaluated for the first time the contribution of geranylgeraniol and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester, isolated from p. pubescens, to the antinociceptive activity using various experimental models to evaluate a distinct pain modulation. calixto et al demonstrated the antiplatelet activity of geranylgeraniol attributed to cyclooxygenase enzyme inhibition, but did not mention data on antinociceptive activity. some vouacapan compounds have been suggested to have a relationship with p. pubescens' antinociceptive activity. nevertheless pain modulation by this species has never been reported, being described herein for the first time. the following screening results demonstrated the activity and general mechanisms involved in antinociception caused by geranylgeraniol and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester. the most relevant additional findings of the present work are that, compounds c and c may present synergistic activity; both intraperitoneal and oral treatment of compounds c and c reduced reactivity to the writhing test demonstrating differences in potency related to the route of administration; both compounds c and c demonstrated possible activity related to vanilloid receptors and/or glutamate peripheral receptors, with c being more potent by the i.p. route; the antinociceptive activity of compounds c and c do not appear to exert their antinociceptive effects in the hot-plate test via opioid receptors. initially, the open field test was performed in order to exclude the possibility that the antinociceptive action of geranylgeraniol and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester could be related to non-specific disturbances in the locomotor activity of the animals. treatment with compounds c and c did not cause any significant change in the ambulation of mice when tested in the open field. however, pentobarbital significantly reduced the locomotor activity of animals in this test. the mean number of crossings was ±, ±, ± and ± for vehicle, pentobarbital, c and c respectively. after these results, the acetic acid writhing test model was employed. this is a convenient stimulus assay for screening, because the intensity of response depends on the interaction of several factors, neurotransmitters and neuromodulators that determine nociception, such as kinines, acetylcholine, substance p and prostaglandins. therefore, this model is responsive to analgesic substances possessing with the most varied action mechanisms, being sensitive to drugs with analgesic activity such as aspirin, kinin receptor antagonists and opioid analgesics with central or peripheral action. this model permitted evaluation of antinociceptive activity caused by both neurogenic and/or inflammatory pain. this assay was used during the initial studies with compounds c and c in order to establish differences in potency using different routes of administration. the antinociceptive activity of compounds c and c were compared with the dichloromethane extract in the writhing test using mg.kg- doses. the reductions in the number of abdominal constrictions were 62%, 64% and 66% for eb, c and c respectively. these results suggested a possible synergistic activity among compounds c and c. accordingly, dose-response curves for i.p. and p.o. administered compounds were determined in the writhing test to calculate ed values and possible differences caused by administration routes. the p.o. administration of compound c showed dose-related activity reducing by 51%, 57%, and 75% the abdominal constrictions with mg.kg- mg.kg- and mg.kg- doses respectively, presenting ed = mg.kg-. the same treatment with compound c reduced constrictions by 43% and 71% with ed = mg.kg- these results showed that compound c was more potent than c when given by p.o. administration. the dose-related activity for compounds administered i.p. in the writhing test, demonstrated that compound c reduced constrictions by 58% and 98% with mg.kg- and mg.kg- doses respectively. compound c reduced constrictions by 84%, 90%, and 98% with mg.kg- mg.kg- and mg.kg- doses respectively, showing ed = mg.kg- more potent than c in the i.p. tests, the numbers of abdominal constrictions of the control group were fewer compared to the control group of the p.o. route, maybe caused by injection-stress of the animals. in order to evaluate the antinociceptive response of compounds c and c in neurogenic pain caused by direct chemical stimulation on nociceptors, the capsaicin test was evaluated. the active principle, capsaicin, from capsicum genus peppers, is the painful substance used to determine if compounds possessing antinociceptive activity act by the vanilloid vr receptors. the animal response is caused by release of neuropeptides, such as substance p, neurokinines, somastotastin, peptide related to the calcitonin gene with participation of nociceptive afferent c fibers, and in part, a fibers. the induced nociceptive process with capsaicin is related to the activation of the tachykininergic system and seems to be mediated by activation of a specific receptor, whose presence has been confirmed with capsazepine, a competitive vanilloid antagonist. evidence also suggests that bradykinin acting through b receptors seems to be involved in the neurogenic nociception caused by capsaicin in mice. in this test, i.p. administration of compound c reduced by 43%, 48%, 58%, and 67% with mg.kg- mg.kg- mg.kg- and mg.kg- doses respectively. compound c showed dose-related activity too, reducing by 51%, 56%, 66%, and 69% the reaction time, with mg.kg- mg.kg- mg.kg- and mg.kg- doses respectively. these results showed that the antinociceptive activity of compounds c and c may be related to the tachykininergic system or vanilloid vr receptors. the same experimental model was tested with p.o. administration of compounds c and c employing morphine as positive control, in order to evaluate the activity of compounds on this route. compound c reduced reaction time by 82% whereas c reduced by 65%, with the positive control demonstrating reduction by 52%. this assay demonstrated that compound c was more potent in neurogenic pain modulated by vanilloid receptors than compound c corroborating the previous result with intraperitoneal administration. once we had demonstrated the activity of compounds geranylgeraniol and 6α,7β-dihydroxyvouacapan-17β-oate methyl ester with the writhing and capsaicin tests, we considered the glutamate test. this model is based on the activation of peripheral glutamate receptors responsible for several types of pain sensation. the nociceptive response induced by glutamate appears to involve peripheral, spinal and supraspinal sites of action and is mediated by both nmda and non-nmda receptors as well as by the release of nitric oxide or by some nitric oxide-related substances. nitric oxide inhibitors and both nmda and non-nmda receptor antagonists, and other drugs have been previously reported to inhibit the acetic-acid and capsaicin- induced nociceptive response. our results showed that i.p. administration of compounds c and c produced a significant dose-related inhibition of the nociceptive response caused by intraplantar injection of glutamate into the mouse's hind paw. in this test, the p.o. route was not evaluated because of possible changes of pharmacokinetic, metabolic or distribution parameters, which could disrupt the interaction among compounds and receptors. compound c, with mg.kg- mg.kg- mg.kg- and mg.kg- doses reduced reaction time by 67%, 72%, 75%, and 80% respectively, corresponding to on ed = mg.kg-. compound c showed ed = mg.kg- reducing by 62%, 83%, and 96% the reaction time with mg.kg- mg.kg- and mg.kg- doses respectively, again more potent than c in peripheral neurogenic modulation. thus, these previous findings and the present results suggested that the antinociceptive action of compounds c and c in the acetic acid, capsaicin, and glutamate tests could be the result of both the inhibition of nos and the blockade of glutamate receptors. further studies are being undertaken to confirm receptors involved on this activity. compounds c and c were evaluated in the hot-plate thermal nociception model by p.o. route. this route was chosen based on preliminary set of experiments which showed to induce less stress to animals. the hot-plate test is a neurogenic-modulated model that produces, at constant temperature, two kinds of behavioral response, which are paw licking and jumping. both of these are considered to be supraspinally- integrated responses. in order to evaluate the antinociceptive activity of compounds c and c mediated by opioid receptors, the hot plate test was carried out using the opioid antagonist naloxone hydrochloride. the p.o. doses for compounds c and c were chosen based on preliminary studies in our laboratory. pre-treatment with naloxone, did not reverse the antinociceptive effect of both compounds when the animals were treated p.o. with mg.kg- doses, especially at minutes after challenge, suggesting that these compounds do not possess similar action to morphine or derivatives when evaluated in this assay. statistical analysis anova duncan's test: *p <; **p < ns: no significant result morphine, naloxone, c c and vehicle given orally.  both i.p. and p.o. treatment with compounds c and c reduced response in the writhing, capsaicin, glutamate, and hot-plate tests demonstrating their contribution to the antinociceptive activity of crude pterodon pubescens benth extracts. compounds, c and c demonstrated activity in models demonstrative of vanilloid receptors and glutamate peripheral receptors. in the hot-plate model, the antinociceptive activity was maintained when naloxone hydrochloride, an opioid antagonist, was administered prior to sample dosing suggesting that compounds c and c do not exert their antinociceptive effects in the hot-plate test via opioid receptors. the findings presented herein also suggested that crude pterodon pubescens benth extract probably is a mixture of substances with synergistic interactive effect, since the crude extract with lower concentrations of compounds c and c presented statistically analogous effects to those of the pure compounds when tested separately with p.o. administration in the writhing experimental model. further studies will be undertaken to establish the mechanisms of action for compounds c and c the synergistic interactions will be evaluated employing the isobole method. 
the crude alcoholic extracts of this plant are used in folk medicine as anti-inflammatory, analgesic, and anti-rheumatic preparations. in hot-plate model, the antinociceptive activity was maintained when naloxone chloride was administered prior to treatment with compounds suggesting that c and c do not exert their antinociceptive effects in the hot-plate test via opioid receptors. to the antinociceptive activity of the crude extract. compounds c and c demonstrated activity against writhing with intraperitoneal and oral routes, capsaicin, glutamate, and in the hot-plate tests, demonstrating their contribution to the antinociceptive activity of crude pterodon pubescens benth extracts. these results allowed us to establish a relationship between the popular use of pterodon pubescens seeds for pain relief and the activity of two major compounds isolated from this species which demonstrated antinociceptive activity. the aim of this study was to evaluate the contribution of geranylgeraniol and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester isolated from pterodon pubescens benth. extract may exert a synergistic interactive effect, since the crude extract containing lower concentrations of compounds c and c gave statistically the same effect to the pure compounds when tested separately in writhing experimental model with p.o. pterodon pubescens benth seeds are commercially available in the brazilian medicinal plant street market. the findings presented herein also suggest that compounds within the crude pterodon pubescens benth. various "in vivo" experimental models corroborate the folk use of this species for different pain and inflammation disorders.
 pterodon pubescens benth seeds are commercially available in the brazilian medicinal plant street market. the crude alcoholic extracts of this plant are used in folk medicine as anti-inflammatory, analgesic, and anti-rheumatic preparations. the aim of this study was to evaluate the contribution of geranylgeraniol and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester isolated from pterodon pubescens benth. to the antinociceptive activity of the crude extract.  compounds c and c demonstrated activity against writhing with intraperitoneal and oral routes, capsaicin, glutamate, and in the hot-plate tests, demonstrating their contribution to the antinociceptive activity of crude pterodon pubescens benth extracts. the observed activity of compounds c and c may be related to vanilloid receptors vr and/or glutamate peripheral receptors. in hot-plate model, the antinociceptive activity was maintained when naloxone chloride was administered prior to treatment with compounds suggesting that c and c do not exert their antinociceptive effects in the hot-plate test via opioid receptors. the findings presented herein also suggest that compounds within the crude pterodon pubescens benth. extract may exert a synergistic interactive effect, since the crude extract containing lower concentrations of compounds c and c gave statistically the same effect to the pure compounds when tested separately in writhing experimental model with p.o. administration. further studies will be undertaken to establish more specifically the mechanisms of action for compounds c and c possible synergistic interactions will be evaluated employing the isobole method.  these results allowed us to establish a relationship between the popular use of pterodon pubescens seeds for pain relief and the activity of two major compounds isolated from this species which demonstrated antinociceptive activity. various "in vivo" experimental models corroborate the folk use of this species for different pain and inflammation disorders. 