Original Paper Antinociception Caused by the Extract of
517
Hedyosmum brasiliense
and its Active Principle, the Sesquiterpene Lactone 13-Hydroxy-8,9-dehydroshizukanolide
Ana P. Trentin1, Adair R. S. Santos1, Alessandro Guedes2, Moacir G. Pizzolatti2, Rosendo A. Yunes2, and Joo B. Calixto1,* 1 Departamento de Farmacologia, Universidade Federal de Santa Catarina, Florianópolis SC, Brazil 2 Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis SC, Brazil
Received: September 30,1999; 1998;Received: Accepted:September April 16, 1999 Revision accepted: April 16, 30, 1998
Abstract:
The hydroalcoholic extract (HE) obtained from stems
and leaves of
Hedyosmum brasiliense,
given
i.p.,
produced signif-
icant inhibition of acetic acid-induced abdominal constriction in mice, with a mean ID50 of 12.7 mg/kg. This effect installed rapidly (0.5 h) and lasted for up to 2 h. Given orally up to 1000 mg/kg, the HE was ineffective. When assessed in the formalin response the HE, given
i.p.,
inhibited the first and second
phase, with ID50s of 31.1 and 21.7 mg/kg for the first and the second phases, respectively. The HE also inhibited capsaicin-induced neurogenic pain with ID50 of 69.0 mg/kg, but, in contrast to morphine, failed to cause analgesia in either the tail-flick or hot-plate models of pain. In addition, its antinociception was
Previous phytochemical investigations carried out on this family have shown the presence of structurally and biogenetically diverse secondary metabolites, such as flavonoids, terpenes, sesquiterpenes, steroids, lignin, and phenols (4). In this study, we have therefore assessed the antinociceptive properties of the hydroalcoholic extract (HE) from the stems and leaves of Hedyosmum brasiliense in different assays of pain. Additionally, we have also described the isolation and chemical identification and also the peripheral, spinal, and supraspinal antinociceptive properties of the sesquiterpene lactone denoted ª13-hydroxy-8,9-dehydroshizukanolide (13-HDS; Fig. 1)º isolated from H. brasiliense.
not reversed by naloxone. The sesquiterpene lactone 13-hydroxy-8,9-dehydroshizukanolide, isolated from
H. brasiliense
already reported in other plant species (given by i.c.v.
and
i.p., i.t.,
or
routes) exhibited graded antinociception against acetic-
acid writhing and capsaicin-induced licking. Additionally, we have corrected some physico-chemical data already reported for this compound. It is concluded that both the extract and the sesquiterpene lactone isolated from
H. brasiliense
produced
marked antinociception in different models of chemical pain. The site of action involved in the antinociception of the 13-hy-
Fig. 1
13-Hydroxy-8,9-dehydroshizukanolide.
droxy-8,9-dehydroshizukanolide remains unclear, but the opioid pathway seems unlikely to be involved in its action. Key words: Hedyosmum brasiliense,
Choranthaceae, sesquiter-
pene, mice, antinociception, acetic acid, formalin and capsaicin tests.
Introduction
Mart. Ex. Miq. is a native plant that grows in central and southern Brazil, belonging to the Chloranthaceae family which consists of 4 genera. In the Americas, only the genus Hedyosmum can be found, and it consists of about 40 species of trees and shrubs (1). The folk medicine in Brazil recommends the infusion of H. brasiliense for the management of headaches, ovary diseases, and rheumatism (2), (3). Hedyosmum brasiliense
Planta Medica 65 (1999) 517±521 Georg Thieme Verlag Stuttgart New York ISSN: 0032-0943 ·
Materials and Methods Preparation of the crude extract, and isolation and chemical identification of the active compound
Botanical material was collected in Ilhota, state of Santa Catarina, Brazil, and was classified by Dr. Ademir Reis as Hedyosmum brasiliense Mart. Ex. Miq. A voucher specimen was deposited at the Barbosa Rodrigues Herbarium (HBR), at Itajaí, SC, under number 2543. Fresh stems and leaves (8 kg) were extracted exhaustively with 4:1 ethanol-water at room temperature (21 3 8C) for 15 days. The ethanol was evaporated and the extract was concentrated to the desired level and stored in the freezer at ±20 8C until use. UV and IR spectra were obtained using Hitachi U-2000 and Perkin Elmer PC FTIR instruments, respectively. The optical rotations were measured on a Polatronic-E Schmidt + Haensh polarimeter. The 1H(200 MHz) and 13C-NMR (50 MHz) spectra were obtained on a Bruker Ac 200F spectrometer, and chemical shifts [d (ppm)] were measured from TMS as an internal standard. HR-GCMS was performed on a Shimadzu GC-MS 0P 2000A (EI-MS).
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Planta Med. 65 (1999)
Ana P. Trentin, Adair R. S. Santos, Alessandro Guedes, Moacir G. Pizzolatti, Rosendo A. Yunes, and Joo B. Calixto
The extract of H. brasilense (450 g) was successively filtered on a column (15 90 cm) over silica gel 60 (900 g) (70± 230 mesh) eluted in a step-wise manner with hexane-AcOEt (100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 50:50, and 0:100, 750 ml each). The crude fractions eluted between hexane-AcOEt (90:10±80:20, 30 g) were chromatographed on a silica gel 60 column (2.5 45 cm) eluted with hexane-AcOEt gradient (100:0, 98:2, 96:4, 92:8, 90:10, 80:20, 70:30, 50:50, and 0:100, 150 ml each). The fractions eluted with hexane-AcOEt (90:10) produced colourless crystals (500 mg) identified as onoseriolide or 13-hydroxy-8,9-dehydroshizukanolide (13-HDS) (Fig. 1), m.p. 91 8C [a]25D: +528 (CHCl3, c 1.0). The molecular formula C15H18C3, was established by HRMS and the covalence and hybridization of the carbon atoms were well corroborated by 13C-NMR and DEPT experiments. Since 13C-NMR data of 13-HDS have not yet been reported in the literature, they are listed below as follows: 13C-NMR (50 MHz CDCl3): d = 17.0 (CH2, C-2), 21.5 (CH2, C-6), 22.0 (CH3, C-14), 22.4 (CH, C-3), 26.4 (CH, C-1), 40.0 (C, C-10), 55.1 (CH2, C-13), 61.9 (CH, C-5), 106.8 (CH2, C15), 123.0 (CH, C-9), 124.2 (C, C-11), 149.3 (C, C-8), 149.6 (C, C4), 150.0 (C, C-7), 170.2 (C=O), C-12). In spite of the fact that 1H-NMR data were shown to correspond with those of onoseriolide isolated from Onoseris albicans (5), the value of [a] is different from that given by Bohlmann et al. (5) for this compound in its oily form. Supporting our value, it should be noted that it is coherent with that proposed by Kawabata et al. (4) for the similar compound 8,9-dehydroshizukanolide, ([a]16D : +59.88, c = 1.02, CHCl3), isolated from Choranthus japonicus (Chloranthaceae), which exhibits the same configuration but lacks the hydroxy group attached to the non-chiral carbon atom located in position 13. We have synthesized the acetate of 13-HDS whose m.p. is 109±110 8C and which is also different to that given in literature (5) in relation to the acetate synthesized from the oily form of the indicated compound. It is important to note that in our study, we have obtained crystals, while Bohlmann et al. (5) obtained an oily compound. We do not have a clear explanation for this difference, but this compound, in consideration of the abovementioned facts, should have the configuration indicated by Bohlmann et al. (5) for onoseriolide. Animals
Male Swiss mice (25±35 g) were kept in a room of automatically-controlled temperature (23 2 8C) under a 12 h light-dark cycle. Food and water were freely available. Animals were acclimatised to the laboratory for at least 1 h before testing and were used once throughout the experiments. Abdominal constriction response caused by intraperitoneal injection of acetic acid
The abdominal constriction induced by acetic acid (0.6%) was carried out according to the procedures described previously (6), (7). Animals were pre-treated with vehicle (0.9% NaCl, 10 ml/kg) or with HE by i.p. (3±60 mg/kg) or p.o. (100± 800 mg/kg) routes, 30 and 60 min before testing, respectively. Other groups of animals were treated with 13-HDS intraperitoneally (3±60 mg/kg), intracerebroventricularly or intrathecally (10±100 mg/site), 30, 10, and 10min before acetic acid injection, respectively, as described previously (8±11). Control
animals received a similar volume of appropriate vehicle i.p. (10 ml/kg), i.c.v. (5 ml/site), or i.t. (5 ml/site). Formalin-induced licking
The procedure was similar to that described previously (7), (12). 20 ml of 2.5 % formalin solution (0.92 % formaldehyde) were injected intraplantarly in the right hindpaw. Animals received the HE (10±800 mg/kg) or vehicle only (0.9% NaCl, 10 ml/kg) given by i.p. or oral routes 30 and 60min before formalin injection, respectively. At the end of the experiments, the animals were killed by cervical dislocation and the paws were cut at the knee joint and weighed on an analytical balance. To assess the possible participation of the opioid system in the antinociceptive effect of HE, animals were pre-treated with naloxone (5 mg/kg, i.p.) 15 min before administration of HE (100 mg/kg, i.p.), morphine (5 mg/kg, s.c.), or vehicle (0.9% NaCl, 10 ml/kg, i.p.). The algesic responses caused by the formalin injection were recorded 30 min after drug administration. The other groups of animals received the naloxone, HE, morphine, or vehicle, 30 min before the formalin injection. Capsaicin-induced licking
The procedure used was similar to that described previously (7), (11), (13). After the adaptation period, 20 ml of capsaicin (1.6 mg/paw) were injected intraplantarly in the right hindpaw. Animals were treated with the HE from H. brasiliense, with 13-HDS (3±300 mg/kg, i.p.; 1±100 mg/i.c.v., or 10± 100 mg/i.t.) as described before. Control animals received a similar volume of the appropriate vehicle intraperitoneally (10 ml/kg), i.c.v. (5 ml/site), or i.t. (5 ml/site), 30, 10, and 10 min before capsaicin injection, respectively. Hot-plate test
The hot-plate test was used to measure response latencies according to the method described by Beirith et al. (10). In these experiments, the hot-plate (Ugo Basile, Model-DS 37) was maintained at 56 1 8C. The reaction time was recorded for control mice, for animals pre-treated with morphine (10 mg/ kg, s.c. used as positive control), or pre-treated with the HE (100 mg/kg, i.p.) or vehicle (0.9% NaCl, 10 ml/kg, i.p.). Animals were selected 24 h previously based on their reactivity in the model. A latency period of 30 s was defined as complete analgesia. Tail-flick test
A radiant heat tail-flick analgesiometer was used to measure response latencies according to the method described by Beirith et al. (10), with minor modifications. The reaction time was recorded for animals pre-treated with morphine (10 mg/ kg, s.c.), with the HE (100 mg/kg, i.p.), or vehicle (0.9% NaCl, 10 ml/kg, i.p.). Animals were selected 24 h previously according to their reactivity in the model. A latency period of 20 s was defined as complete analgesia.
Antinociception Caused by the Extract of Hedyosmum brasiliense and its Active Principle
Planta Med. 65 (1999)
Measurement of motor coordination
In order to evaluate the possible non-specific muscle-relaxant or central sedative effects of HE, mice were tested on the rotarod assay as reported previously (10). In these experiments the rota-rod (Ugo Basile, Model 7600) was used. The bar rotated at a constant speed of 22 revolutions per minute, and animals were selected 24 h previously by eliminating those mice which did not remain on the bar for two consecutive periods of 60 s. Animals were treated with HE (100 mg/kg, i.p.) or with vehicle (0.9% NaCl, 10 ml/kg, i.p.) 30 min before being tested. Results are expressed as the time (s) for which animals remained on the rota-rod. The cut-off time used was 60 s. Drugs
The drugs used were: acetic acid, formalin, and morphine hydrochloride (Merck AG, Darmstadt, Germany), capsaicin (Calbiochem, San Diego, California, USA) and naloxone hydrochloride (Dupont, Garden City, USA). The HE of H. brasiliense, morphine, and naloxone were dissolved in 0.9% NaCl solution just before use, except capsaicin and 13-HDS which were dissolved in ethanol and tween 80 plus 0.9% NaCl solution, respectively. The final concentration of tween 80 or ethanol did not exceed 5% and did not cause any effect per se. Statistical analysis
Results are presented as mean s.e. mean, except the ID50 values (i.e., the dose of the extract or compound that reduced the pain responses by 50% in relation to control group values), which are reported as geometric means accompanied by their respective 95% confidence limits. The ID50 values were determined by graphical interpolation from individual experiments using the least squares method. The statistical significance between groups was calculated by means of analysis of variance followed by Dunnett s multiple comparison test or Newman Kuels test. P values less than 0.05 (P < 0.05) were considered as indicative of significance. Results
The results of Figure 2 (A) show that the HE of H. brasiliense produced significant and graded antinociception when assessed against acetic acid-induced abdominal constrictions. The calculated mean ID50 value is shown in Table 1 and the inhibition observed was 91 4%. The antinociceptive action of the HE installed rapidly (30 min) and lasted for up to 120 min (results not shown). The HE caused no antinociception when administered orally up to 800 mg/kg (results not shown). The HE given by i.p. (but not by oral) route produced doserelated inhibition of both phases of the formalin test (Fig. 3, A and B). The estimated mean ID50 values for the first and second phases are presented in Table 1. The inhibitions obtained when the HE was administered by i.p. route were 71 4 and 91 9% for the first and second phases, respectively. However, the HE failed to affect formalin-induced edema formation (results not shown). The HE, given i.p., produced dose-related inhibition of capsaicin-induced pain (Fig. 2 B). The estimated mean ID50 value is presented in Table 1 and inhibition was 70 7%.
Effect of i.p. (*) or p.o. (l) injection of the hydroalcoholic extract of H. brasiliense on acetic acid-induced writhing (A) or capsaicin-induced licking (B). Each point represents the mean of 6 to 8 animals and the vertical bars indicate the S.E.M. the point (0) indicates the control values (animals injected with the 0.9% NaCl solution) and the asterisks denote the significance levels, when compared with control groups (ANOVA), ** p < .01.
Fig. 2
Comparison of the mean ID50 values for the antinociceptive actions of HE, 13-hydroxy-8,9-dehydroshizukanolide (13-HDS), aspirin, morphine, and dipyrone in several models of nociception in mice.
Table 1
Writhing Test HE HDS
Route
Aspirina
(mg/kg) (mg/kg) i.c.v. (mg/site) i.t. (mg/site) i.p. (mg/kg)
Formalin test
Route
HE
i.p.
(mg/kg)
Aspirina
i.p.
(mg/kg)
Dipyroneb
i.p.
(mg/kg)
Capsaicin test
Route
i.p. i.p.
HE HDS
First Phase (ID50) 31.1 (22.2±43.5) >200.0 54.3 (35.1±83.9)
Second Phase (ID50) 21.7 (13.6±34.6) 22.1 (13.8±37.6) 92.6 (82.3±104.3)
ID50
(mg/kg) 69.1 ( 40.6±117.7) (mg/kg) 20.9 ( 8.1±54.0) i.c.v. (mg/site) 34.5 ( 24.6±48.1) i.t. (mg/site) 7.5 ( 4.0±14.0) s.c. (mg/kg) 0.8 ( 0.6±1.1) i.c.v. (mg/site) 0.4 ( 0.3±1.0) i.p. (mg/kg) 72.9 ( 63.0±84.3) 49.2 ( 38.6±66.7) i.c.v (mg/site) i.t. (mg/site) 140.5 (105.4±210.8) i.p.
Dipyroneb
b Data
12.7 ( 8.7±19.0) 14.8 ( 9.6±22.7) 11.9 (10.0±14.1) 43.0 (34.2±54.0) 23.9 (13.1±43.8)
i.p.
Morphinea
a Data
ID50
from Vaz et al. (9). from Beirith et al. (10)
The HE did not affect the increase of latency response in either the hot-plate or the tail-flick tests (Table 2). Under similar conditions, morphine caused a significant and marked latency increase in the hot-plate and tail-flick assay (Table 2). The results of Table 3 show that the pre-treatment of animals with naloxone had no significant effect on the antinociception
519
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Planta Med. 65 (1999)
Ana P. Trentin, Adair R. S. Santos, Alessandro Guedes, Moacir G. Pizzolatti, Rosendo A. Yunes, and Joo B. Calixto Effect of morphine (s.c.) and HE (i.p.) in the hot-plate and tail-flick tests in mice.
Table 2
Fig. 3 Effect of i.p. (*) or p.o (l) injection of the hydroalcoholic extract of H. brasiliense on formalin-induced nociception in mice. The total time (mean S.E.M.) spent licking the hindpaw was measured in the first phase (0±5 min, A) and against the second phase (15± 30 min, B) after s.c. injection of formalin in the hindpaw. Each point represents the mean of six to eight animals and the vertical bars indicate the S.E.M. The point (0) indicates the control values (animals injected with the 0.9% NaCl solution) and the asterisks denote the significance levels, when compared with control groups (ANOVA), *p < .05, **p < .01.
Drugs
Dose (mg/kg)
Latency (s) Tail-flick
Hot-plate
Control Morphine HE
0 10.0 100.0
7.2 0.7 19.5 0.5** 9.6 1.0
5.7 0.5 24.0 1.5** 7.5 0.9
Each group represents mean S.E.M. of 6 to 10 animals. **P < 0.01 compared with the corresponding control values.
Effect of naloxone on the antinociception induced by morphine and the HE of H. brasiliense against formalin-induced pain in mice.
Table 3
Drugs Control Naloxone Morphine
Dose (mg/kg)
0 5 5 H. brasiliense 100 Naloxone + Morphine 5+5 Naloxone + H. brasiliense 5 + 100
Licking (s) 0±5 min
15±30 min
66.0 2.0 171.6 6.3 66.7 1.4 151.2 8.8 8.8 4.14** 0 0** 33.9 2.9** 20.1 11.4** 62.5 1.3+ 119.2 10.1+ NS 25.8 4.0 0 0NS
Each group represents mean s.e.m. of 6 to 10 animals. **P < 0.01 when compared to control value; +P < 0.01 when compared to agonist plus antagonists vs. agonist alone. NS, not significant statistically when compared to agonist plus antagonists vs. agonist alone.
Effect of i.c.v. (*), i.t. (&), or i.p. (l) injection of the 13-hydroxy-8,9-dehydroshizukanolide isolated from H. brasiliense against acetic acid-induced writhing (A) or capsaicin-induced licking (B). Each point represents the mean of 6 to 8 animals and the vertical bars indicate the S.E.M. The point (0) indicates the control values (animals injected with the 0.5% tween 80) and the asterisks denote the significance levels, when compared with control groups (ANOVA), **p < .01. Fig. 4
caused by the HE, but reversed the effect(s) caused by morphine against both phases of formalin-induced licking. The 13-HDS, administered by i.p., i.c.v., or i.t. routes, produced significant and graded antinociception when assessed against acetic acid-induced abdominal constriction (Fig. 4 A). The calculated mean ID50 values are shown in Table 1 and the inhibitions observed were: 89 2, 57 4 and 52 5% according to i.p., i.c.v., and i.t. routes, respectively. The same treatment with 13-HDS, given by i.p., i.c.v., or i.t. routes, also caused a doserelated and significant inhibition of the capsaicin-induced licking (Fig. 4 B). The calculated mean ID50 values are shown in Table 1 and the inhibitions observed were: 60 5, 94 4, and 61 5% when the compound was given i.p., i.c.v., or i.t., respectively. Because of the limited amount of the compound, it was not possible to test it orally against either acetic acidinduced abdominal constrictions or capsaicin-induced licking. Discussion
The results presented in the current study show, for the first time to our knowledge, that the HE and the main sesquiter-
pene lactone isolated from stems and leaves of H. brasiliense, administered systemically to mice, produce pronounced and dose-related antinociception when assessed against acetic acid-induced writhing response, capsaicin, and both phases of formalin-induced licking. However, the HE had no antinociceptive action when tested in two models of thermal pain, the tail-flick and hot-plate assays. Surprisingly, our results demonstrated that the active principle(s) present in the H. brasiliense failed to produce antinociception when given orally, suggesting its (their) poor oral absorption. The antinociceptive action caused by the HE of H. brasiliense set in rapidly and lasted for at least 2 h when assessed by acetic acid-induced abdominal constriction. Furthermore, the main active constituent present in this plant, the sesquiterpene lactone identified as 13-HDS, produced marked and dose-related spinal and supraspinal antinociceptive action. When compared with known standard antinociceptive drugs, the sesquiterpene 13-HDS, depending on the route of administration employed, was about 1.2- to 18.7-fold more active than aspirin, acetaminophen, or dipyrone. However, it was about 26- to 86-fold less active than the potent alkaloid morphine (9), (10). Our results confirm and also extend previous studies carried out with a plant belonging to the same genus, H. bonplandianum (1), indicating that the n-butanol extract and two glycosyl flavonoids isolated from H. bonplandianum, kaenpherol 3-O-[a-L-rhamnopyranosyl(1®6)b-D-glucopyranoside] and kaemferol 3-O-[b-D-glucopyranoside], exhibit analgesic action in mice. The possibility that such flavonoids may also be present in H. brasiliense remains to be seen.
Antinociception Caused by the Extract of Hedyosmum brasiliense and its Active Principle
Another interesting characteristic of the effect of the sesquiterpene lactone was its marked and dose-related antinociception when tested through systemic, spinal, and supraspinal routes against the neurogenic pain response caused by intraplantar injection of capsaicin into mice. The capsaicin test has been used to evaluate new analgesic drugs (11), (14±16). Usually, this model of nociception is quite resistant to the great majority of non-steroidal anti-inflammatory drugs, but it is very sensitive to morphine, dipyrone, and drugs that antagonise substance P or glutamate receptors (9±11), (14), (15). Also relevant are the findings showing that HE dose-dependently antagonises both phases of the formalin test. There is now a substantial amount of evidence indicating that most non-steroidal anti-inflammatory drugs are completely inactive in preventing the first phases of the formalin test (9), (10). Several reports now suggest that both capsaicin and formalin-induced licking are mediated by release of glutamate and substance P from sensorial neurons at the spinal cord (11), (13), (17). All the antinociceptive effects for the HE of H. brasiliense were observed at doses which had no effect on motor function of animals, as revealed by the complete lack of effect when the rota-rod apparatus was used for testing. The mechanism underlying the antinociceptive action of the HE seems to be unrelated to activation of the opioid system. The antinociceptive action of the HE, in contrast to that reported for morphine, was not reversed by naloxone, a non-selective opioid antagonist. Secondly, the HE of H. brasiliense was devoid of analgesic action when assessed in the thermal model of nociception, the tail flick, and hot-plate tests, under conditions where morphine had a marked antinociceptive effect. Furthermore, the hot-plate and tail-flick tests are commonly used to assess narcotic analgesics or other centrally-acting drugs, including sedatives and muscle relaxants or psychotomimetics (9), (10). In summary, we have demonstrated that the HE and the sesquiterpene lactone 13-HDS, isolated from the leaves and stem of H. brasiliense, exhibit dose-related antinociception when assessed in chemical but not thermal models of nociception in mice. The mechanisms involved in their action are not completely understood, but the involvement of the opioid pathway seems unlikely. Acknowledgements
The authors are indebted to Dr. Ademir Reis for botanical classification of H. brasiliense. This study was supported by grants from CNPq and FINEP (Brazil). A.P.T. is a undergraduate Medical student and A.R.S.S. and A.G. are PhD students in Pharmacology and Chemistry. They thank CNPq and CAPES for fellowship support. References
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Prof. Dr. Joo B. Calixto
Departamento de Farmacologia Universidade Federal de Santa Catarina Rua Ferreira Lima, 82 88015-420 Florianópolis SC Brazil E-mail:
[email protected] Fax: +55 48 2224164
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