q Birkha¨user Verlag, Basel, 1997 Inflamm. res. 46 (1997) 509–514 1023-3830/97/120509-06 $ 1.50+0.20/0
Inflammation Research
Anti-allergic effects and oedema inhibition caused by the extract of Drymis winteri K. S. Tratsk1 , M. M. Campos1 , Z. R. Vaz1 , V. C. Filho3 , V. Schlemper3 , R. A. Yunes2 and J. B. Calixto1 1
Department of Pharmacology, Universidade Federal de Santa Catarina, Rua Ferreira Lima 82, 88015-420 Floriano´polis, Brazil, Fax þ55 482 224164, e-mail:
[email protected] 2 Department of Chemistry, Universidade Federal de Santa Catarina, Rua Ferreira Lima 82, 88015-420 Floriano´polis, Brazil 3 Department of Chemistry, Nu´cleo de Investigac¸o˜es Quı´mico-Farmaceˆuticas, Universidade do Vale do Itajaı´, 88302-202 Itajaı´, Brazil Received 24 June 1997; returned for revision 19 August 1997; accepted by E. Neugebauer 24 September 1997
Key words: Drymis winteri extract – Medicinal plant – Allergy – Anti-inflammatory – Paw oedema
Abstract. Objective: To study the acute anti-inflammatory and anti-allergic properties of an extract of D. winteri. Material and Methods: Paw oedema induced in rats with various stimuli and anaphylactic shock in mice. Results: The hydroalcoholic extract (HE) of D. winteri (Winteraceae) (30 to 100 mg/kg, p.o., 1 h prior) inhibited carrageenan (300 mg/paw) and dextran (100 mg/paw)induced paw oedema formation in a dose-dependent manner, with mean ID50 values of 49 and < 30 mg/kg, respectively. The HE of D. winteri (30 to 100 mg/kg) also inhibited paw oedema induced by bradykinin (BK) (3 nmol), substance P (SP) (10 nmol) and PAF-acether (PAF) (10 nmol), in a dose-dependent manner, with mean ID50 values of 56, 63, and 58 mg/kg, respectively. However, the HE inhibited the rat paw oedema induced by prostaglandin E2 (PGE2 ) (10 nmol) (29 6 7 and 33 6 2% at 60 and 240 min) to a smaller extent, and had no effect on oedema elicited by histamine (100 nmol). In adrenalectomized animals, the inhibition by the HE of D. winteri (100 mg/kg, p.o., 1 h prior) of BK-elicited oedema (3 nmol/paw) was significantly smaller when compared with that observed in control animals. When assessed in rats actively sensitised to ovalbumin (OVO), the oedema caused by OVO (6 mg/paw) was significantly inhibited by HE of D. winteri (30 to 100 mg/kg, p.o.), with a mean ID50 of about 65 mg/kg. The HE of D. winteri (100 and 200 mg/kg, p.o.) significantly increased survival rate when assessed in anaphylactic shock in mice actively sensitised to the antigen. The protective effect was long-lasting, being observed for up to 15 h. Dexamethasone, used as positive control (0.5, 1 and 2 mg/kg) produced a long-lasting (up to 24 h) increase in the survival rate of the animals. Conclusions: These results confirm and extend our previous studies, and demonstrate the clear oral anti-inflammatory and anti-allergic properties of the active principle(s) present in the barks of D. winteri, thus confirming its reported medicinal use in folk medicine for the management of airway diseases.
Introduction Drymis winteri is a plant belonging to the Winteraceae family, native to the south of Brazil and some other countries of South America. Folk medicine recommends the infusion of its barks or leaves for the treatment of different inflammatory diseases, such as asthma, allergy and bronchitis. This plant is also used as an antispasmodic and antipyretic [1], and is sometimes used for the treatment of cancer [2]. Although a great amount of evidence supports the traditional use of this plant in folk medicine, allied to chemical isolation and identification of several constituents in this and other species of the genus such as flavonoids [3], sesquiterpenoids [4–6] and terpenoids [7, 8], very little information is available regarding the pharmacological actions of its extract or active principles. In a recent study [9] we showed that the HE of barks of D. Winteri antagonis, in a concentration-dependent and reversible manner, the trachea contractions induced by several mediators involved in asthma and allergy, namely tachykinins (through NK2 but not NK1 receptors), bradykinin, the stable analogue of thromboxane A2 , U46619 and prostaglandin E2 and partially contraction elicited by histamine, without affecting the contractions caused by acetylcholine. In addition, the HE of D. winteri inhibited, in a concentration-dependent manner, contractions induced by ovalbumin and compound 48/80 in guinea pig trachea from actively-sensitised and normal animals, respectively [9]. Such data confirms, at least partly, the medicinal properties of this plant reported in folk medicine, and strongly suggests a useful potential therapeutic application of its active principle(s) as an anti-inflammatory, anti-allergic and antiasthmatic preparation. We therefore decided to investigate, in the present series of experiments, the in vivo oral effect of
Correspondence to: J. B. Calixto
m
K. S. Tratsk et al.
510
Inflamm. res.
the HE of D. Winteri on rat paw oedema induced by several mediators involved in the inflammatory and allergic processes, and also some phlogistic substances such as carrageenan and dextran. Attempts have also been made to determine whether the active principle(s) present in the HE of this plant exhibit in vivo anti-allergic properties in animals which have been actively sensitised to ovalbumin.
(0.25 g/kg, i.p.), the dorsal region was incised (approximately 2 cm), and both adrenal glands were removed [13–15]. After surgery, animals were returned to their cages, with free access to food and drink, but water was substituted by 0.9% NaCl solution to maintain physiological sodium plasma concentrations. After seven days, animals received the HE of D. winteri (100 mg/kg, p.o.) or saline solution (10 ml/kg, p.o.) 60 min before the experiments. The oedema induced by bradykinin (3 nmol/paw) was managed and evaluated as described above.
Materials and methods
Evaluation of anti-allergic activity
Plant material and preparation of hydroalcoholic extract
Anaphylatic shock in sensitised mice caused by chicken egg albumin. Male Swiss mice (18–20 g) were actively sensitised by subcutaneous injection of 0.2 ml of saline solution (0.9%) containing 20 mg of OVO dispersed in 1 mg of Al(OH)3 [12]. Fourteen days after this, animals received a new injection of 20 mg of OVO. Seven days after the last injection of OVO, animals were treated with different doses of HE of D. winteri (100 to 200 mg/kg p.o.) or with saline solution (0.1 ml/10 g, control group). In another group of experiments, the animals were treated with HE of D. winteri (100 mg/kg), 1, 4, 15, or 24 h before i.v. injection of OVO to analyse the time-course of the anti-allergic effect of this plant. Animal death was defined as the instant at which respiration ceased. The number of animals surviving was quantified after a period of 60 min after injection of OVO (500 mg/kg, i.v.). In a separate series of experiments, the animals were treated with dexamethasone (0.5, 1 and 2 mg/kg, s.c.) 2, 24 or 48 h before, and it was used as a positive control.
Botanical material was collected in Bom Retiro, State of Santa Catarina, Brazil, and was classified by Prof. Leila da Grac¸a Amaral (Department of Botany, Universidade Federal de Santa Catarina). Samples of the plant were deposited in the Herbarium Flor at this University. The barks of D. winteri were minced and extracted with 50% ethanol-water in the proportion of 1:3 (W/V), being maintained at room temperature (21 6 3 8C) for 15 days. The solvent was fully evaporated and the extract was concentrated to the desired level and stored at ¹20 8C. The extract was dissolved in 0.9% NaCl solution to the desired concentration just before use.
Animals Non-fasted male Wistar rats (120–150 g) or Swiss mice (20–30 g) from our department, housed at 22 6 2 8C under a 12 h:12 h light-dark cycle, were used. Food and water were freely available. The experiments reported were carried out in accordance with current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals [10].
Drugs The following drugs were used: bradykinin, prostaglandin E2 , lambda carrageenan grade IV, dextran, substance P, histamine, captopril, chicken egg albumin (ovalbumin), 2,2,2-tribromoethanol, dexamethasone, compound 48/80 (Sigma Chemical Co., St. Louis, MO, USA) and platelet aggregating factor (PAF) (Bachem, Switzerland). The stock solutions for all peptides and drugs used were prepared in PBS (1–10 mM) in siliconized plastic tubes, maintained at ¹18 8C, diluted to the desired concentration just before use. The other drugs were prepared daily in 0.9% w/v NaCl.
Measurement of rat paw oedema Experiments were conducted using non-fasted male Wistar rats (140– 180 g) kept in a room controlled for temperature (22 6 2 8C) and illumination (12 h on and 12 h off). Under anaesthesia with 2,2,2tribromoethanol (0.25 g/kg, i.p.), animals received a 0.1 ml intraplantar injection, in one hindpaw, of phosphate-buffered saline (PBS; composition: NaCl 137 mmol/l; KCl 2.7 mmol/l and phosphate buffer 10 mmol/l) containing carrageenan (300 mg/paw), dextran (100 mg/paw), compound 48/80 (12 mg/paw), bradykinin (BK, 3 nmol/paw), substance P (SP, 10 nmol/paw), platelet-aggregating factor (PAF, 10 nmol/paw), prostaglandin E2 (PGE2 , 30 nmol/paw) or histamine (His, 100 nmol/ paw). The contralateral paw received the same volume of PBS alone and served as a control. In experiments with bradykinin, animals were pre-treated with captopril (5 mg/kg, s.c.) 1 h prior to prevent the action of kininases [11]. For allergic oedema, the animals were actively sensitised by subcutaneous injection of 50 mg of OVO dispersed in 5 mg of Al(OH)3 dissolved in 0.1 ml of the saline solution [12]. After 14 days, oedema was induced by injection of 6 mg of OVO into the left hindpaw. Animals were treated orally with HE of D. winteri (30 to 100 mg/kg) 60 min before. In other group of experiments, the animals were treated with dexamethasone (0.5 mg/kg, s.c., 24 h) that was used as a positive control. Oedema was measured by use of a plethysmometer (Ugo Basile) at several time-points after injection of irritant substances or inflammatory mediators, and was expressed as the difference in ml between both paws.
Statistical analysis The results are presented as the mean 6 SEM, except for the ID50 values (i.e. the dose of the extract that reduced oedema formation by 50% relative to control value) which are reported as geometric means accompanied by their respective 95% confidence limits. The ID50 values were determined by use of the least-square method for individual experiments. Statistical comparison of the data was carried out by the use of analysis of variance followed by Dunnett’s test or unpaired Student’s t-test, when indicated. The x2 -test was used to compare the survival rate in the group treated with the vehicle and the groups treated with drugs. p-Values of less than 0.05 were considered significant [16].
Results The results of Figure 1 show the effect of HE of D. Winteri (30 to 100 mg/kg p.o.) on rat paw oedema caused by intraplantar injection of carrageenan at different intervals of time. The treatment of animals with HE of D. winteri (30 to 100 mg/kg, p.o.), 1 h beforehand, inhibited in a dose- and time-dependent manner, carrageenan (300 mg/paw)-induced oedema (percentage of inhibitions of 72 6 1, 88 6 1, 73 6 3, 67 6 2% at 30, 60, 120 and 240 min after the irritant injection, respectively). The calculated mean ID50 value (and 95% confidence limits) determined at the 4 h interval point,
Adrenalectomy In order to investigate the possible participation of endogenous glucocorticoids in the oedema inhibition caused by the HE of D. winteri, animals were anaesthetised with 2,2,2-tribromoethanol
m
Vol. 46, 1997
Anti-allergy and oedema inhibition by Drymis winteri
511
Fig. 1. Effect of treatment with HE of Drymis winteri (B 30; A 60 and X 100 mg/kg, p.o.) on rat paw oedema induced by intraplantar injection of 300 mg of carrageenan 1 h (A), 2 h (B) or 4 h beforehand (C). Panels D and E represent the same treatment 1 h before, but in the rat paw oedema induced by dextran (100 mg) or by compound 48/80 (12 mg). The control responses are shown in all panels (W). Each point represents the mean of 6 animals and the vertical lines the SEM. Significantly different from respective control value *p < 0.05.
Fig. 2. Effect of treatment with HE of Drymis winteri (B 30; A 60 and X 100 mg/kg, p.o.) 1 h before, on rat paw oedema induced by intraplantar injection of bradykinin (3 nmol, (A); substance P (10 nmol, (B); Paf-acether (10 nmol, (C); prostaglandin E2 (30 nmol, (D) or histamine (100 nmol, (E). The control responses are shown in all panels (W). Each point represents the mean of 6 animals and the vertical lines the SEM. Significantly different from respective control value *p < 0.05.
by the treatment of animals with HE of D. winteri, having a mean ID50 value (1 h) of < 30 mg/kg. However, these effects were not dose-dependent. The percentages of inhibitions of the responses were 44 6 1, 48 6 1, 64 6 5 and 64 6 2% at 30, 60, 120 and 240 min, respectively (Fig. 1D). In contrast, the rat paw oedema induced by compound 48/80 (12 mg/ paw) was not inhibited at any time by treatment of animals with D. winteri extract (100 mg/kg, p.o.) (Fig. 1E).
was 49 (37–64) mg/kg (Fig. 1A). When the HE of D. winteri was administered 2 and 4 h before the injection of carrageenan, it still inhibited the oedema. The percentages of inhibitions observed in animals treated 2 h prior were: 32 6 5, 34 6 5 and 32 6 3% at 60, 120 and 240 min, respectively (Fig. 1B), and for 4 h they were 35 6 4, 30 6 3 and 19 6 2 for 30, 60 and 120 min (Fig. 1C). Also, dextran (100 mg)-induced rat paw oedema was significantly inhibited
m
K. S. Tratsk et al.
512
Inflamm. res.
was inhibited significantly by treatment with HE of D. winteri (100 mg/kg, p.o., 1 h beforehand) (Fig. 3). However, the inhibition caused by HE of D. winteri was significantly reduced (22 6 3, 35 6 4, 36 6 2 for 20, 30 and 60 min) when compared with inhibition caused by the HE in control animals. When assessed in rats actively sensitised to OVO, the oedema caused by intraplantar injection of the antigen (OVO, 6 mg/paw) was significantly inhibited by the HE of D. winteri (30 to 100 mg/kg, p.o.) (Fig. 4A). The calculated mean ID50 value (calculated at 30 min) was 65 (61.0–70.0). Dexamethasone (0.5 mg/kg, s.c., 24 h prior), used as a positive control, largely prevented (77:5 6 2%) OVOinduced paw oedema in animals sensitised to OVO (Fig. 4B). The HE of D. winteri (100 mg/kg, p.o.), administered 1 to 24 h beforehand, caused an increase in the survival rate of the animals. However, it was more effective when given 15 h before anaphylatic shock induced by OVO (500 mg/kg, i.v.) in mice (p < 0.05) (Fig. 5A). The protective effect caused by the HE of D. winteri was long-lasting, being observed up to 15 h after its administration (70% of protection). The treatment of animals with HE of D. winteri (200 mg/kg, p.o.), 15 h after its administration, also increased significantly the survival rate of animals (Fig. 5B). The treatment of animals with the positive control drug dexamethasone (0.5, 1 and 2 mg/kg, 24 h beforehand) significantly increased the survival rate of animals (Fig. 5D), an effect that lasted for up to 24 h (Fig. 5C).
The treatment of animals with HE of D. winteri (30 to 100 mg/kg, p.o. 1 h beforehand) significantly inhibited the oedema induced by several mediators of inflammation such as BK (3 nmol/paw) (Fig. 2A), substance P (10 nmol/paw) (Fig. 2B) or PAF-acether (10 nmol/paw) (Fig. 2C). The calculated mean ID50 values (and the 95% confidence limits) for these effects were 56 (47–67) mg/kg for BK (at 20 min), 63 (56–70) mg/kg for SP (at 1 h) and 58 (44–72) mg/kg for PAF (at 30 min). However, the same treatment with HE of D. winteri inhibited, to a lesser extent, PGE2 (10 nmol)-induced oedema (maximal inhibitions of 29 6 7 and 33 6 2% at 60 and 240 min) (Fig. 2D), but did not significantly affect the oedema caused by histamine (100 nmol) (Fig. 2E). In animals that had been adrenalectomized bilaterally seven days prior, the oedema caused by BK (3 nmol/paw)
Discussion The results of the present study confirm and extend our previous data [9] demonstrating that the active principle(s) present in the HE of the barks of D. winteri exhibit significant oral inhibition in oedema formation, showing significant anti-allergic properties when assessed in activelysensitised animals. Evidence has suggested that the pro-inflammatory peptides [17, 18], such as tachykinins [17, 19, 20], kinins [19, 21] and some metabolites derived from arachidonic acid
Fig. 3. Effect of treatment with HE of Drymis winteri on rat paw oedema induced by 3 nmol of bradykinin in bilaterally adrenalectomized rats (X 100 mg/kg, p.o.) or in saline treated animals (O). Each point represents the mean of 6 animals and the vertical lines the SEM. Significantly different from respective control value *p < 0.05.
Fig. 4. Effect of treatment with HE of Drymis winteri (B 30; A 60 and X 100 mg/kg, p.o.) on rat paw oedema, induced by 6 mg of ovalbumin in sensitized animals (A). (B) Effect with 0.5 mg/kg s.c. of dexamethasone (24 h prior). The control responses are shown in all panels (W). Each point represents the mean of 6 animals and the vertical lines the SEM. Significantly different from respective control value *p < 0.05.
m
Vol. 46, 1997
Anti-allergy and oedema inhibition by Drymis winteri
513
interaction with histamine receptors and/or its action. In an attempt to provide additional evidence to demonstrate that active principle(s) present in the HE of D. winteri might be interacting with histamine release, as reported previously for the guinea pig trachea in vitro [9], we have assessed its effect on oedema caused by compound 48/80, a known histamine releaser [26, 27]. Results of the present study clearly demonstrate that the HE of D. winteri did not affect the oedematogenic response induced by compound 48/80. Such findings contrast somewhat with our in vitro study [9], and reveal that when given in vivo the active principle(s) of D. winteri is unable to attenuate 48/80-mediated oedema formation. In our previous study [9], we demonstrated that the HE of D. winteri produced a concentration-dependent inhibition of OVO-mediated contraction in guinea pig trachea from animals that had been actively sensitised to the antigen. These findings led us to examine in the present study whether the active principle(s) of D. winteri also have anti-allergic properties in vivo. At a similar dose where the HE inhibited paw oedemas induced by several inflammatory mediators, it consistently attenuated ovalbumin-induced oedema formation in the paw of rats that had been actively sensitised to this antigen. The HE of D. winteri given orally to mice, like dexamethasone (used as a positive control), produced a significant increase in survival rate when assessed in anaphylactic shock in mice actively sensitised to OVO. Taken together, these and our previous in vitro findings demonstrate that the HE of this plant exhibits a very interesting anti-allergic action. The mechanisms by which the active principle(s) of D. winteri exert its (their) action are currently unclear. However, it seems reasonable to suggest an action of the active principle(s) at the receptor and/or second messenger level, related to the mechanism of proinflammatory mediators [9], and interaction with certain cytokines or nitric oxide pathways, among others, might be involved. However, additional studies are clearly necessary to confirm such possibilities. The difference observed in the short duration of action for the HE in inhibiting rat paw oedema (up to 4 h) compared with the long-lasting (up to 15 h) inhibition in the anaphylactic shock in mice is intriguing. However, several differences could account for these results, for example: 1) the difference in the animal species used, 2) involvement of different chemical mediators in these models, and 3) while the rat paw oedema is an acute and local phenomenon, the anaphylactic shock in mice involves a systemic action. Regarding the active constituent(s) responsible for the anti-oedema and anti-allergic actions of the HE of D. winteri, we have recently identified and analysed in vitro some secondary metabolites of this species [28]. When tested in the guinea pig trachea in vitro, the sesquiterpene polygodial at micromolar concentrations shows essentially the same pharmacological profile reported earlier for the HE of D. winteri. Therefore, polygodial [30] and possibly other minor terpenes present in the barks of D. winteri which have not yet been chemically characterised, seem to be the main constituence responsible for the reported antagonistic action of the extract against contraction elicited by tachykinins, kinins and prostanoids. Unpublished results from our group have demonstrated that polygodial, given either systemically or locally, inhibits
Fig. 5. Time (A, C) and dose-dependent (B at 15 h, D at 24 h) effects of treatment with HE of Drymis winteri (100 mg/kg, p.o.) (A, B) or with dexamethasone (C, D) on anaphylactic shock in sensitized mice. Each column represents the mean of 8 animals. Significantly different from respective control value *p < 0.05.
pathway [22, 23], are intimately implicated in the pathogenesis of the airway diseases, particularly asthma and allergy [23, 24]. In line with our previous findings reported for the guinea pig trachea in vitro [9], the HE of D. winteri (given orally) consistently attenuated, in a dose-dependent manner, the paw oedema induced by several mediators known to participate in inflammatory and allergic processes [21], including substance P, BK, PAF-acether and, to a lesser extent, prostaglandin E2 . Also relevant are the results showing that at a similar dose the HE of D. winteri also significantly attenuated carrageenan and dextran-induced paw oedema. It has been well documented that several inflammatory mediators, such as kinins, prostaglandins, histamine and PAF-acether, are involved in the oedematogenic responses elicited by carrageenan and dextran [25]. Taken together, such results show that the active principle(s) present in the HE of D. winteri inhibited paw oedema formation dependent on neutrophil mobilization [28, 29]. The reason for the extensive inhibition reported for the HE of D. winteri against oedema induced by bradykinin, substance P and PAF is not clear at this stage of our work, and deserves further investigation through the use of the isolated active principle(s) present in this plant. Our results also demonstrate that the anti-oedema action of the HE of D. winteri seems to be, at least in part, indirect, involving the release of endogenous glucocorticoids from adrenal glands, as the previous adrenalectomy of animals partially attenuated its anti-oedema action. Also relevant in the present study is the fact that, in contrast to the results reported in our previous studies on the guinea pig trachea in vitro [9], the HE of D. winteri, at doses which markedly prevented the paw oedema induced by various mediators, failed to affect the oedema caused by histamine. Such findings are consistent with the view that its pharmacological actions in vivo are probably independent of
m
K. S. Tratsk et al.
514
paw oedema induced by carrageenan, substance P and bradykinin, and the ear oedema formation induced by croton oil and capsaicin in mice. Furthermore, polygodial reveals systemic, spinal and supraspinal antinoception when assessed in thermal and chemical models of pain in mice, being about 6 to 154-fold less potent than morphine. The mechanisms of analgesic action of polygodial involve, at least in part, an interaction with an opioid system, since its analgesic response is largely reversed by the non-selective opioid antagonist naloxone (unpublished results). In conclusion, results of the present study are in line with our previous findings [9, 30], proving that the active principle(s) present in the barks of D. winteri, a reported medicinal plant employed for the treatment of asthma and allergy, demonstrate marked and relatively long-lasting oral paw oedema inhibition induced by several mediators involved in asthma and allergy, such as the pro-inflammatory peptides substance P and bradykinin, as well as PAF-acether, and to a lesser extent, prostaglandin E2 . Also the HE of D. winteri, given orally, showed anti-allergic action in both rats and mice actively sensitised to ovalbumin. Thus, the active constituent(s) present in the barks of D. winteri may be useful for the development of a new oral drug of potential relevance to the management of asthma and allergy.
Inflamm. res.
[10] Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109. [11] Campos MM, Calixto JB. Involvement of B1 and B2 receptors in bradykinin-induced rat paw oedema. Br J Pharmacol 1995;114: 1005–13. [12] Amorim CZ, Cordeiro RSB, Vargaftig BB. Involvement of platelet activating factor in death following anaphylactic shock in boosted and in unboosted mice. Eur J Pharmacol 1993;235: 17–22. [13] Flower RJ, Parente L, Persico P, Salmon JA. A comparison of the acute inflammatory response in adrenalectomized and shamoperated rats. Br J Pharmacol 1989;87:57–62. [14] Campos MM, Mata LV, Calixto JB. Expression of B1 kinin receptors mediating paw edema and formalin-induced nociception. Modulation by glucocorticoids. Can J Physiol Pharmacol 1995;73:812–9. [15] Landucci ET, Antunes E, Donato JL, Faro R, Hyslop S, Marangoni S, et al. Inhibition of carrageenin-induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2 . Br J Pharmacol 1995;114:578–83. [16] Nair NGK. Erroneous applications of the chi-square test. Indian J Med Res 1987;86:537–45. [17] Maggi CA, Gianchetti A, Dey RD, Said SI. Neuropeptides as regulators of airway function: Vasoactive intestinal peptide and the tachykinins. Physiol Rev 1995;75:277–322. [18] Frossard N, Fajac. Airway neuropeptides – what is their role in physiopathology? Allerg Immunol (Paris) 1995;27:74–9. [19] Bertrand C, Geppetti P. Tachykinin and kinin receptor antagonists: Therapeutic perspectives in allergic airway disease. Trends Pharmacol Sci 1996;17:255–9. [20] Joos GF, Kips JC, Peleman RA, Pauwels RA. Tachykinin antagonists and the airways. Arch Int Pharmacodyn Ther 1995; 329:205–19. [21] Goldie RG, Pedersen KE. Mechanisms of increased airway microvascular permeability: Role in airway inflammation and obstruction. Clin Exp Pharmacol Physiol 1995;22:387–96. [22] DuBuske LM. Mediator assays and modulation of inflammation in asthma: Introduction. Allergy Proc 1995;16:55–8. [23] Thien FCK, Walters EH. Eicosanoids and asthma: An update. Prostaglandins Leukot Essent Fatty Acids 1995;52:271–88. [24] Emanuel MB, Howarth PH. Asthma and anaphylaxi: A relevant model for chronic disease? An historical analysis of directions in asthma research. Clin Exp Allerg 1995;25:15–26. [25] Brito FB. Pleurisy and pouch models of acute inflammation. In: Alan AR, editor. Pharmacological methods in the control of inflammation. Dagenhem: Rhone Poulenc LTC, 1989:173–228. [26] Vinegar R, Truax Jf, Selph JI. Johnston P, Venable AI, Mc Kenzie KK. Pathway to carrageenan-induced inflammation in the hind limb of the rat. Fed Proc 1987;46:118–26. [27] Fechter M, Egger D, Auer H, Popper H. Experimental eosinophilia and inflammation the effect of various mediators and chemoattractants. Exp Pathol 1986;29:153–8. [28] Paton WDM. Compound 48/80: A potent histamine liberator. Br J Pharmacol 1951;6:499–508. [29] Maling HM, Webster ME, Williams MA, Saul W, Anderson W. Inflammation induced by histamine, serotonin, bradykinin and compound 48/80 in the rat: Antagonists and mechanisms of action. J Pharmacol Exp Ther 1974;191:300–10. [30] El Sayah M, Cechinel Filho V, Yunes RA, Pinheiro TR, Calixto JB. Action of polygodial, a sesquiterpene isolated from the bark Drymis winteri, on agonist-induced contractions of the guinea pig ileum and trachea in vitro. Eur J Pharmacol (In Press).
Acknowledgements. The authors are indebted to Leila da Grac¸a Amaral for the botanical classification of Drymis winteri. This study was supported by grants from Conselho Nacional de Pesquisa (CNPq) and Financiadora de Estudos e Projetos (FINEP), Brazil. K. S. Tratsk and M. M. Campos are undergraduate and postgraduate students receiving a grant from CNPq.
References [1] Morton JF. Atlas of medicinal plants in middle America-Bahamas to Yucatan. Springfield Thomas, 1981:457–63. [2] Riccieri TMN. Bibliografia de plantas medicinais. Jardim Botaˆnico do Rio de Janeiro 1981;1:224. [3] Torres R, Pardo F, Velasco MV. Flavonoid from Drymis winteri. Fitoterapia 1992;6:553–8. [4] Appel HH, Brooks CJW, Overton KH. The constitution and stereochemistry of drimenol, a novel bicyclic sesquiterpenoid. J Chem Soc 1959;673:3322–32. [5] Corte´s MM, Ovarzu´n LM. Tadeonal and isotadeonal from Drymis winteri. Fitoterapia 1981;52:33–5. [6] Brown GD. Drimendiol, a sesquiterpene from Drymis winteri. Phytochemistry 1994;35:975–7. [7] Apple HH, Dohr R. Terpenos del canelo chileno III. Scientia 1958;25:135–42. [8] Cruz A, Silva M. Further terpenoids and phenolics of Drymis winteri. Phytochemistry 1973;12:2549–50. [9] El Sayah M, Cechinel Filho V, Yunes RA, Calixto JB. Action of the extract of Drymis winteri on contraction induced by inflammatory mediators, compound 48/80 and ovalbumin of the guinea pig trachea in vitro. Gen Pharmacol 1997;28:699–704.
m
