Analgesic and antiinflammatory effects of chalcones isolated from Myracrodruon urundeuva Allemao.

Summary

The present work showed analgesic and antiinflammatory activities from a fraction containing three dimeric chalcones (chalcone enriched fraction -- CEF), isolated from the stem-hark ethyl acetate extract of Myracrodruon urundeuva Allemao (Anacardiaceae). M. urundeuva is a popular medicinal plant used widely in Northeast Brazil, mainly as a topical female genital tract antiinflammatory. We observed that the CEF (5 and 10 mg/kg body wt., i.p. or p.o.) inhibited acetic acid-induced abdominal contractions in mice. In the formalin test, the CEF (5 and 10 mg/kg body wt.) was more effective intraperitoneally and inhibited predominantly the second phase of response. Naloxone reversed this effect, indicating an involvement of the opioid system. The CEF (10 and 20 mg/kg body wt.) also increased the reaction time to thermal stimuli in the hot-plate test in mice, after i.p. but not after p.o. administration. In the carrageenan-induced paw edema test in mice, the CEF (20 and 40 mg/kg body wt.) decreased paw volume signifi cantly, after i.p. administration 2-4 hours after carrageenan injection. The CEF (40 mg/kg body wt.) was also active orally during the same period of time. The present work is the first report on peripheral and central analgesic effects and antiinflammatory activity of natural dimeric chalcones.

Key words: Myracrodruon urundeuva, dimeric chalcones, analgesic and antiinflammatory effects

Introduction

Myracrodruon urundeuva Allemao (Anacardiaceae) is a 10- to 15-m high tree common in the Brazilian Northeastern "caatinga" (shrub forest), especially in Ceara state. It is also found in the Brazilian states of Bahia, Minas Gerais, and Goias. An aqueous extract from the stem bark of this plant is used popularly in gynecology as a female genital tract antiinflammatory. The plant is also used in the treatment of respiratory and urinary tract diseases, hemoptysis, metrorrhagias and diarrheas, as an infusion or decoction, But is mostly used topically in post-partum gynecological treatment. It is also used in the treatment of skin wounds.

Previous work (Menezes, 1986) showed that aqueous as well as hydroalcoholic extracts from M. urundeuva stem bark exert potent antiinflammatory activity in experimental models of acute and sub-acute inflammation. The hydroalcoholic extract also showed hepatoprotective, anti-diarrheal and anti-ulcer activities (Morais et al., 1999). Chemical fractionation of the ethyl acetate extract yielded seven fractions, among which two showed pharmacological activity. One of them was a chalcone-enriched fraction (CEF), while the other presented catechic tannins as its main constituents. We have shown that both fractions are somewhat involved in the pharmacological activity of the plant (Viana et al., 1997).

Chalcones and dihydrochalcones belong to the class of flavonoids which, apart from their antioxidant activity, are known for their ability to strengthen capillary walls, thus assisting circulation and helping to prevent and treat bruising, varicose veins, bleeding gums and nosebleeds. They may also be useful in the treatment of heavy menstrual bleeding with no apparent cause. A third beneficial effect of some flavonoids, such as quercetin, rutin, curcumin, silymarin and green tea polyphenols, is their reputed antiinflammatory effect, which may be related to their ability to inhibit cyclooxygenase and lipooxygenase enzymes. Cyclooxygenase and lipooxygenase enzymes act on arachidonic acid metabolism in cell membranes to form potent inflammatory prostaglandins, some of which promote swelling and, possibly, symptoms such as headaches, rashes and joint pains (Zuanazzi, 1999).

Since phytomedicines from M. urundeuva are popular remedies in Northeast Brazil, and considering that clinical studies (Melo et al., 1998) revealed an absence of significant toxic effects in the plant elixir, the objectives of the present work were to study analgesic and anti-inflammatory activities of chalcones isolated from the stem bark of M. urundeuva, in order to clarify their mechanism of action.

Materials and Methods

Plant Material

Myracrodruon urundeuva Allemao was originally collected near the city of Iguatu, Ceara, and identified by Dr. Afranio Fernandes, of the Department of Biology of the Federal University of Ceara. It is presentely cultivated in the Medicinal Plant Garden of the same University. A voucher specimen is deposited at the Prisco Bezerra Herbarium under the number 14.999.

Isolation of Chalcones The chalcone-enriched fraction (CEF) was obtained from the ethyl acetate extract prepared with 5 kg of M. urundeuva ground stem bark, as described previonsly (Viana et al., 1995). The material was treated previously with hexane to remove lipid substances. The chromatographic fractionation of the ethyl acetate extract on a silica gel column resulted in seven fractions, after elution with chloroform, chroloform-acetone (9:1; 8:2; 7:3; 1:1), acetone, and methanol. Two fractions presented anti-inflammatory activity according to the pharmacological monitoring. Preliminary chemical tests showed that one fraction presented a predominance of chalcone type of compounds and the other mainly catechic tannins. The isolation of the chalcone type fraction compounds was performed using chromatographic techniques developed for that purpose (Bandeira, 2002), through the utilization of corn starch as the column support and elution with a mixture of dichloromethane/methanol (95:5). Through chromatographic procedure, three compo unds were isolated from that fraction (CEF). The analytical procedure used for structural identification of these compounds involved RMN [H.sup.1] and RMN [C.sup.13] (HBBD and DEPT [theta] = 135), with the contribution from bidimensional spectra of a homonuclear correlation of hydrogen and hydrogen ([H.sup.1], [H.sup.1]-COSY), as well as a heteronuclear correlation of hydrogen and carbon H, [C.sup.13]-[COSY-.sup.n][J.sub.CH] [n = 1 (HMQC) and n = 2 and n = 3, corresponding to HMBC and [H.sup.1], [H.sup.1]-NOESY, respectively. The precise attribution of chemical displacements of carbon and hydrogen atoms revealed, for the first time, that the CEF is composed of three dimeric chalcones, named urundeuvine A, B and C, the chemical structures of which are shown in Figure 1.

Pharmacological tests

Acetic acid-induced writhing: in this model (Koster et al., 1959), groups of 8-29 female mice each were administered 0.6% acetic acid (10 ml/kg body wt., i.p.), and the number of abdominal contractions was registered over 20 min, starting 10 min after acetic acid injection. Animals were treated intraperitoneally or orally with GEF (5 and 10 mg/kg body wt.), 30 or 60 mm before acetic acid administration, respectively. Distilled water (vehicle) was used i.p. or p.o. in control animals.

* Formalin test: in this test, licking time in seconds was registered from 0-5 min (first phase) and from 20-25 min (second phase), after the intraplantar administration of formalin (20 [micro]l of a 1% v/v solution) in the right hind paw (Hunskaar et al., 1985; Hunskaar and Hole, 1987). A total of 102 female Swiss mice (20-25 g each) was treated intraperitoneally or orally with the CEF, 30 or 60 mm before formalin injection, respectively. The possible involvement of the opioid receptor in CEF antinociceptive action was determined by the subcutaneous administration of naloxone (2 mg/kg body wt.), 15 mm before the CEF (10 mg/kg body wt., i.p.) or morphine (5 mg/kg body wt., i.p.) injections, followed by the administration of formalin 30 min later.

* Hot plate test: for the hot plate test (Eddy and Leimbach, 1953), groups of 9 to 18 female Swiss mice (20-25 g each) were treated with CEF (5-20 mg/kg body wt., i.p. or p.o.) or morphine (5 mg/kg body wt., i.p.) as standard. Measurements were performed before (0 time) and 30, 60 and 90 min after drug administration, with a cut-off time of 30 sec to avoid paw lesions.

* Carrageenan-induced paw edema: a total of 80 female Swiss mice (20-25 g each) was treated with the intraplantar injection of carrageenan 1% (0.1 ml/paw) 30 or 60 min before the intraperitoneal or oral administration of CEF (5 to 40 mg/kg body wt., i.p. or p.o.), respectively. Indomethacin (10 mg/kg body wt., p.o.) and vehicle (distilled water, 1 ml/l00 p.o. or i.p.) were also used as standard and control, respectively. Paw volume was measured using a plethysmometer (Ugo Basile, Italy) before and 1, 2, 3 and 4 h after the intraplantar injection of carrageenan (Winter et al., 1962).

* Statistical Analysis: all values are expressed as means [+ or -] SEM. Data were analyzed by ANOVA followed by Student-Newman-Keuls as the post hoc test. Results were considered statistically significant at p < 0.05.

Results

Table 1 shows the antinociceptive effect of the CEF on acetic acid-induced abdominal contractions in mice. The compounds caused 57 and 70% inhibition of contractions at doses of 5 and 10 mg/kg body wt., i.p., respectively. Oral administration was less efficient, and the absorption was somewhat irregular, producing 50 and 33% inhibitions at doses of 5 and 10 mg/kg body wt., respectively.

In the formalin test (Table 2), inhibition occurred predominantly during the second phase of the response. Thus, after i.p. administration of 5 and 10 mg/kg body wt. CEF caused a 76% response inhibition of the second phase. Pretreatment with naloxone partially but non-significantly reversed the CEF antinociceptive effect. As expected, morphine was very efficient during both the first (35% inhibition) and second (77% inhibition) phases and its effect was totally reversed by naloxone. Although 18 and 21% inhibition of the second phase was observed after the administration of 5 and 10 mg/kg body wt. of the CEF, this fraction was less efficient orally.

In the hot-plate test (Table 3), although no significant effect was observed at CEF doses of 5 mg/kg body wt., i.p., 73 and 43% increases in the latency to thermal stimuli were observed at doses of 10 and 20 mg/kg body wt., i.p., after 30 and 60 mm of drug administration, respectively. Increases of around 54 and 69% were also observed after administration at the dose of 20 mg/kg body wt., i.p., in the same observation periods. Interestingly, in both cases, the effect was long lasting, and 95 and 53% increases in thermal stimuli could still be observed 90 min after drug administration, at the doses of 10 and 20 mg/kg body wt., i.p., respectively. The CEF effect (10 mg/kg body wt., i.p.) was partially reversed by naloxone pretreatment after 30 min and reversed totally after 60 min of drug administration. No significant effect was observed after oral administration of CEF. The central analgesic effect of morphine was reversed totally by pretreatment with naloxone.

The CEF also showed an antiinflammatory effect on the rat paw edema induced by carrageenan (Table 4), after oral and intraperitoneal administration, in a dose-dependent manner. After intraperitoneal administration, significant results were observed at the highest dose, 40 mg/kg, at the third and fourth hour after carrageenan injection, with 48 and 35% reduction in paw volume, respectively. A small but significant decrease (22%) was also seen at the third hour, after administration of 10 mg/kg body wt., i.p. Significant antiinflammatory effects were also detected after the oral administration of 40 mg/kg body wt. of CEF, at the second, third and fourth hour, with decreases of paw volume on the order of 27, 59 and 50%, respectively. Indomethacin (10 mg/kg body wt., po) used as standard, decreased paw volume by 30% at the third hour.

Discussion

Flavonoids are a large group of naturally occurring compounds found in fruits, vegetables, grains, bark, roots, stems, flowers, tea and wine. A variety of in vitro and in vivo experiments have shown that selected flavonoids possess antiallergic, antiinflammatory, antiviral and antioxidant activities. Certain flavonoids possess potent inhibitory activity against a wide array of enzymes such as protein kinase C, protein tyrosine kinases, phospholipase A2 and others (Middleton, 1998). Other flavonoids (Manthey, 2000) potently inhibit prostaglandins, a group of powerful pro-inflammatory signaling molecules. Studies have shown that this effect is due to flavonoid inhibition of key enzymes involved in prostaglandin biosynthesis (i.e., lipoxygenase, phospholipase, and cyclooxygenase). Flavonoids also inhibit phosphodiesterases involved in cell activation. Much of this effect is upon the biosynthesis of protein cytokines that mediate adhesion of circulating leukocytes to sites of injury. Protein kinases are another c lass of regulatory enzymes affected by flavonoids. Inhibition of these key enzymes provides the mechanism by which flavonoids inhibit inflammatory processes (Manthey et al., 2001).

In addition, the deleterious effects of excessive release of nitric oxide (NO) have been implicated in tissue damage and inflammation. Recent results (Srivastava et al., 2000) suggest that tannic acid and polyphenols are potent inhibitors of NO synthase activity and NO production, and that these effects are independent of their antioxidant activity.

Chalcones isolated from natural products are known to possess several important biological properties, including antifungal (ElSohly et al., 2001; Lopez et al., 2001), leishmanicidal (Kayser and Kiderlen, 2001; Chen et al., 2001; Nielsen et al, 1998), and antimalarial (Dominguez er al., 2001; Ram et al., 2000; Li et al., 1995). Some other researchers also discuss the ability of chalcones to inhibit lipid peroxidation in rat liver microsomes (Rodriguez et al., 2001; Cos et al., 2001) and mouse liver (Machala et al., 2001) and to inhibit in vitro oxidation of low-density lipoproteins (Miranda et al., 2000). Another recent study (Phan et al., 2001) showed that several compounds including chalcones isolated from Chrornolaena odorata are powerful antioxidants, protecting cultured skin cells against oxidative damage.

It has been shown that reactive oxygen and nitrogen species contribute to the pathophysiology of inflammatory conditions. A recent report indicates that a chalcone derivative inhibited chemiluminescence in vitro, cell migration and eicosanoid and TNF-alpha in mouse air pouch injected with zymos an. This chalcone derivative also exerted inflammatory effects in the carrageenan paw edema (Herencia et al., 2001).

Hsieh et al. (1998) showed that 2',5'-dihydroxychalcone may be a promising antiinflammatory agent, because it is a potent mediator and cyclooxygenase inhibitor. Their results indicated that the andinflammatory effects of these compounds are mediated, at least partly, through the suppression of chemical mediators released from mast cells and neutrophils. Recently, Nakamura et al. (2002) synthesized fluorinated 3,4-dihydroxychalcones which were shown to be 5-lipoxygenase inhibitors and inhibitors of peroxidation in rat liver microsomes.

Correa et al. (2001) synthesized eleven chalcones which were tested for their antinociceptive activity using the writhing test in mice. The authors showed that some of these compounds, after intraperitoneal administration, caused potent and dose-related antinociception. Among the compounds tested, 3,4-dichlorochalcone was the most effective one and, in the formalin test, inhibited only the inflammatory pain (second phase of the test).

In the present work, we studied antinociceptive and antiedematogenic activities of a chalcone-enriched fraction (CEF) isolated from M. urundeuva, a medicinal plant largely used in Northeast Brazil for the treatment of several diseases, especially as an antiinflammatory in gynecological disorders. We showed a significant and dose-dependent antinociceptive effect of chalcones at low doses, after intraperitoneal administration, in the writhing test in mice. These compounds were less effective orally, probably due to a somewhat erratic absorption.

In the formalin test, which measures pain of both neurogenic (first phase) and inflammatory (second phase) origins, the CEF inhibited preferentially the second phase of the response, and the opioid system seems to have a role in this effect. A small but significant decrease in carrageenan-induced paw edema was also detected, after intraperitoneal as well as after oral administration of a higher dose.

Antiinflammatory effects have already been demonstrated with chalcones (Hsieh et al., 1998), and some chalcone derivatives have been reported to be potent chemical mediators and cyclooxygenase inhibitors. Hsieh et al. (1998) showed that some chalcones present strong inhibitory effects on the release of [beta]-glucuronidase and histamine from rat peritoneal mast cells stimulated with compound 48/80. Other chalcones exhibited potent inhibitory effects on the release of [beta]-glucuronidase and lysozyme from rat neutrophils stimulated with fMLP. Also, Lin et al. (1997) tested some chalcone derivatives for their inhibitory effects on platelet aggregation and activation of mast cells and neutrophils. They reported that the arachidonic acid-induced platelet aggregation was inhibited potently by almost all those compounds, and some also showed a potent inhibitory effect on collagen-induced platelet aggregation and cyclooxygenase. The authors suggest that the anti-platelet effects of chalcones are mainly a result of the inhibition of thromboxane formation. It is possible that at least part of the antiinflammatory effect observed in the present work may also be due to platelet anti-aggregation, as we observed that CEF inhibited around 20% of ADP-induced-aggregation in human platelets (data not shown).

Analgesic and anti-inflammatory effects have already been observed in flavonoids as well as in tannins (Ahmadiani et al., 1998; Ahmadiani et al., 2000). In the present work, the observed CEF antinociceptive effect is probably related to the opioid system. Although antiinflammatory effects have also been detected in chalcones, the present results are, as far as we know, the first report of peripheral and central analgesic as well as antiinflammatory effects of dimeric chalcones from Myracrodruon urundeuva.

Table 1

Effects of the chalcone enriched fraction (CEF) of Myracrodruon
urundeuva Allemao on acetic acid-induced abdominal constructions in
mice.

Group                          No. abdominal         %
                               contractions/         inhibi-
                               20 min                tions

Control, i.p. (29)             42.6 [+ or -] 2.27    -
CEF
 5 mg/kg body wt., i.p. (13)   18.5 [+ or -] 2.40 *  56.6
 10 mg/kg body wt., ip. (12)   12.6 [+ or -] 2.66 *  70.4
Control, p.o. (16)             42.8 [+ or -] 2.33    -
CEF
 5 mg/kg body wt., p.o. (8)    21.4 [+ or -] 2.72 *  50.0
 10 mg/kg body wt., p.o. (12)  28.8 [+ or -] 3.97 *  33.0

Values are means [+ or -] SEM of the number of animals (in parentheses).
Mice were treated with CEF 30 (i.p.) or 60 min (p.o.) before acetic acid
injection. The number of contractions was measured for 20 min, 5 min
after acetic acid administrations

* P<0.05 vs. control group.

Table 2

Effects of the chalcone enriched fraction (CEF) of Myracrodruon
urundeuva Allemao in the formalin test in mice.

Group                               Licking Time (sec)
                                    1st6 phase

Control, i.p. (19)                  45.6 [+ or -] 17.8
CEF
 5 mg/kg body wt., i.p. (8)         47.3 [+ or -] 2.49
 10 mg/kg body wt., i.p. (12)       48.7 [+ or -] 2.38
Nal+CEF(7)                          54.9 [+ or -] 5.92
Morphine, 5 mg/kg body wt., ip (7)  29.7 [+ or -] 3.82 *
Nal+Mor (7)                         60.1 [+ or -] 6.52
Control, p.o. (7)                   57.2 [+ or -] 8.13
CEF
 5 mg/kg body wt., p.o. (7)         43.0 [+ or -] 4.04
 10mg/kg body wt., p.o. (7)         46.1 [+ or -] 4.45

Group                               Licking Time (sec)
                                    2nd phase

Control, i.p. (19)                  18.8 [+ or -] 2.32
CEF
 5 mg/kg body wt., i.p. (8)         4.4 [+ or -] 1.73 *
 10 mg/kg body wt., i.p. (12)       4.5 [+ or -] 1.82 *
Nal+CEF(7)                          13.2 [+ or -] 4.19
Morphine, 5 mg/kg body wt., ip (7)  4.4 [+ or -] 1.97
Nal+Mor (7)                         13.6 [+ or -] 4.20
Control, p.o. (7)                   16.4 [+ or -] 4.53
CEF
 5 mg/kg body wt., p.o. (7)         20.6 [+ or -] 6.74
 10mg/kg body wt., p.o. (7)         14.9 [+ or -] 3.58

Values are means [+ or -] SEM of the number of animals (in parentheses).
CEF was administered 30 min (i.p.) or 60 min (p.o.) before formalin
injection, and licking time was measured as described in the Text. Nal -
nalaxone, 2 mg/kg body wt., s.c.; Mor-morphine, 5 mg/kg body wt., i.p.

* p <0.05 vs. control group.

Table 3

Effects of the chalcone enriched fraction (CEF) of Myracrodroun
urundeuva Allemao in the hot-plate test in mice.

Group                           T0                  T30

Control(8)                      14.3 [+ or -] 1.35  13.0 [+ or -] 1.01
CEF
  5 mg/kg body wt., i.p. (9)    13.1 [+ or -] 1.56  15.5 [+ or -] 2.09
 10 mg/kg body wt., i.p. (9)    15.6 [+ or -] 1.29  22.5 [+ or -] 3.76 *
 20 mg/kg body wt., i.p. (10)   13.1 [+ or -] 1.05  20.0 [+ or -] 2.04 *
Nal+CEF, 10 mg/kg body wt., ip  13.6 [+ or -] 1.36  17.9 [+ or -] 2.50
Mor 5 mg/kg body wt., i.p. (9)  16.3 [+ or -] 2.27  35.4 [+ or -] 2.30 *
Nal+Mor (9)                     13.8 [+ or -] 2.14  12.1 [+ or -] 1.64
CEF
 10 mg/kg body wt., p.o. (9)    13.4 [+ or -] 1.85  15.1 [+ or -] 1.23
 20 mg/kg body wt., p.o. (9)    10.3 [+ or -] 1.14  11.9 [+ or -] 1.27

Group                           T60

Control(8)                      12.7 [+ or -] 0.76
CEF
  5 mg/kg body wt., i.p. (9)    12.5 [+ or -] 1.33
 10 mg/kg body wt., i.p. (9)    18.1 [+ or -] 3.16 *
 20 mg/kg body wt., i.p. (10)   21.5 [+ or -] 1.78 *
Nal+CEF, 10 mg/kg body wt., ip  14.8 [+ or -] 1.82
Mor 5 mg/kg body wt., i.p. (9)  30.7 [+ or -] 3.38 *
Nal+Mor (9)                     11.5 [+ or -] 1.27
CEF
 10 mg/kg body wt., p.o. (9)    11.4 [+ or -] 1.88
 20 mg/kg body wt., p.o. (9)    12.9 [+ or -] 0.97

Group                           T90

Control(8)                      11.7 [+ or -] 0.94
CEF
  5 mg/kg body wt., i.p. (9)    12.5 [+ or -] 1.16
 10 mg/kg body wt., i.p. (9)    22.4 [+ or -] 1.61 *
 20 mg/kg body wt., i.p. (10)   17.9 [+ or -] 2.28 *
Nal+CEF, 10 mg/kg body wt., ip  8.1 [+ or -] 1.60
Mor 5 mg/kg body wt., i.p. (9)  20.7 [+ or -] 2.80 *
Nal+Mor (9)                     13.3 [+ or -] 2.15
CEF
 10 mg/kg body wt., p.o. (9)    12.5 [+ or -] 1.27
 20 mg/kg body wt., p.o. (9)    10.4 [+ or -] 1.04

Values are means [+ or -] SEM of teh number of animals (in parentheses).
Latencies to thermal stimuli were measured at several times (immediatcly
(T0), and at 30 (T30), 60 (T60) and 90 (T90)min), 30 min (i.p.) or 60
min (p.o.) after drug administration. Mor - morphine; Nal - nalozone, 2
mg/kg body wt., s.c.

* P<0.05 vs. control group at thesame period of time.

Table 4

Effect of teh chalcone enriched fraction (CEF) of Myracrodruon urundeuva
Allemao in carrageenan-induced paw edema in mice.

Group                                    Paw volume (ml) at the
                               1st hour             2nd hour

Control, i.p. (22)             0.15 [+ or -] 0.01   0.20 [+ or -] 0.013
CEF
  5 mg/kg body wt., i.p. (8)   0.15 [+ or -] 0.015  0.20 [+ or -] 0.009
 10 mg/kg body wt., i.p. (14)  0.14 [+ or -] 0.013  0.19 [+ or -] 0.011
 40 mg/kg body wt., i.p. (8)   0.11 [+ or -] 0.014  0.18 [+ or -] 0.016
CEF
 10 mg/kg body wt., p.o. (6)   0.19 [+ or -] 0.016  0.26 [+ or -] 0.024
 20 mg/kg body wt., p.o. (6)   0.16 [+ or -] 0.013  0.23 [+ or -] 0.023
 40 mg/kg body wt., p.o. (8)   0.12 [+ or -] 0.012  0.16 [+ or -] 0.009
Indomethacin
 10 mg/kg body wt., p.o. (6)   0.10 [+ or -] 0.011  0.20 [+ or -] 0.013

Group                          Paw volume (ml) at the
                               3rd hour

Control, i.p. (22)             0.27 [+ or -] 0.013
CEF
  5 mg/kg body wt., i.p. (8)   0.22 [+ or -] 0.01
 10 mg/kg body wt., i.p. (14)  0.21 [+ or -] 0.013 *
 40 mg/kg body wt., i.p. (8)   0.14 [+ or -] 0.013 *
CEF
 10 mg/kg body wt., p.o. (6)   0.33 [+ or -] 0.024
 20 mg/kg body wt., p.o. (6)   0.23 [+ or -] 0.013
 40 mg/kg body wt., p.o. (8)   0.11 [+ or -] 0.009 *
Indomethacin
 10 mg/kg body wt., p.o. (6)   0.19 [+ or -] 0.02 *

Group                          Paw volume (ml) at the
                               4th hour

Control, i.p. (22)             0.22 [+ or -] 0.014
CEF
  5 mg/kg body wt., i.p. (8)   0.17 [+ or -] 0.016
 10 mg/kg body wt., i.p. (14)  0.17 [+ or -] 0.013
 40 mg/kg body wt., i.p. (8)   0.13 [+ or -] 0.014 *
CEF
 10 mg/kg body wt., p.o. (6)   0.28 [+ or -] 0.018
 20 mg/kg body wt., p.o. (6)   0.21 [+ or -] 0.021
 40 mg/kg body wt., p.o. (8)   0.10 [+ or -] 0.009 *
Indomethacin
 10 mg/kg body wt., p.o. (6)   0.18 [+ or -] 0.018 *

Values are means [+ or -] SEM of the number of animals in parentheses.
CEF was administered 30(i.p.) or 60 min (p.o.) before carrageenan
injection.Edema volume was measured before and at 1, 2, 3 and 4 h after
carrageenan injection. Control -- carrageenan alone.

* P<0.05 vs. control group over the same period of time.

Acknowledgments

Authors are grateful to the technical assistance of Ms. M. Vilani Rodrigues Bastos. The work had the financial support of the Brazilian National Research Council (CNPq) and the Research Support Foundation (FUNCAP) of the State of Ceara, Brazil.

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G. Viana, Federal University of Ceara, Rua Barbosa de Freitas, 130/1100, 60.170-020 Fortaleza, Brazil Fax: ++55-85-242-3064; e-mail: [e-mail]